GENE EXPRESSION PROF'ILING OF W-INDUCED SKJN CANCER USING cDNA MICROARRAY TECHNOLOGY
Brandon George Howeii, Hon.B.Sc.
A thesis submitted in confodty with the requïrements for the degree of Master of Science (M.Sc.), Graduate Department of Medical Biophysics,
in the University of Toronto.
@ CopHght by Brandon George Howell, 2001
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Canada
Thesis for Master of Science (M.Sc.) degree November 2001 convocation
By : Brandon George Howeli (Hon.B.Sc.) Graduate Department of Medical Biophysics
University of Toronto
ABSTRACT:
Ultranolet (UV) light represents a complete carcinogen, and it is implicated in
causing the most prevalent form of skin cancer, basal ceiî carcinoma @CC). Revious
studies have show that W cm induce profound changes in cutaneous gene expression
leading to immune suppression, which contributes to cutaneous maiignanc y. This
nsearch thesis was undertaken to establish the use of cDNA rnicroarray technology for
examining gene expression profiles in the sicin. To establish the technique, we h t
examineci W effects on keratinocytes. Several W-regulated genes were discovered
including the endogenous angiogenesis inhibitor thrombospondin-1, wâich is potentiaîîy
relevant to W-ioduced carchogenesis. To examine genes regdateci in skin cancer more
directly, biopsies h m 50 BCC patients were analyzed by microarray. The differential
gene expression data obtained for these patients, in addition to the in vitro W-inducible
genes. may promote further understanding of genetic processes involved in non-
melanoxna skin cancer.
I would finit like to thanir my supervisor Dr. Danie1 N. Sauder, and supervisory committce members Dr. Robert Kabel, Dr. Dm Dumont and Dr.'~age Jothy for their invaluable guidance and support.
A speciai thank you to dl of my colleagues in the Damatology Resesrch laboratory at Sunnybrook & Women's Coilege Health Sciences Centre. Shabana, Wascem, Invin, Brandon Tuvel, Binghe. Hideaici, Qinchao, Hiro and Shige.. . 1 sincaely appreciate ail of your efforts, and u\anir you for crcating a wonderhl working enVu01~cnt.
nisnk you Dr. NoweU Solish for your surgicai utparise in providing the BCC specimens. You have a wondcrful seme of humour, and 1 am gram for your support end teachings. 1 also thank you Dr. From for your assistance with the BCC pathology photographs.
People at the Ontario Micmprray Centre: M d t h , Susan, Eric, Bryan and Neil, thanks for all of your help. I especially thank Chao Lu for aestiDg the OQ AMAD database, and spcnding COUII~~CSS hours with me discussing micraarray data
nisnlt you Patrick for your clustcring advice. Your &stance was most appnciated.
And 1 must not forget my wondahil family for thtir contiauous love and support: Yvonnc, Mom, Dad, David, Nanna, Clinton, Karen, Timothy, Kailey, Orna, John, Majory, Derek, Tcrtcnce, Keiiey, Todà, Zoya, Aunt Yvonne, Cht, Aunt hwL. Aunt Donna, Konrad, Adrieme, Mina, Bill, CMs, Donna, and the rest of the Meyer, Shtfiin, Howell and Gaston families.. . 1 love you all v a y much.
My d w Snends Mariano and Cecilia, and Clins... nia& you so much for your late-night philosopbicd discussions, srniles and laughtcr. 1 couid not have done it without you.
Thanks to my exallent microbiology fricnds Isabella, Kdy, Dareyl, Robert, Greg, men, Sbitley, Mario and Dr. McGavin. You make the fÏrst floor of the rrsearch wing a fun place to be.
To El Elena, Chris, Dareyl and Roben.. . nia& for keeping my dnems of beiig a professional tennis player live and smng.
Thank you Garry for the latcaight debates and motivation when 1 truly nceded it.
My othcr Sunnybrook colleagucs, 1 thank you as well for al1 of your assistance: Isabelle, Shawna, Dana, Wen, Ceha, Voskas, Jarnie, Nina, Stephcn, Asim, Paul, Zubin, Nicole, Shane, J-C, Barbara, Cap, Giuiio, Juin, John, Guido, Shan, Ssmmy, Davey, Rachel, Brian, Amy, Mina, Homa, Thaddcus, Micheiie MacPherson, Michefle Maaney, Cassmclra, Sue, Tas... and the list goes on.
And those Erit~lds et the Ontaio Cancer Instituie: Ivan, Lynn, Stcpbaiiie, Blair, Grant, Rusty, Jason, Kevin.. . and this list gacs on as well.
If 1 m i d anyone, 1 apologize. Thank you 9 for making graduate school an enjoyable timc, and fm helping me thn,ugh the ups and downs toward achieving îhis d e p .
ABSTRACï.. . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .ii ACKNOWLEDGEMENTS.. . . . . . . .. * . . . . . . . . , . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . *a. . . . , , . . ., , , . . , . . . . . , . , , , , . *. , . ,, iii TABLE OF CONTENTS.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .iv LET OF TABLES .................................................. . ............... ..................... . ...... ..v LiSTOFFIGURES ............................................... .. ......... .......... .......................... vi . . DEDICATION.. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . ..vu
Chapter 2 - LITERATUREREVIEW..........- .................. ... .... . ................ . ............ 3-20 The üitraviolet Spectrum Skin Cancer Epidtmiology UV Effects on Cutaneow Immune Function W Effects on Cytokine Biology W-Induced Apoptosis Oncogenes and Tumour Initiation CeIl Cycle, DNA Repair and W Effects Hedgehog Sigaalling in Basai Ceîi Carcinoma Twnour Angiogenesis and the nirombospondins S v Statement
Chapter3-
Chapter 4 -
Chapter 5 -
HYPOTHESIS ................................ . . . *.****.*.... *..*.*....*.. ........ 2 -22
MATERIALS AND METHODS .... .......... ..................................... ...... 23 -35 Keratinocyte Ceil Culaire In Vitro W B Irradiation Tissue Coilcction and Reservation RNA Extraction Reverse Transcription Semi-Quantitative RT-PCR Analysis In Viîm Transcription cDNA Mictoarray Analysis
Chapter 6 - DISCUSSION ......... ............. ........ ..................................... ...... 76- 102 Chapte; 7 - FüTUREDIRECl'lONS ................................... . . . . . . . . ........ . 103- 105
LIST OF TABLES
......................... TABLE 1A: Uprcgulation in Keratincytcs Aftcr UVB Exposure 53 - 54 TABLE 1B: Domgdation in Kcratinocytes Mer W B Exposure ..................... 55
TABLE 2A: Consistent Uprcgulaîion in BCC by Cluster Analysis ........................ 56 - 58 TABLE 2B: Sclective Upregdation in BCC by Cluster Analysis .......................... 59 - 60
TABLE 3: Consistent Downregulation in BCC by Cluster Analysis .................... .6 1- 62
TABLE 4A: Upregulation in at h t 25% of the BCC Patient Sample ..................... 63 O 64 . TABLE 4B: Upregdation in at least 30% of the BCC Patient Sample ..................... 65 66
..................... TABLE 4C: Uprcgulatioa in at llcpt 40% of the BCC Patient Sample 67 TABLE 4D: Upregulation in at least 50% of the BCC Patient Sample ..................... 68
TABLE SA: Domgdation in at least 25% of the BCC Patient Smple ................. 69 - 70 ................. TABLE SB: Dowwgulation in at least 30% of the BCC Patient Sample 71
TABLE SC: Downrcgdation in at least 40% of the BCC Patient SPmple ................. 72 TABLE 5D: Domgdation in at least 50% of the BCC Patient Sample ................. 73
.................................. TABLE 6: Other Potentiliy Uptegulatcd Guoes in BCC 74
.............................. TABLE 7: Other Potcntially Domgulated Gcncs in BCC 75
LIST OF FIGURES
FlGURE
FIGURE 1:
FIGURE 2:
FIGURE 3:
FIGURE 4:
FIGURE 5:
FIGURE 6:
FIGURE 7:
FIGURE 8:
FIGURE 9:
FIGURE 10:
PAGE . UVB Dose ûptîmization Using RT=PCR ....................................... 43
........................ In Vitro Transcription of Artincial Arabadopsis RNA 44
...................................... The Human 1700 Gene (1.7k) Microarray 45
.... Microarray Reveais Two-Fold Downreguiation of Thrombospondin4 46
.............................. ûptimization of RTIPCR for Tbrombo~pondin~l 47
Connrming Dowllteguiation of Thrombospondin- 1 using RT-PCR ........ 48
.............................. Histopathology of Nodular and Sclerosing BCC 49
....................... Hicrarchical Patient Cluster for BCC Microarray Data 50
Upregdaud Gcne CIusters for BCC Microarray Data ........................ 51
Domguiated Gene Clustas for BCC Microarray Data .................... 52
1 dedicate this thesis to my beautinil girirnend Y v o ~ e , and to rny beloved motber Linda. Both of you provide me endless love and support each and every day, and thae is no way 1 could have completed this work without your cherished miles and laughter. Thank you so much for everyîhing you do. 1 love you.
Non-melanoma skin cancer, namely basal ce11 carcinoma (BCC), is the most
cornmon form of cancer in humans, and its incidence continuously increases each yeat '. The major cause of BCC is ultraviolet 0 light emitted by the sun; the reason king
that UV is mutagenic (creating harmful DNA mutations in the skin), and it can induce
profound epi-genetic changes leading to suppression of the periphed arm of the immune
sy stem.
The work of this nsearch thesis has been to advance the understanding of UVB-
induced gene expression, and gene replation in BCC, by discoverhg new genes
involved in these processes. In a two-phase study we have utilizeâ a molecular biology
technique known as cDNA microarray technology to screen for modulated gene
expression of appmximately 1700 human genes relevant to immunity and cancer. To
establish this technique we first examineci W-inducible genes in vitro using a
keratinocyte cell culture model. Once the technique was established we sought to utilize
rnicroarrays to directly examine genes regulated in BCC.
cDNA rnicroarray technology is a relatively new technique in the field of
molecuiar biology that has revo1utionized the study of gene expression in biological
systems. Briefly, this technique ailows one to assess the gene expression levels for
thousamis of genes at one time when comparing a biological test sample to a normal
control. Conclusions can then be made about what genes are tumed 'on' or 'off in an
experimental system such as BCC. Microarrays have undergone extensive rehement
during the past few yean to the point where they are wi&ly used in molecular biology
research 2-8. Fortunately, we have been able to obtain a large number of cDNA
- mimmays fmm the Ontario Micmgrray Centre (Oiitano Cancer Institutes University of
Tmnto), which has aiîowed us to opthize the rniaoanay assay for andyzing the &in,
and to examine a large sample of 50 BCC patients.
Attempts have been made to ident0 genes that are potentiaiiy involved in the
pathogenesis of BCC. Some of the molccuies elucidated by this work may evenniaily
becorne therapeutic targets for the treamient of BCC and other forms of &in cancer.
Importantly, we are the first to report these types of shdies using cDNA microarrays to
analyze gene expression patterns related to human non-melanoma skin cancer.
The foilowing literature review offers an intuitive summary of the cumnt
knowledge on ultraviolet (ITV)-induced carcinogenesis, aiid skin tumour biology, to
provide a basis for interpreting the data of this research thesis. The topics reviewed
include: the W specmim, skin cancer epiàemiology, cutaneous immune function and
cytokine biology, UV-induced apoptosis, oncogenes and tumour initiation, ceil cycle and
DNA repair, hedgehog signalhg in basal ceii carcinoma, and tumour angiogenesis.
The Ultraviolet Smtruxn:
The photons of sunlight begin a series of genetic events in skin leading to cancer.
This has triggered concem surrounding decreases in the ozone layer of Earth's
atmosphere. A depletion of ozone permits more W radiation to reach Earth's surface.
The ultraviolet radiation spectrum is subdivided into three bands h m the longer to
shorter wavelengths: W A (32e400 nm), W B (280-320nm) and W C (<280 nm).
UVC is used in gehcidal larnps, but is not naturaliy found at Earth's surface due to
ozone abs0rpt;on in the stratosphm. UVB and W A are partiaiiy absorbed by the ozone
layer, however WB is most aîTected by changes in ozone concentration ? A major
biological effect of UVB is to induce dypyrimidine photoproduct mutations in DNA (e.g.
cyclobutane dimers and 6,4 photoproducts) by a direct photochernical mechanism,
whereas W A is absorbed by other cellular constituents and induces mainly oxidative
damage indirectly 'O. Excess exposure to UVB can have numemus adverse affects on
buman health. Some examples of this are sunbum, sLin cancer and catiuacts 9:i1;'2. Even
though UVB radiation has a direct carcinogenic effect (by causing DNA mutations), it
has ais0 becorne clear in the h t 20 years that exposure to WB can induce profound
_immunalogicai alterations was iniriall. sh~m uing animai stuâies. ~t bas
been àemomtrated that exposure of mice to W B radiation can cause development of
immunogenic s h cancers which grow in theV hosts because of WB-induced down-
15;16 regdation of immune surveillance . Alttiough the mechanimu of this dom-
regdation have not been hilly elucidated, there is evidence that suppressor T ceiis inhibit
anti-tumow immune responses '"19. Additionaiiy, cenain patterns of pst-UVB cytokine
signahg in the outer skia layer (epidemis) can hinder tumour antigen presentation in
the skia This is discussed further below.
Skin Cancer Enidemiolom:
Over the past decade there has been a ciramatic increase in the incidence of all
types of sicin cancer; including the potentially fatai malignant melanoma. Despite the
proven protectivc effects of topical sunscreens and extensive information campaigns, the
incidence of sunlight-induced skin cancer is expected to increase m e r in the future due
to the cumulative nature of factors induchg photocarcinogenesis, incnasing life
expectancy, participation in remational outdoor activities and decreasing atmospheric
ozone levels li12. An estimatecl one in six Canadians will develop skin cancer in hisher
lifetime, and this numba is incnasing by four to five percent each year. Skin cancer
currently represents one out of every thm new cancer diagnoses in North America
A newbom in Canada has a one in a hundnd chance of developing melanoma, and the
incidence of melanoma is doubling every years 12. Recently, Canada was
estimated to have p a t e r than fifty thousand new cases of skin cancer per year. UV-
induced skin tumeurs such as basal ceU carcinomas (BCC) and squamous ce1 carcinomas
(SCC) currently exhibit the most rapidly nshg incidence of al1 human tumours with an
esthgtgl rate of 600,000-800,000 aew cases per year in the United States alone "". Appmximately 95% of these can be attributed to excess UV exposure.
W Eilects on Ciitaneaus Immune Ii'crrnction:
One hallmark of acquired inmiunity is the ability to pnsent foreip antigens to T
ceils. Without antigen presentation, adaptive immune responses cannot be initiated nor
cm immunological memory be created. nie denciritic cell family is the most specialized
in antigen presentation 14. For this m o n , dendritic ceils are ofken mferred to as
'profcssional' antigen presenting cells (APC). Langerhans cells (LC), the most
peripherd member of the denàritic celî family, play a crucial role in antigen presentation
in the skin. LCs occupy the epidermis where Urey encomter an ooslaught of
environmental challenges. Therefore, proper LC function is an essential facet of the
pexipheral immune response. To fulfill the d e m d s of peripheral immunity, LCs
represent one of the most potent APC types in the body 25. LC function is drarnatically
impaired upon exposure to W iight. This is m e even for sub-erythemal doses of UVB
'6. For example, UVB d u c e s LC density and hinders LC maturation, migration, antigen
presentation, and pro-infiammatory signalling in the epidermis and regional lymphoid
tissue It is important to note that each of these consequences an likely
attributable to UV-induced alterations in gene expression, and have the potential for
accommodating malignant growth in the skin. Because LCs are located in the suprabasai
layer of the e p i d d s , where UVB cm mily penetrate, they are considend to be one of
the major targets of UVB irradiation 14. UVB cm induce a profound decrease in the
number of LCs present in the epidermis. Additionally, those LCs which survive UV
assault possess abnormal morphology (e.g. elongated dendrites) with deficient dendntic
- - - - - -- p s e y? UVB reduces the expr_tssion of costit@atory and accessory molecules on
the surface of LCs including: CD80 (B7-l), CD86 (B7-2) and CD54 OCAM-1) ''-? Evidence has shown that this hinders the abîiity of LCs to stimulate syngeneic and
aiiogeneic T ceils "". WB-irradiated LCs also fail to induce proper activation of
antigen-specific Thl clones, leading to T celi anergy Collectively, impaireci LC
function is largely responsible for the imrnunosuppression observed after UVB exposure
in vivo. This phenornenon reduces the capability of LCs to present tumout-associated
20-24 antigens, potentiaîly aiding in the onset of cutmeous maüpancy .
Keratinocytes (KC), the preàominant ce11 type in the epidermis, fwiction as a
prottctivc barricr against exogenous enviro~~mentai stimuli by producing keratin proteins
3? Similar to LCs, W light alters KC morphology and hct ion ". KCs can indirectly
Muence W-induced immune suppression via relcase of LC-inhibitory cytokines 20-23;40
Therefore, the effects of UV light on KC gene expression are equaiiy relevant to UV-
induced carcinogenesis. M a y of the KC-expressed molecules involved in this process
are discussed below.
A wide variety of cell types release polypeptides known as cytokines. These
patent molecules cm serve autocrine, paracrine and at times, endocrine funciions in ceiî
to cell communication 41'42. Cytokines that are produceci by leukocytes (white blood
cells) an often tcrnicd interleukins ". These mo1ecules interact with specifîc cytokine
teceptors in the membranes of their target celis. By interacting with these comsponding
rcceptors, cytokines can induce different responses (e.g. growth, differentiation or even
celi death). There are a number of differcnt familes of cytokines including: intaleukinû
,. - - --- - . (Il& hematopoietic colony-stimulahg factors CCSF). iaterfctons (IFN), immunogiobplin
supcrfamily moldes , p w t h factors, transfonning p w t h factors (TGF), tumour
necrosis factors 0 and chemohIes ". The role of cytokines in immune responses
has been an intense area of investigation over the past two decades. Our laboratory in
particultu has ken active in delineating the role of cytokines in cutaneous inflammation.
Endence has implicated cytokine dysreguiation in causing a variety of cutaneous
4345 diseases including psoriasis, contact dennatitis and W-induced carcinogenesis . Importantly, recombinant cytokines have also been used therapeutically to treat these
dematological conditions.
Cytokines, as mentioncd above, play a significant role in cell-to-ceU
communication. WB irradiation of hurnan epidermal KCs alters their expression of
cytokines. The creation of cyclobutane pyrimidine dimm by UVB plays an instigative
role in UUs process 'O. Profiles of cytokine expression may then modulate the actinty of
ceils of the immune system. Cytokines which have documnted expression by KCs
include: IL-1 (a and B), IL-3, IL-6, IL-7, ILS, IL-IO, IL- 12, TNF-a, TGF-BI, IFN (a, 8,
and y), leukaemia inhibitory factor 0, macrophage hhibitory factor (ME), nerve
growth factor granulocyte macrophage-colony stimulating factor (GM-CSF) and
platelet activating factor (PAF) In certain cases, when the expression of cytokines is
increased in KCs, it is for a gene that does not have constitutive expression. However, it
is often the case that W light induces cytokine expression in KCs to a much higher level
than base-line constitutive expression. Various stimuli such as UVB, LPS aad cytokines
themstlves cm induce expression of the above-listed cytokines significantly ". UVB irradiation upregulates almost ali cytokines analyzed to date. IL7 is one exception to
f- & iszdowp@td by WB. Bt~8~use IL7 i s a gpwth factor for epidamal T
cells, its downreguiation can be implicated in UVB-induced immune suppression M.
Many KC-exprcssed cytokines are mediators of cutaneous immune function. For
example, modulation of IL-1 and the L1 naptor (IL-IR) by UVB are important in
4t;S meàiating autocrine cytokine networks in KCs leading to upregulation of GM-CSF . GM-CSF c m then activate cells of the immune system such as monocytes and
macrophages. IL1 also plays part in the UV-induced exacerbation of the autoimmune
disease systemic lupus erythemtosus '*. IL-8 h a b a n shown to mediate inflammation
in psoriasis by creating expression of inducible nitric oxide synthase (NOS) in KCs of
pscwiatic skin lesions '%'. Certain KC-expresseci cytokines (e.g. TNF-a) are also
associateci with the development of so -ded 'sunburn cells', which are now known to be
KCs undergoing apoptosis. Apoptosis is aucial for eliminating WB-damaged cells in
the epidennis, and it plays an important role in preventing skin cancer.
UV-Induced A m ~ t ~ :
Induction of apoptosis following UV exposm appears to be a protective
mechanism to delete severely damaged cells that bea. the risk of malignant
transformation. W-mediatecl apoptosis is a complex process, and severai different
molecular pathways are involved 62. Some shidies have established associations between
certain molecules and apoptosis of KCs. For example, TNF-a can induce KC apoptosis
via signalhg through the TNP receptor (R)-1 (i.e. TNFRp55) ". The TNF-R
superf.amily includes a number of type4 transmembrane glycoproteins including: TNF-
Rp55, TMrRp75, Fm, nerve p w t h factor rcceptor (NGFR), CD27 and CD40 ".
Uiiportantly, expression of the Fas death nceptor hm been demonstrateci for KCs and
other sIM components ". Recent evidence has shown that Fas-induced KC apoptosis in
responst to ultraviolet iight, pnvents the accumulation of pro-carcinogenic p53
mutations by deleting W-mutated KCs ". Fas induces apoptosis in KCs via its
intracellula. death domain, and this has b a n supported by nceptor cross-iinking studies
using anti-Fas monoclonal mtibodies @. UV also seems to dkctly stimulate cross-
linking of Far 66:67. Fas-associated death domain (FADD) acts as an adapter protein in
bath the TNF-RpS5 and Fas apoptotic cascades, and is responsible for downstnam signal
transduction by d t i n g caspases a. Caspase-3, for example, cap then cleave the
catelytic subunit of DNA-dependant protein kinase @NA-PKcs) which is an important
end result in WB-induced apoptosis ". Lkewise, protein kinase C-delta (PKC-delta)
can be activatecl by caspase-dependent proteolysis during W-induced KC apoptosis .
This indicates the complex branching of signal transduction pathways which can occur
during the apoptotic process. Signahg via p53 contributes to the induction of apoptosis
by uprepuiating cell-surface expression of Fas, and also by early transcriptional
regdation of galectin-7, and by regulating expression of molecules in the Bcl-2 family
66" Galectin-7 is a beta-galactoside binding protein specifically expressed in
stratified epitheiia and notably in epidermis, but barely detectable in epidermal tumours
due to loss-of-function mutations in p53 ". Dyscgulation of the Fas/FasL-IL-1p
converthg enzyme (ICE) apoptosis signalhg pathway mentioned above is also believed
to be important in skin cancer etiology because inadquate apoptosis in the epidermis can
promote clonal expansion of KCs which have accruecl DNA mutations ". A recent study
using chronic W irradiation in a hairlcss SKH-hrl mouse mode1 demonstrated complete
loss of FasL expnssion after 4 weeks, with detection of p53 mutations in the epidermis as
- ---- d y - 1 we& aftcr irradiation began. Ttg ours which developed in this mode1
subscquently lacked expression of Fas-L indicating how important this pathway may be
to the initiation of s i ch cancer 74. An opposhg study has nprteù FasL expression in
vivo by BCC tumour cells, and this bas been associated with paitumoral T lymphocytes
that are undggoing apoptosis ''. Therefore, FasL may also play an important role in
aiiowing BCC to escape immune surveillance by deletion of effecuir T cells.
Onconenes and Tumour Initiation:
Extensive documentation has validated the role of UV irradiation as a tumour
initiator and promotcr, induchg both squamws and basal ceIl carcinomas. Certain WB-
indu& events can oppose the apoptotic cascade, and promote cell survival. For
example, the transcriptional activation and proper regdation of NF-kappa is knom to
be important to the apoptotic-mistant phenotype of epiâermal-derived KCs 76'6". The
major consequeilce of this is that cells which develop DNA mutations aud oncogenic
expression patterns may be permitted to proliferate and transfom. Studies have shown
overexpression andor activation of proto-oncogenes c-fodc-jun (AP-1) md c-myc in
KCs foliowing W B treatment 7" The upregulation of c-fos is mediated by WB-
induced activation of p38 mitogen activated protein base @38 MAPK) and extracellular
signal-related kinase @RK) 'l. Overexpression of c-myc in the epideds induces
proMeration, inhibits terminal differentiation and demeases the sensitivity of KCs to
WB-inducad apoptosis 82. Another W - i n d u d mechanism of immediate early gene
activation is via a dose-dependant induction of a putative apoptosis inhibitor temed 1W[-
llp22 in KCs "? EX-l/p22 is involved in NF-kappaB-mediated cell swival by
blocking Fas- or TNF-a-induced apoptosis in various cells incluciing KCs u-87. UV can
also stimulate agivation of the phosphatidyl-inositol 3-kinase (PU-K)/AICT/PKB
pathway through the EGF receptor @GF-R) M. AKT functions, in part, to promote ce11
survival by phosphorylating the Bcl-2 family member BAD and the ceil death pathway
enzyme, caspape-9 M. W-induced activation of the PU-WAICT pathway may enhance
survival of mutated KCs, thereby promoting skin cancer- This role for PU-K/AKT has
been found in s e v d other types of cancer. Hepatocyte growth factorlscatter factor
(HGFBF), a cytokine produceci by mesenchymal ceîis, cm also prevent WB-induced
apoptosis of primary human KCs via PU-K signaIlhg through the c-met receptor,
uitimatcly inhibithg caspape-3 activity ". In addition, moderate doses of W B cm
induce 6-fold inmeases in cyclooxygenase-2 (COX-2) expression in KCs, and this causes
a potent induction of the eicosanoid prostaglandin E2 (PGE2) W. COX-2 has received
enormous attention in recent years as a potential pharmacological target for preventing
tumour development ". Eicosanoids, mainly of the PGE2 species, have p w t h
promoting activity and they also induce regdators of angiogenesis (discussed M e r
below), including vascular endothelid growth factor (VEGW, basic fibroblast growth
factor (bFGF), PDGF, and endothelin-1 91. In addition, PGE2 has been implicated in
systemic W-induced immune suppression for its ability to induce an IL-4AL-IO Th2
cytokine cascade 92. The induction of COX-2 by üVB also implicates involvement of
members of the MAPK pathway including the MEKKI, SEKlIMKK4, p38 MAPK
caca& indu& by IL-l$ 93. Lastly, it has been shown that W B cm activate COX-2
expression by an indirect oxidative paracrine mechanism relatcrl to intracellular
giutathione levels 9?
a DNA Re- and UV F B ' :
Besides the reqhment for WB-damaged cells to delete themselves by
apoptosis, c d cells in the skin must repair themselves following UVB assault*
Howeva, if a cell that is geneticaiiy mutated by UVB does not adequately repair its
DNA, and d a s not undergo apoptosis, it may continue to prolif'rate causing mutations to
accumulate every tirne it undergoes mitosis. Dependhg on the normal differentiated
hct ion of a ccii, it will be programmecl to eiüm repair itself, or terminate itself. For
example, the basal KC ceil layer of the epidermis is comprised of stem ceils which give
rise to KCs that M e r differentiate themselves as they traverse the stratified epithelium.
Following WB-induced damage, basal KCs undergo ce11 cycle m s t and nucleotide
excision npair (NER) of their DNA in order to prolong their d e as stem celis. In
contrast, differcntiated KCs undergo apoptosis foIIowhg UVB-induced demage 95s.
Both of these processes arc reguiated by p53 ". Thus, it becomes important to
understand aspects of cell cycle conml followhg UVB exposusc. Studies have indicated
that W B can cause aitered gene expression of certain molecules involved in the celi
cycle pmcess including: p16, p2 1, p27 and p53 95*,98-1û4 , . ese molecules are important
in their interaction with cyclinlcyclin-dependant kinase (cdk) complexes. They function
to inhibit cdk enzymatic activity, and this prevents phosphorylation of the retinoblastoma
protein (Rb) leading to cell cycle amst 'O2. Several studies have suggested that the p53
tumour suppressor gene is involved in development of non-melanoma skin cancer
":lm. Very early on in the development of BCC and SCC p53 is mutated in a UV-
specific manner, or p53 is lost ":'Oo. This suggests that p53 is crucial in protecting
normal KCs h m the hamiful effwts of W. p53 transgeaic mice develop SCCs
-;- foIlo~&~g -W radiation 'y The c ~ n t understanding is
suppressor activity by promothg ce11 cycle anest at the GlIS
that p53 elicits tumour
phase barcier. Once this
occurs, p53 can rcgulate NER of the DNA. or pmmote apoptosis lm. However, many
other tumou suppssor genes act downstrtam of p53, and independent of p53, in these
signaihg processes. For example, p21 and pl6 have bccn implicated as having p53-
independent d e s in NER for KCs of the epidermal basal ce11 layer It has also
beai shown that W B can increase the levels of pl6 and p21 protein in normal human
epidermis and cultured KC models p27 has been shown to induce G1 m s t in
melaaocytes following W B irradiation 'O3. The recently cloned tumow suppnssor
candidate p33 (lNG1) can also be induced by W B in a marner independe~t of p53 status
'". p63, a p53 homologue highly expresseci in the basal layer of the epidamis, also
appcars to mediate ceîi cycle amst and WB-induccd apoptosis as show by stuàies in
p63 transgenic mice. These mice expuience a 40-45% decrease in e p i d e d apoptosis
following W B expasun as comparecl with nontransgenic littermates 'O9.
Cancer development requins the accumulation of numerous genetic changes
which are usually bclieved to occur through the presence of unrepaired DNA lesions. As
mentioned above, exogenous DNA-damaghg agents such as UVB can lead to mutations
in the absence of efficient enor-fkee DNA repair. Several DNA repair pathways are
present in living cells and are weU conserveci from prokaryotes to eukaryotes "O. These
inchde mismatch -air (MhrlR), photolyases, base excision, pst-replication repair and
NER. NER is the most versatile of the DNA repair systems and it recognizes and
eljminates a wide vaxiety of DNA lesiom; particulady those induced by WB "O.
Ceaain MMR genes are also relevant to BCC and these are discussed M e r below.
Time course expaimtnts for edy adaptive responses to W B in hairless Sal mice
have shown that DNA strand breaks in the basal layer of the epidermis wiii inmase
steadiiy up until6 hours after trcatment with an acute single dose of UVB "'. However,
understanding the significance of DNA npair to BCC has corne h m phenotypic
evidence in Xeroderma Pigmentosum (XP) patients. People with XP possess inheriteà
somatic mutations (autosomal recessive) for DNA repair enzymes in the NER system.
Persistence of unrepaired DNA damage produceci by exposure to UV light is associated,
in the XP syndrome, with an extremely high level of BCCs in sun-exposed sights "O.
Molecules involved in NER include XPA, XPB, XPC, XPD, XPF, XPG, CSB and
ERCCl Il2. 'Ihere is limited evidence to suggest that XPB, XPD and ERCCl are most
important for DNA repair capacity in BCC patients Il2. Certain polymorphisms in the
XPD gene at the exon 6 alle have been designated as a potential risk factor for both
sporadic and famiiiai BCC 113'1'4. Severai studies have shown that the presence of
genetic instability is associated with carcinagenesis. Genetic instability can be an
indication of MMR defects, and it is detected by changes in microsatellite regions in
DNA. One nsult of DNA replication emrs (RER) is a phenornenon calleci microsatellite
instability o. MI is characterized by length changes at repetitive loci scattereà
throughout the genome and/or loss of hetemygosity &OH), and it is a recently
rccognized genetic mechanism important in the development of some human cancers '15.
MI may also constitute a sensitive mark= for the presence of gene mutations Il6. MI has
been demonstratd in aggrrssive forms of cancer inc1uding colorectal carcinom and
malignant melauoma Il7. On the contrary, one study has revealtd that less than 5% of
non-melanoma skin cancers possess MI l15. Anothtr study has shown that some BCC
patiPltswdllbitMlinthc~afth~MSII2MMRgme(chrornosomc2p16loc~~)
and p53 (chromosome 17p21 locus) '16. However. it is of importance that W B can
augment expression of MSH2 by at least 8-fold implicating a d e for MMR in repairhg
UVB-induced DNA damage ll'. This has been subsmtiated by an
immunohistochexnistry sîudy cornparhg MSHî protein levels between normal human
skin and BCCs. Expression of MSH2 is consistentiy and strongly upregulated in BCC as
compand to unaffecteci epideds 'la. Intuitively, this abundant expression of MSH2
may be of importance for the genetic stability of BCC when compared to more aggressive
carcinomas, and this could potentiaily contribute &O the non-invasive phenotype of BCC.
Hedgeho~ Sienal i in~ in Bssll Ceii Cardn011~:
In search for the molecular basis of BCC, several hown oncogenes and tumour
suppnssor genes have becn analyzed. For exemple, there is a low incidence of activating
RAS mutations llsl" and p53 mutations exist in approx. 50% of BCCs However,
neither of these have aay correlation with the clinical fatures of the himours 1~1"'127.
The GTPase activating protein rasGAP has also been found mutated in a smaü subset of
BCCs that hold an aggressiw phenotype ? Interestingly, LOH studies on BCC
suggested that an important himaur suppressor gene was located on chromosome 9q
126127:1b132. Isolation of this gene in question eventually came from work on a familial
autosomal domiriant fom of BCC known as nevoid basal cell carcinoma syndrome
(NBCCS) or G o r b syndrome '". This tumour suppressor gene was cloned by two
independent groups and subsequently nameà PTCH, afta the drosophiîa developmental
gene 'patdied' lY:'? In addition to NBCCS, PTCH loss-of-fwiction mutations have
since been identified in sporadic BCG and BCCs of XP patients 12~127;1"'1321"'39. It is
also a p-siwty that the PTCH gent be inyolved in a &procal translocation with an
unknown gene on chromosome 16~13 la. Compiled h m this &ta it has been
estabfished that the pathogenesis of BCC involves constitutive activation of the Sonic
hedgehog (Shh) signaihg pathway, because the PTCH tumour suppressor gene encodes
a trammembrane Shh receptor and antagonist "'-lu. Most PTCH mutations produce
mcated proteins '", and the location of mutations dong the PT'CH protein has
delincateci s e v d important hioctional d o h s involved in interactions with Shh, the
oncogene 'Smwthened' (SMO), and the GLI family of transcription factors 13';14'. As
mutations in PTCH have been linkcd to BCC, it follows that mutations which have the
same effect as inactivation of PTCH shouid also lead to BCC fo~mtion. This is where
SM, S M 0 and GLI enter the picture. Overexpnssion of Shh lads to formation of BCC
in both mice and transgtnic human slcin Activating SM0 mutations have been
detecud in sporadic BCC 147'149. Members of the GLI family arc also involved as
foilows: Vertebrates have evolved at least thne separate GLI genes temed Glil, Gli2
and Gii3 These moledes are among the final targets of the Shh signai
transduction pathway 133. Expression studies on Gli3 have shown that it is not
overexpressed in BCC '":"';'? Glil expression is tightiy correlated with Shh pathway
activity under normal and pathologie conditions, however skin-targeted overexpression of
Giil in mice reportediy does not givc nse to BCCs ld3. In contrast, IU-Gli2 transgenic
mice possessing targeted GU overexpression in the basai ceiî layer of the epidermis
exhibit multiple sLin tumours characteristic of human BCCs The resemblance of
these muMe tumom to human BCC occurs at multiple levels. For example, they
contain activation of multiple Shh target genes incluchg PTCH, Glil and Gl.2. These
_ tumom have a shiny surface with a transiucent or peady appearance. Lady, the BCCs
in K5-Gli2 transgenic mice have prominent telangicctases, or tumour angiogenesis
characteristic of ceriain subtypes of human BCC '". Tumour Ando~enesis and the Tbiombosrn,ndins:
Skin tumours, and other solid tunom, are thwght to induce an 'angiogenic
switch' that attracts foxmation of immature spmuting blood vessels h m pre-existing
mature vessels. This process must occur in order for solid tumours to progress beyond
the size of a 2mm pinhead '". Inhibition of angiogenesis is currentiy one of the most
exciting areas of research in cancer therapy. This is, in large part, due to the fact that
tumour-specific anti-angiogenic therapy appears immune to the development of dmg
resistance by tumour cells lS6. Studies of the tumour microcirculation in BCC have
shown that the blood vessels found within BCCs have markedly abnormal architecture,
157 forming bizam, disorgarhcd aetworks characteristic of other solid tumours .
Moreover, increased microvessel density cornlates with a more aggressive phenotype in
BCC patients "*. Thus, it has been proposecl that malignant s b tumours may be
appropriate thenpeutic targets for experimental angiogenesis inbitors "? In normal
tissues, vascular quiescence is maintained by the dominant innuence of endogenous
augiogenesis inhibitors over angiogenic stimuli. In contrast, tumour angiogenesis (via
tnggering of the angiogenic switch) is induced by increased secretion of angiogenic
factors anà/or by downrcgulation of angiogenesis inhibitors 16'. One c m t
understanding is that tumour angiogenesis c m k initiated as a result of the hypoxic
microenvironment which develops in growing tumour masses. This hypoxic state lads
to activity of the hypoxia inducible factor @IR?)-la family of transcription factors which
lead tc-upnguiated wpressi~n of various isohms d vasculat endothelial mwth factor
(VEGF) VEGF, in conjunction with the regdation of numerous other angiogenic
activators and inhibitors, promotes vascuiarization of a tumour. Several molecules
nlated to this process have been discovend during the past decade '&. In terms of non-melanoma skin cancer, one fsmily of endogenous angiogenesis
inhibitors lcnown as the thrombospondins has attracted ment attention 15*1".
Thrombospondin-1 (TSP-1) was first discoverai in 1978 as a high molecular weight
glycoprotein released by platelets in respo~lsc to thrombin stimulation '". TSP-1 was
subsequently mapped to chromosome 15 via translocation studies in SCC cell lines
TSP-1 has ben ncognized as an endogenous angiogenesis inhibitor for about ten years
now, and extensive rcsearch on this molecule has placed it in an evolving group of
molecules termeci 'matricellular proteins' 16'. 'Ihe term 'matricellular proteins' was
coined by Dr. Paul Bomtein to describe secretcd macromolecules that interact with ceil-
surface receptors, extracellular matrix @O, p w t h factors, and/or proteases but do not
in themselves subserve strictly or exclusively structurai roles la. Other matricellular
proteins include TSP-2, SPARC (seccetecl protein acidic and nch in cysteine), tenascins C
-- and X, osteopontin and the syndecans '? An important role for matricelîuiar proteins is
that they are capable of disrupting cell-ECM interactions by counter-adhesion
, mechanism to induce the intermediate adhesion state la. For example, TSP-1 and TSP-
2 cm stimulate reorganilrition of actin stress fibas and disassemble focal achesion
complexes, but they only have minimai or negligible effects on celi shape. This fom of
ECM de-adhesion is associated with tissue rcmodeiling, morphogenesis, and vascular
gmwth The N-terminal heparin-binding domain (HBD) of TSP-1 and TSP-2
- - - containa the- focai a ~ ~ ~ ~ ~ g m h b g a c t i n t y lil, ltlowevert it is not ceaaia
TSP-2 hm indepmdent de-adhesive activity, or if it just reiies on its ability to
ma& metalloproteinase 2 2 - 2 ) '";ln. hning this past decade, severai studies have
taken place to ascertain the mechdsms of action that nsult in angiogenic inhibition by
TSP-1, as weii as to identify functiond xegions of the molede worth simulating ia the
form of chernical mimetics for treatment of cancer '"l". The TSP family cumntly
consists of five polypeptides termed thrombospondins 1-5 ln. TSP-1 and TSP-2 are
homotrimcric proteins that are similar in structure whercas TSP-3-5 exist as pentamers
anci ciiffer h m TSP-1 and TSF2 in key structural domains that have functional
relevance with respect to angiogenesis '? TSP-1 hm been more extensively studied, and
it has been shown that the biologicai fuaction of TSP-1 can be ceU type-specific
depcnding on the relative expression or activation of several TSP-1 receptors.
Calrcticuiin is one receptor that mediates the TSP-1 de-adhcsive activity mentioned
above 17'. However, certain receptors on endotheliai celis (EC) propagate specific
angioinhibitory signals h m TSP-1 andor TSP-2. For example, the WXXWXXW type 1
rtpeats in TSP-1 and TSP-2 bind to heparin sulfate proteoglycan (HSPG) and indirectly
inhibit binding of bFGF to ECs l". The CSvTCG type 1 repeats bind to the CD36
receptor which d t s the src-related kinase p59-fyn and p38MAPK to activate caspase-
3 and induce apoptosis of ECs lm. The TSP N-terminal NVR sequences cm interact with
a3B1 integrin to inhibit EC proliferation 18'. Interaction between inteph-associated
protein (IAP) and the RWWMWK motif in the TSP-1 and TSP-2 C-terminus inhibits
tube formation by ECs and blocks integin-dependent tyrosine phosphorylation of focal
adhesion hiriase (FAK) lm. It is also possible that TSP-mediated organization of coliagen
fitililp anbMMP-2 activiîg could regrilate SngiOgcPeSiS 'y. TSP-1 has a hQh binding
affiinity for the pro-angiogenic chemokine HGWSF, and SF-TSP-1 interaction inhibits
chernotaxis of ECs la. Lastly, it ha9 been demonstrateci in vitro and in vivo that TSP-1 is
a major activator of latent TGF-pl by releasing it h m its latency-associated protein
(LAP) In human breast carcinoma cell lines this occm through interaction with
the TSP-1 receptors aVB3 integrin and IAP lM. TGF-p signaIlhg through Smad4/DPC4
is then capable of inhibithg angiogenesis by an unlaiown mechanism '87-'89. Evidence
now suggests that TSP-1 and TSP-2 hold several functions in the skin. For instance,
these molecules play a very potent d e in skin tissue repair and wound healing, both of
which are proccsses involving vascular remodelling 'm. Importantly, TSP-1 and TSP-2
are capable of causing regrasion of SCCs in expuimental xenograft models (using A431
and SCC-13 cdl lines) via their potent inhibitory action on mgiogenesis The role
of these molecules is presumably similar in BCC pathogenesis, however this remains to
be studieci.
Swnmaw Statement:
Coilectively, the highlights of cutaneous photobiology and cancer research that
have been introduced above represent pieces of a broader molecular network involved in
malignant transformation of the s b . This research project was performed in attempt to
adjoh new molecules to this story. Using cDNA microarray technology to examine
WB-induced gene expression, and gene regulation in BCC, several candidate genes
have been identifid These genes (discussed in the following chapters) may aid in better
understanding the pathogenesis of BCC.
Living orgdsms musr integrate numemus molecular pathways in order to control
the expression levels of every gene in the genome. It is this dynamic process which
governs the fine line between health and disease, because a properly functioning cellular
system must maintain its phenotypic identity and homeostasis via gene expression.
Variation in gene expression accounts for much of the biological cüversity of humaii ceiis
and tumours. Non-mehoma sicin tumom. for example, are diverse in th& clinicai
prcsentation lg'. Ova the past n u m k of years gene expression has been investigated in
several disciplines, including tumour irnmunity, cytokine biology, cell cycle regdation,
apoptosis sipal transduction and angiogenesis (see Chapter 2). Candidate genes âom
each of these areas of research were prescrit on the cDNA rnicroarmys utiiized for this
thesis. With this in mW1, the following List of hypotheses was made for these
experiments:
1) Microarray analysis will elucidate several new genes modulated by WB.
2) Certain UVB-ngulated genes discovereà by microarray may be relevant to the
pathogenesis of non-melanoma skin cancer.
3) Dinerential gene expression data h m BCC patients will M e r improve upon the
cumnt molecular understanding of this disease, and provide insight into the
biolopical relevance of candidate genes in cutanwus malignancy.
4) Hierarchicai gene clusta analysis of the BCC microamy data will illustrate cornmon
a n d s in gene expression that exist for dinerent histological subtypes of BCC (Le.
noàdar BCC versus sclerosing BCC).
- 5)Ihc~v~of.stui;riatrg~aRUpvidea~sfafufureMesrch,dsidb
delincating potential therapeutic targets for skin cancer.
. - C
Keratlnocvte CeN Cul-:
Rimary ceii cultures of normal human keratinocytes (KC) were prepareâ as
pviously &sMbed with some modifications. Briefîy, nematal foreskin
specimens were washed five timss with phosphate-buffered saline (PBS) containing 2.0%
antibiotidantimycotic solution [ 10,000 units/mi peniciliin, 10,0Oûpg/ml streptomycin,
25pg/ml amphotcricin BI (Invitmgen, Groningen, Netherlands). Foreskins were cut flat
with connective tissue removed, and then digesteci overnight at 4OC in a 0 . 2 ~ filter-
sterilized 1% Dispase-II protease (Roche Molecular Biochemicals, Laval, Quebec)
solution containing 9.6mglml Dispase-II in Dulbecco's Modined Eagles Medium @-
MEM) supplemented with 10% heat-inactivated fetal calf s e n i m (FCS) (Invitmgen), and
1.0% antibiotic solution [10,000 unitdm1 penicillin, lO,ûûûpg/rnl smptomycin]
(Invitmgen). Epidamis sheets were then peeled fiom the dermis and stirred in 1X
ûypsin-EDTA (0.05% trypsin and 0.53mM EDTA) for 20 min. at m m temperature.
Trypsin was inhibited with q u a i volume of D-MEM supplemented with 10% FCS
(complete D-MEM). The tissuekell suspensions were passed through a sterile nylon celi
filter, end centrifuged fot 10 min. at 1000 X g. CeU pebts wem rssuspended in complete
D-MEM. These cells were seeded at 2-4 x 106 cells per lOcm pehi dish, overlaid with
cornpiete D-MEM, and cultund for 3-5 days (37OC and 5.0% C a ) to allow for ceIl
attachment. CeUs were then trypsinized (1X trypsin-EDTA), washed with PBS to
nmove residual FCS, and resuspended in KC serum-fiee medium (K-SFM)
supplemented with bovine pituitary extract (BPE) (2@3Opg/rnl) and recombinant human
epidermal gmwth factor (rhEGF) (0.1-0.2ng/ml) (Invitmgen) for passage into tissue
cPlma aasLn ICCs werr then maintainrAinKSEMcuhre d u m (37OC and 5.0%
Ca) with mtdium npiaceà every 2 days. Third-passage KCs were utilized for al i
experiments.
In Vitro W B Irradiation:
The in vitro treatment of KCs with UVB was performed as previously desdbed
l" with some modifications. Briefîy, third-passage KCs were seeded at 2-4 x 106 celis
per lûcm petri dish and utüued for wrpaiments at subconfiuence (80-8596 density).
Media was removed, celis were washed twice with pre-wanned PBS, and overlaid with
lml of PBS to maintain mois- while irradiated. Li& were removed the petri
dishes and the KCs were exposed to minimm erythcxm doses (lWD) of UVB (20-50
mkrn2) using a four-tube fluorescent W B lamp that etnits energy in the UVB range
(290-32ûnm) with an emission peak at 313nm. UVB dose was quantitated using an
International Light Inc. radiometer/photometa (Ealing Scientinc, Saint-Laurent,
Quebec). Following W B tnatment, PBS was removed, and KCs were cultured in K-
SFM (37OC and 5.0% C a ) for specified amounts of time priot to RNA extraction
procedures. Sham-treatecl conml KCs undement the same procedure, however normal
visible light was used instcad of UVB.
Tissue Collection and Preservation:
Human basal ceU carcinoma (BCC) and normal skin tissue speciwns were
obtained via Mohs' micrographie surgery (Women's College Hospital, Toronto, Ontario).
%CC tissue was collectcà upon prhnary excision of the skin ~mours. Nomial skin tissue
was eitha site-matchcd aml coliected pnor to corrective su~gical procedures, or a biopsy
of normal sltia h m an ana of simüar sun exposure to the BCC was obtained. Oncc the
- tissue was cxcised hm the patient it was h m e d k l y pleccd in RNAUeP RNA
preservation fluid (Ambioa, Austin, Texas) as per the manufacturer's instructions.
B M y , tissue specimens were cut into pieces less than 0.5cm3 and placed in 3rd of
RNAlaterTM in sealeà coiiection vials. Once placed in this solution the samples were
stond at -30°C until RNA extraction procedures were performed. Rior to RNA
extraction, tissue samples were thawcd at m m temperature, removed h m the
presewation fiuid, and RNA was extracteci aa pcr the method described below. For each
patient, routine pathology reports were reviewed to delineate the histological subtype of
BCC king analyzed (see Figure 7).
RNA Extraction:
Total RNA was extracted h m ceIl culture and skin tissue specimens by two
different methods. RNA extraction h m ceIi culture was performed using the RNeasym
mini spin column kit (Qiagen, Mississauga, Ontario) as pu the manufacturer's
instructions. Bnefly, media was removed h m the petri dishes, ceU monolayers were
overlaid with Qiagen RLT lysis buf5er. and ceiî lysates were collected using a sterile cell
scraper. Cell iysates were then passed ten times through a 20-G (0.9mm) needle and
oyrin8e to shcPr genomic DNA. The resulting wspensionr were mixed with 70% ethanol
by gentle pipetting, and then loaded into RNeasyTM columns for sequential washing of the
RNA. Total RNA was eluted h m the columns using nbonuclease (RNAse)-fke,
diethylpyrocarbonate @EX)-treated water.
RNA extraction h m skia tissue was paIormed ushg the RNAzol (guanidinium
thiocyanate-phenol-chloroform) method of Chomczynski and Sacchi Ig5 with miwr
modifications. Briefly, skin tissue specimms were removed h m RNAlaterm
..~&mW~sterilef~andplacedin~hnrtnmusttuksdesigriedfa
homogenization. Tissue specimns were kept below 2 û h g in weight to avoid saturation
of the RNAzol reagcnt. lm1 of RNA-Stat-6ûnl (Tel-Test '3" Inc., Frienàswood, Texas)
RNAml was then added to the specimens, and the tissue was homogenizeâ using a rotor-
stator homogenizer blade. Standard procedures were then foilowed for phenol-
chlomfonn-bascà extraction of total RNA. Al1 RNA pellets were dissolveà in DEPC-
treated water.
RNA preparations intendeci for reverse transcription - polymerase chah reaction
(RT-PCR) were tnated with 1-2 uni& 0 of RNAse-fhe dcoxyribonuclease @NAse)-1
(Novagen, Madison, Wisconsin) to remove residual genomic DNA contamination. The
amount of total RNA in each preparation was measured using a Beckman DU-70m
WNisible Spectrophotometer (VWR CanLab, Mississauga, Ontario) to calculate
absorbante at 260nm (&O)* RNA concentration = (A2~)(4O~g/mt)(dilution factor). The
A d A m was dso calcuiated for dl RNA preparations to indicate consistent purity of
total RNA.
Revene Transcri~tkn 0:
the RT protoc01 was as follows: 2pg of total RNA was incubated for 5 min. at 6S°C in
1Opl Dm-treatcd water containhg 30U of RNAsin RNAse inhibitor (Phannacia
Biotech, Dorval, Quebec) and 1.Opg of oligo(dT) primer [dT18] (Invitrogen) foilowed by
rapid coolhg on ice. The RT reaction mixture was then added to each sample. This
containcd 1Opî of 5X RT bMer [ 2 5 W Tris-HCl (pH 8.3). 3ûûmM KCl, 15-
MgClI] (Pharmaci8 Biotech), 25pi of lOmM dNTP mix [ l W each dATP, d m ,
dGWF dCrP], 5pL of 0.lM ditbkcitol 8xKL 500U of M o l o ~ t y MUMC
Leukemia Virus (MMLV)-Reverse Transcriptase (Pharmacia Biotech) reconstituted with
DE-treated wam to a total rcaction volume of 50pi. The reaction was incubated for
60 min. at 37OC, and then terminateci by a 5-min. heat inactivation at 9S°C. 2pl of the
resulting cDNA preparation was then utüized for each subsequent PCR reaction.
For the thrombospondin-1 (TSP-1) micromy confirmation experiments (see
F i p s 5 and 6) a différent RT protocol was utiiized to mimic the RT cDNA-labelling O
reaction conditions utilized for cDNA microanay analysis (described below). This
modifled RT protocol was as follows: 5pg of total RNA was combined with a RT
reaction mixture containing 8pl of SX RT bufk [25ûmM Tris-HC1 (pH 8.3). 375mM
Ka, 1SmM MgCid (Invitrogen), Z p l of lûmM dïWP mix [lOmM each dAT]P, d m ,
dGTP, dCTP], 1.5pl of 100pmoYpi (100~&M) AncT oligo primer [dTU)VN] (Co- DNA
Services, Kingston, Ontario). 4pl of 0.1M Dm, and 30U of RNAsin RNAse inhibitor
(Pharmacia Biotech) rcconstituted with DEPC-treated water to a total reaction volume of
40@. This mixture was then pre-warxned for 5 min. at 6J°C followed by 5 min. at 42OC.
400U of SuperScriptnlI Reverse Transcriptase (Invitrogen) was then addeci, and the
reaction was incubated for 2 hours at 4Z°C. The reaction was tcrminated by a 15-min.
heat inactivation at 70°C. 5pi of the resuiting cDNA preparation was then utilued for
each subsequent PCR reaction.
3 Oiisntitative RT-PCR Analvsis:
After reverse transcription to cDNA, amplification was c d e d out by PCR '%.
R i m r sets for human TNF-a and human glyceraldehyde-3-phosphate dehyhgenase
(G3PDH) w a e purchased h m Clontech Laboratories (Pdo Alto, Caiifomia). The
~ p x i r n a queace f a TNF-cw~IL 5'-TCrEY3GAACCCGAGTGACAAA-3' and
the downstnam primer sequence was 3'-TATCTCïCAGCTCCACACCA-5t to yield a
124 base pair (bp) product. The upstream primer sequence for G3PDH was
5'-TGAAGGTCGGAGTCAACGGAmGGT-3' and the downstream primer sequence
was 3'-CATGTGGGCCATGAGGTCCACCAC-5' to yield a 983 bp product. Primer
sets for human TSP-1 were purchased h m C m DNA SeMces baseâ on the work of
Bunone et al. '" with a minor modification. The upstream prima sequence for TSP-1
was 5'-CGTCCTG'ïTCCTGATGCATG3' and the downstream primer sequence was
3'-AACACGTCCTTCTGTCCCGG-5' to yield a 472 bp product. These TSP-1 primers
are iatron-spanning. This makes the 472 bp PCR product specific to CDNA exon
sequences, and genomic DNA contamination easy to identify (see Figure 5).
For the TNF-a UVB dose optimization experiments (se Figure 1) 8pl of PCR
cocktail mix was added to 2pl of cDNA RT template to fom a 10pi total miction
volume. This PCR mix contained 1p.I of 10X PCR bu&r [2OOmM Tris-HCl (pH 8.3),
5- KCl, 15mM MgCl21 (Invitmgen), 1.6pl of 1.25mM âNTP mix [1.25mM each
dATP, dTTP, dGTP, dCTP], lpl of 10-SM tetramethy~ammoniumchlonde ('MAC), OSpl
of 1ûûpM upstream primer, O S @ of 100pM downstream primer, and 0.5U of T q DNA
polymerase (Invitmgen) reconstitutcù with DEPC-trcated water to a PCR mix volume of
For the TSP-1 microamay coufimation experiments (see Figures 5 and 6) 45pl of
PCR cockteil was added to 5pl of cDNA RT template to form a 50pl total reaction
volume. 'Ibis PCR mix contained 5pl of 10X FCR bu&r [ZûûmM Tris-HCi (pH 8.3),
SO(IPaMKQ, 15mM M W (lavitrogai), INof 1 0 e ù M m mix [lW each dATP,
arrP. dGTP, dCïP], 1pl of 10% TMAC, 1pl of lOOpM upsûeam primer, 1pi of
1ûOpM downstream primer, and 2 . N of PlatinumTM Tuq DNA polymeme (Invitmgen)
reconstituted with DEPC-treatcd water to a PCR mix volume of 454.
AU PCR nactions were overlaid with 15pi of mineral oil to prevent evaporation
during high temperature incubations. Positive cDNA template controls were used in each
RT-PCR experimmt. Negative controls (RNA template instead of cDNA) were also
utiiizcd to ensun that the malyztd FCF2 products reprcsented mRNA gene expression
levels in the mode1 system. PCR cycles were performed in a Perkin-Elmer/Cetus
Thermal Cycler 480 (Perkin-Eimer/Cetus, Noma&, Comeciicut) using a 5-min. warm
up at 94OC foilowed by denaturation, mealhg and extension temperatures of 94OC (1
min.), 5840°C (1 min.) and 72OC (1 min.), respectively. Cycle number ranged fkom 25-
35 cycles depending on the optimized amplincation and saturation limits for each gene.
FCR products for the bouse-keeping gene (G3PDH), and the candidate WB-modulated
genes (TNF-a and TSP-1). were size-ftactioned (with qua1 loading) by electrophoresis
in a 1.0% agarose gel (made with 1X TAE buffer) and visualized using ethidium bromide
staining. Gels were imaged using Gel Doc 2000 (BioRad, Mississauga, Ontario), and
relative intensities for the PCR products were determincd using a Molecular Dynamics
Personal Demitometer SI and ImagtQumtrn software system (Molecular Dynamics,
Sunnyvale, California). Densitometer readings werc normalizcd to G3PDH to generate
relative gene expression levels for the candidate gaies. Experiments were repeated thne
times, and a two-taiîed Student's t-Test was usai to idenw statistical significance.
arabadopsis thulium (plant) RNA transcripts as a positive l
hybridization control for cDNA microamy analysis a saies of steps were nquired.
Arabadopsis plasmid @ARAB) was obtained h m the Ontaiio Microamay Centre
(Toronto, Onteno). pAMB maxiprep DNA was prepand h m competent DHSa e.coli
ceiis grown ovemight at 37OC in 200nil of LB plus Ampicillin growth media. A
QIAfilter plasmid maxiprep kit (Qiagen) was udlized to isolate pARAB DNA as per the
manufacturer's instructions. pARAB was then linearized with lOOU of the restriction
enzyme Sad (New Englmd Biolabs, Mississauga, Ontario) in a 20pl reaction mixture
containing NEBuffer 1 [IOmM Bis-Tris Propane-HCI (pH 7.0), lûmM MgC12, ImM
Dm supplemented with 100pghl bovine serum albumin (BSA) (New England
Biolabs). Linearizeà pARAB (approx. 4kb in length) was cleaned by
phenol:chloroform:isoamyl alcohol (25:24:1) extraction, ethanol precipitated, and
resuspendeâ in DEPC-treated water. A T7 polymerase run-off transcription reaction was
then performed. T&e template orabadopsis gene encodeci in pARAB is downstream of a
TI promoter, and RNA transcripts were gencrated using the T7 Riboprobem kit
- (Promega, Madison, Wisconsin). Bricfly, approx. 2pg of hcarized PARAB wss placed
in a 25pl reaction volume containing SpI of 5X transcription b d e r [2ûûmM Tris-HCI
(pH 7.9). 30mM MgC12, lûmM spermidine, 50mM NaCl] (Romega), 2.5@ of 0.1M
DIT, 4pl of lûmM rNTP mix [lOmM each rATP, rUTP, IGTP, CI?] (Romega), and
4ûU of recombinant RNAsin RNAse iabibitor (Romega). 15U of T7 RNA polymerase
(Romega) was added to the reaction for a 30 min. incubation at 37OC, and this was
repeated once to mdce a total incubation time of 1 hou with a cumulative enzymatic
activity of 30U of T7 RNA polymtrzt~e. The transcription naction was then treated with
lu of RNAse-frte RQ1 DNAse (Romega) to &grade the ün«LnZed PARAB template
and yield arcrbodopsis RNA transcripts (appmx. lkb in length). The in vitro transcripts
were then cleaned by phenol:chlorofom:isoarnyl aicohol (2524: 1) extraction, ethanol
prccipitated, and resuspended in DEPC-treated water. To avoid wasting the transcripts
via UV spectrophotometer Azso meesurements, an approximate concentration for the
purificd arobodopsis transcripts was determined by agarose gel electrophoresis in a 1.0%
agarose mini-gel with etbidium bromiàe staining. The band intensity for a smaU aiiquot
of arakibpsis -scripts was compared to bands of hown concentration in a 21
fisgxnent GeneRuierfM DNA molecuiar weight ladder (#SM0331, MBI Fermentas,
Vilnius, Lithuania). The concentration of the traiiscript solution was then adjusteci to
approx. 2ng/pî with DEPC-treated water pior to use in the RT mixture for cDNA
xniaoarray analysis (described below).
cDNA Microarrav Analvsis:
Total RNA h m test and control specimens was obtained as desaibed above. 5-
lOpg of each RNA preparation was utilued in separate RT reactions to incorporate
fluorescent labels into cDNA. The same appximate quantity of test and control RNA
was utilized pcr individual comparative hybndization experiment (Le. 10pg test RNA vs.
10pg control RNA). The two fluorescent labels used werr cyanine 3 (Cy3)- and cyanine
5 (Cy5) conjugated to dCTP (Mandel. Guelph, Ontario). R e c i p d labelling was
perfomed in dl experiments to aid in identification of true-positive hybriàization results
( i r each pair of test and control RNA preps was utilued in two separate mimanay
hybridizations with the fiuorcscent labels reverseci for the second hybndization). The RT
labei-g miction was as follows: 5-10pg of total RNA syspended in 20pi of DEPC- - -- -
treated water was combined witb a 19pl RT reaction mixture containing 8pi of 5X RT
bUna [ 2 5 W Tris-HC1 (pH 8.3). 375- KCl, lSmM MgCl21 (Invitrogen), 3pi of
2ûmM àNTP mix (without dCTP) [6.7mM each dATP, d m , dGTP], 1p.i of 2mM
d m , 1.5pl of lOopmoi/pl (100pM) AncT oligo primer [dTpoWVJ (Cortec DNA
Services), 4pi of 0.1M D'iT, 1pl of 2ng/pl orabadopsis thuliunu control RNA (Ontario
Micromy Centre), and 15U of RNAsin RNAse inhibitor (Pharmacia Biotech). 1pï of
eithcr 1mM Cy3-dCTP or 1mM Cy5-dCTP was then added to the RT reaction mixture
(dependhg on the label requinment) to make a total reaction volume of 40M. The
reaction was pre-warmed for 5 min. at 65'C followed by 5 min. at 42OC. 40ûü of
SupcrScriptnlI Reverse Transcriptase (Invitmgen) was then aàded, and the miction was
incubated for 2 hours at 4S°C. The naction was tenninated, and midual RNA
hydrolyseci, by addition of 4pl of 50mM EDTA and 2pi of ION NaOH (respectively) for
a 20-min. incubation at 65OC. The pH and salt concentration were then adjusted using
4pi of SM acetic acid. Robes were combined at this point ta malre a 100pl solution
(containirg both test and conml cDNA). The cDNA was precipitated with lûûpi of
isopmpanol for 30 min. at -30°C, centrifugeci for 10 min. at 12,ooOrpm, and washed with
70% ethanol. Aftcr air-drying the pellet, it was resuspended in 5pl of DEPC-treated
water and combwd with 35pl of hyôridization solution [20:1:1 mixture of DIG Easy
Hyb b e e r (Roche Molecuîar Biochemicals), lOmg/ml yeast tRNA (Roche Molecular
Biochemicals), lûmgiml sonicated caîf thymus DNA (Sigma-Aldrich, Oaidle, ûntario),
incubated for 2 min. at 6S°C and c w l d to m m temperature @or to use]. This 40)rl
hybridization nnxhae (containing both cDNA probes) was hmibmâ for 2 min. at 6S°C,
cooled to m m temperature, and loaded onto a human 1700 gene/fmtwe ( 12 ) spotteci
cDNA mimarray (Ontario Microarray Centre) with a 24x30m.m coverslip overlay.
Microarrays were ûansfemd immediately to pre-warmed 37OC humid hybridization
chambers equilibrated with DIG Easy Hyb (Roche Molecular Biochernicais) and kept in a
dark 37OC dry incubator for 15-18 hours. Microarray cover slips were removed with
gentle agitation in lx SSC bufEer at m m temperature. The microarrays were then
washeà thne consecutive tirnes for 10 min. (30 min. total) in stiining dishes containing
37OC lx SSUO. 1% SDS solution to remove non-specifically bound material. Residual
SDS was rcmoved by a final quick wash in 1X SSC at room temperatwe, aiid the
mictoarrays were dried over whatman papa (inside microscope slide boxes) via
Beclmian conter-top centrifugation [ m m temperature, 700rpm, 5 min.]. AU procedures
were kept out of light to prevent photobleaching of the Cy3 and Cy5 fluorophores.
Microarrays h m the KC-WB experiments were scatllled using the confocal GSI
Lumonics ScauArraym Microarray Analysis System (Packard BioScience, Biiîerica,
Massachusetts). Laser power and photo-multiplier tube (PMT) setting s were equilibrated
ushg fluorescence intensities f b m the arakbpsis congd features on the micromys.
ldbit TIFF microamay images were quantitateci using QuantArraytY ADalysis Software
(Packard BioScience), and global normalization of the Cy5/Cy3 feature fluorescence
intensity ratios (CySICy3 ratios) was pertormed using Quantarray Data Handler mams
(Ontario Microarxay Centre) for Microsoft Excel. Microarray hybridizations were
repeat6d at least three times to identify consistent gene expression changes.
Reproducible Cy5lCy3 ratios greater than or qua1 to 2, or less than or equal to 0.5, w m
used to idmtify gmes upreguïated or downreguiatcd by at lcast two-fold followhg UVB - - --% -
exposm.
Micmamys h m the BCC tissue study were scanncd using a GenePixm 4000
Microarray Scanner (Axon Instruments, Foster City, California), and GenePuN Pm 3.0
software (Axon instruments) was utiiizeà for data acquisition. P m settings were set as
high as possible without generating pixel saturation, and then equiiibrated using global
fiuorcsaace inteasities in the histogram function of the GenePixm Pro 3.0 software. A
line average of 4 was useà for data scans. l6-bit TIFF microarray images were
quantitatecl using GenePixTM Pro 3.0 software, and raw deta files for aî i BCC patients
were uploaded into the ncently developed Ontario Cancer Institute ( (XI) AMAD
micmamy database (Ontario Microamy Centre) for normalization and statistid
anaiysis. Globd nonnalization was p e r f o d on the ratio of the median net intensity
(background subtracted; CySICy3 ratio) for each gene. As a conmol, 12 self-vs.-self
microamay hybridizations with a normatized global CySlCy3 ratio of 1.000 were utilized
to defint false-negative nonnaliaxi Cy5/Cy3 ratio cut-offs for the 50 BCC patient data
set (see Chapter 5 and Chapter 6). The self-vs.-self hybridizations comprised of two co-
hybridized cDNA probes which were generated h m the same RNA @ooled BCC anci
normal s h RNA); one probe labeiled with Cy3-dCTP and the other with CyS-dCTP.
Norxnabd data was filted by signal to noise ratio and net fluorescence charnel
intensity (background subtracted) tbrrsholds in order to discard features near the noise
dettction levels. F-test (ANOVA) and t-test statistical methods were u t i l i d to perform
gene by gene comparisons between the BCC patient and self-vs.-self data sets. A
statistidy significant list of genes which arc upregdated or downreguiated in BCC was
denned. Similaritics among patients-edBCC histologic~ subtypes were then visualizeâ -=-&A--& - - --
using Quster and TneView software (Stanford University, www.miaoarra~s.org) for
hierarchical gene cluster malysis. Clusta anaiysis was pcrformed using base 2 logmithm
transfomm of the ratio data to linearize the gene expression changes. Uncentreù Pearson
correlation and average linkage clustering were then performed using agglomeraîive
hierarchical distance meiric processing in TreeView.
t u S - R E * S u L ~
ümization for N o d Rnmaa Keratinacvte Madel:
in preparation to examine UVB-induced gene expression in nomLal human
keratinocytes (KC) with cDNA microanay technology, it was first necessary to determine
what dose of UVB should be used. There were two nasons for this: a) different doses of
W B can cause dinmnt changes in gene expression, and b) it was postulateci that
microarray analysis would yield a large amount of data. Therefore, it was best to have a
weîi-de- dose of üVB before attempting to optimize the microarray assay. Using
primary cultured KCs (sa Matmals and Methods), a UVB dose-course experiment was
pe~ormed in the minimum erythema dose (MED) range between 20-50 mJ/cm2. At W B
doses of 20,30,40 and 50 d/cm2, the expression level of TNF-a was measured 12 hours
pst-UVB treatmcnt using semiquantitative RT-PCR. TNF-a expression was chosen
due to its hown regulation by WB. We hypothtsized that whatever dose of UVB
induceci the greatest expression of TNF-a would be an adequte dose for microarray
d y s i s . The results of these WB dose-course expcriments are shown in Figure 1.
Briefiy, the W e s t induction of TNF-a expression was seen folîowing exposure of KCs
ta 20 or 30 mIfcm2 of UVB (Figure 1; lanes 4 and 5). From this experiment, the 20
ml/cm2 dose was chosen for subsequent m i m m y malysis to elucidate new genes that
are regulated by W B in KCs.
EstsbUsbin~ the cDNA M i c m v Techniau%:
As a positive control for microarray hybnâization, artificial arubudopsis RNA
transaipts w m generated by in vitro transcription. This was done using a T7
polymerase ~IQ-off transcription reaction as dcsmibed in Materials and Metbods. Results
- - - frpm these transrription experiments are
plasmid comtruct @ARAB) was cut by
shown in Fi- 2.
restriction digestion
Briefîy, an arabodopsis
into a linear 4kb DNA
fragment (Figurc 2; lane 2). This linearizcd plasmid then acted as a template for
orabadopsis RNA transcription initiatecl at a Tï promoter upstream of the arabadopsis
codiiig region. Tmcripts of approx. lkb in length were generated (Figure 2; lane 3).
The saturated Ikb band in lane 3 indicates the efficiency of the transcription reaction (i.e.
a large amount of product was proàuced). The smeared appearance of lane 3 is Uely a
=suit of aruhdbpsis RNA transcript degradation while migrating through the agarose
DNA gel. The phenoYchlomfform extractcd arubu&psLr transcnpts (protein removed
h m the solution) are shown in the adjacent laiie (Figure 2; lane 4), and they migrated as
a c h lkb band. These puxifïed transcripts were then utilized in the labelling reactions
for cDNA microarray analysis.
A mies of microarray hybriciization expaimcnts were perfomed to optimize the
assay in terms of the cDNA probe labelhg reaction, hybriàization and washing
conditions, and parameters to be useâ for scaaniDg a human 1.7k microarray (see
Materials and Methods). An optimized mimarray image is shown in Figure 3. Also
describecl in Figure 3 are various aspects within the micromay image incluàing the grid
arrangement of the features, arabadapsis positive controls, and rabbit globin negative
conmls. Cy5 and Cy3 each have distinct excitation and emission spectra which dow
for àetection of the differential fluorescent labeiüng betwcen a test and control sample.
Upon close examination of the microamay image in Figure 3, one can see that three
different colours (mi, green and yellow) aise fbm the fluorescence emissions of the
miaoanay féanires. This pseudacolour repnsentation shows Cy5 enrissions as mi, Cy3
combination of the two as yeilow. Computcrized
these micnierr~y images yields a &CS of Cy5
quantitation of the
aad Cy3 emission
intensities for each gene. The fluorescence intensities are then n o d z e d (to globally
quilbrate the Cy5 and Cy3 fmture signals m s s the entire m i m m y ) , and Cy51Cy3
gene expression ratios arc obtained. Base two logarithms of the Cy5ICy3 ratios convert
the gene expression data to a hear scale (Le. a CySlCy3 ratio of 2 becomes equal to 1
a h base two logarithm conversion, and a Cy51Cy3 ratio of 0.5 becomes equai to -1).
Cy5/Cy3 ratios quai to 1 or -1 indicate that genes have either been turned on
(uprcguiated) or turncd off (downngdated) by two-fold in the test specimen,
respectively. Reciprocal labeiiing experiments (i.e. reversing the fluorescent labels used
for treatamt and control RNA sampks) were performed in alî cases to assist in
identifying mie-positive hybriàization resuits. in other words, reciprocal labeiîing was
used to ciinhate the chance that high CySICy3 ratios (Le. absolute value greater than or
equal to 1) were an artifact of different labels being incorporatecl into two comparative
cDNA specimens (sec Figure 4; described below).
Microarrav Analvsis to Detennine UVB-Inducible Genes:
Using the 20 mllcm2 W B dose detennined in Figure 1, RNA was extracteci fiom
KCs 12 hours after UVB- or sham-treatrnent. Mimarray analysis was then performed to
discover genes which are upreguiated or domgulated by at least two-fold after UVB
exposun. Genes which appeared to be upreguiated by WB are Listed in Table lA, and
those which appeared to be domgulated by UVB are listed in Table 1B.
In addition to Ugag the KC mode1 for estabïishing the micromy technique in our
laboratory, the WB-modulated gene lis& were utiiizeà to begh delincating candidate
- - -- -gents which may be relevant to W-induced carchogenesis. One gene in particular, a
potent angiogenesis inhibitor known as thrombospondin-l (TSP-1), was shown to be
dowmgulateù by two-fold following UVB treatment (Figure 4). The true-positive
micmamy hybriàization for TSF1 was supportecl by a reciprocal labelling experiment
(Figure 4; panel B). The potential nlevance of this TSP-1 downngulation to W-
induced cminogenesis is discussed later in this manuscript (see Chapter 6).
TSP-1 was chosen as a candidate gene to be utilized in conhmtion experiments
for validation of the micromy assay by semi-quantitative RT-PCR. Microarray
technology is an excellent way to discem which genes may be turned on or off in an
experimental model, however we felt it was important to show that the results of
micmamy hybidization cm be confirmai by a potentidy more sensitive method such
as RT-PCR. Optimization of the RT-PCR rraction for TSF-1 is shown in Figure 5. KCs
were treated with the same UV regimen as in the mimarray experiments, however the
extracteci RNA was treated with RNAse-fke DNAseI to remove minor DNA
contamjnation prior to the RT reaction. This yielâs RT-PCR experiments with proper
negative controls to iridicate that PCR products are a refïection of mRNA gene
expression levels (Figure 5; lanes 3,s. 7 and 9). To validate the two-fold downregulation
of TSP-1 identlfied by microarray anaiysis, an 18-hou the-course RT-PCR expriment
was performed wherc RNA was extrpctcd from KCs evay 3 hours foîîowing exposure to
20 mJ/cm2 WB. The results of this experimtnt are s u m d z e d in Figun 6. Averaged
TSPD1/G3PDH densitometer ratios from four separate expaiments confirmeci the two-
fold downregulation of TSP-1 a the 12-hour time point (Figure 6; lane 6). In addition, it
- -. .-..._ _-. .was .hm that UVB=indullld & w p r c ~ of TSP-1 began a h 6 houn, and
continue. up to the 18 hou tirne point (Figure 6; lanes 4-8). Having estabfished the
efficacy of the mimamy technique we sought to àirecdy address the gene expression
profiles of UV-induced tumours, by comparing human BCC specimens to nonnal skin.
To analyn BCC gent expression via microarray. surgical specimens were
obtaincd by a microscopie surgical procedure hown as Mohs' Micrograpbic Surgery.
BCC tumour specimens were compareâ to patient-matchcd normal skin specimens h m a
region of similar sun exposure (see Materials and Methods). Cihicai pathology reports
confinneci that two dinercnt histological subtypes of BCC were analyzed; nodular BCC
and sclcrosing BCC. Pathologicai sections of these two subtypes of BCC are shown in
Figure 7. From these images, the distinct pathology of nodular and sclemsing BCC can
be not id . Both forms of BCC arise h m the same celi type (Le. basal KCs), however
nodular and sclemsing BCC invade the dmnis and regulate their extracellular
environment much differcntly. Samples were colicctcd h m a total of 31 nodular
patients and 19 sclcrosing patients for microamy analysis.
Anaiysis of each patient was performed using reciprocal labelling (described
above) to yield duplicate data for each patient. Since each gene is spotted in duplicate on
the l.7k microarrays, this provided four pieces of ratio data per gene per patient (unless
fcatures reqlPind fiagging due to random spotting and hybridization defects). Duplicate
ratio data (four ratios per gene) h m each patient was averaged to yield one data set per
patient. The gene expression ratio data from cach patient was tbcn subjected to two-
dimensional hierarchical clusterhg to observe similarities between the patients' gene
expression profiles. The patient cluster is shown in Figure 8. The self vs. self control
hybridizations ail clustemi togeth (Figure 8; cluster A) to suggest that a valid statistical
clustering process was used. The patients appeared to cluster into 5 groups (Figure 8;
clusters B-F). Certain patient clusters appear to be enriched with either nodular or
sclerosing BCC patients, however clusters C-F aii contain both BCC subtypes.
Thrnfore; b m these experiments (and the genes spotted on the 1.7k microarray) it rnay
be Wfîcult to elucidate gene expression promes that are specific to either nodular or
sclemsing BCC. Figures 9 and 10 continue to iliustratc similarities in gene expression
across the patient sample. One cluster region revealed a set of 116 genes which appear to
have consistent upregulation across the patient sample (Figure 9; panel G) @sted in Table
2A). Within this set of genes there also appears to be a sub-cluster of 43 genes that are
more abundantly uprcgulated (Table 2A; bold print). 14 genes appear to have selective
uprcgulation in both noduiar and sclmsing patients (Figure 9; panel H) (iisted in Table
2B; bold print). Another subset of genes is potcntially more upregulated in sclerosing
BCC (Figure 9; J) (Table 2B). however this is only based on a few patients and requires
m e r investigation. In temis of downrcguiation, there appeared to k a consistent
ciuster region of genes across the patient sample (Figure 10; panel G) (listed in Table 3).
Another way to npresent the gene lists denved from cluster analysis is to display
the genes according to what percentage of patients showed upregulation or
downrcgdation of those genes. This type of analysis is shown in Table 4 (A-D) and
Table 5 (A-D). From the consistently upregulated gene list (Table 2A). the genes which
showed upregdation in at least 25% of the patients are listed in Table 4 A Those genes
upregulatcd in at least 30% of the patients are listed in Table 48, and genes upregulated
in at lcast 4û96 and 50% of patients arc listed in Table 4C and Table 4D, respectively.
From the consistently downrcgulatcd gene list (Table 3). the gens which showed
downregulation in at least 25% of the patients are listed in Table SA. Simüarly, genes
which appeared to be downregulated in at least 30%, Eb.96 and 50% of patients are listed
in Table SB, Table SC and Table SD, nspectively. This approach to analyzing the data
condenses the gene lis& in an attempt to delineate the most consistent gene expression
changes across the patient sample.
It is also of interest to note a short list of genes, some of which did not pass the
statistical ANOVA (F-test) Cnteria (@.OS) for inclusion in the hierarchical clustering
process, but did appear to be upregulated or downreguiated in a select number of patients.
These upregulated and downngulated genes are listed in Table 6 and able 7,
respectively. Further investigation will be quired to delineate whether these genes may
also be relevant to the pathogenesis of BCC.
UVB Dose (m J/cm2)
Finun 1 : UVB dose optimi zation usi ng RT-PCR. Normal hum an keratinocy tes were treated with vaiying doses of UVB as described in Materials and Methods. RNA was extracted 12 hours post-UVB treatrnent, and RT-PCR was performed to measure expression level s for G3PDH and the UVB-inducible gene TNF-a. PCR products were sire-fmctioned in a 1 .O% agarose gel and stained wi th ethidium bromide. Lanes 1 and 2 are negative and positive controls, respectively . Lanes 3 to 7 are the observed gene products dter the doses of UVB indicated (0-50 mllcm2). Densitometer anal ysis (normal ized to G3PDH) confi rmed the hi ghest induction of TNF-a fol lowi ng exposure to 20 and 30 mJlcm2 W B . This is shown by the bright TNF-a bands in lanes 4 and 5.
Fhun 2: In vitro transcription of artificial arabadopsis tha/ium RNA. ïhe mubudopsis plasmid constnict (PARAB) was ünemized by Sac1 restriction digestion, and utilimd in a T7 RNA polymerase nin-off transcription reaction as described in Material s and Methods. Prducts from each step of the protocol were size-fractioned i n a 1 .CF% agarose gel and stained with ethi diurn bromide. The agarose gel was run at hi gh voltage to m in im ize tim e for RNA degradation. Lane I shows a DNA m d ecul ar wei ght ladder. Lane 2 shows l ineari ted pARAB DNA template (approx. 4 kb in length). Lane 3 shows the outcome of the in vitro transcription reaction before RNAse-free DNAseI treatment to remove the pARAB template. The arabudopsis RNA transcnpts are approx. 1 kb in length. Lane 4 shows punfied mabadopsis RNA recovered fol lowi ng RNAse-fke DNAseI treatment of the transcn ption reaction, and phenol /ch1 orofonn extraction.
Hum an 1.7k Microarray (16 blocks)
- 110 Gene Block /(contains 220 Featum)
2 FeaturesIGene (Du plicate Spotting)
- Ara badopsis Tbaliana (yellow)
(Positive Con ttol Fei t u m )
- Rabbit Globin (Negative Control Features)
Figure 3: The hum an 1 700 gene (1.7k) m icroarray . This i s a spotted cDNA mi croarray containhg approx. 1 700 hum an cDNA gene clones spotted in dupl icate to give a total of approx. 3400 spots (fatures). To understand mi croarray temi nology , certain items are label led. The microarray contains a total of 16 subarray s tenned 'blocks' . Each block contain s approx. 220 features, representi ng 1 10 genes. Red features indicate hy bn dization by a cDNA probe (test sam ple) labd led with cyariine (Cy)-5, and green features indicate hybridization by a cDNA probe laôelld with Cy-3. Arabadrbpsis Thaltiana (plant) positive control features are present in the bottom row of each bl ock, and these fluoresce yeilow indicating equal hybridization of both Cy5- and Cy3-labelled tarabadopsis cDNA probes to the mi croamay (see Material s and Methods). Rabbi t g ld in negative contrds are dso present in the bottan row of each Mock, and the absence of hy bn dization to these features indicates mi ni mal non-speci fi c bi ndi ng (i . e. the positive test features throughout the ns t of the array resul t fiom speci fic bi ndi ng between human cDNA probes and human cDNA features).
Gene Expression Level
TSP- 1 (CyS/red)
Figure 4: Microarray anal ysis of WB-treated nomal human keratinocy tes reveal s 2- fol d downregul ati on of thrombospondi n- l (TSP- 1). Nonnal h m an keratin ocytes were treated with 20 m3/cm2 W B . RNA was extractcd 12 houn post-UVB treatment, and cDNA mi croarray hybri diration was perfonned to compare UVB-induced gene expression to a sham-treated control. In panel (A), shm control cDNA was label led with Cy 3 (green), and WB-induced cDNA was labelled with Cy 5 (red). The green features for TSP-1 were quantitated to show a 2-fold downregulation of TSP-1. Adjacent is a scatter plot of the gene expression ratios (base two logarithrn) for every gene fiom this microarray hybridization. TSP-I is indicated with an arrow to be among a d e c t group of genes that are downregulated by at least 2-fold. Reci procal labding experi men ts with the Cy 3 and Cy 5 dy es reverseci y ield the sam e resul t (B). In this case, the red features for TSP-1 quanti tate to show a 2-fd d downregulation of TSP-1. The scatter plot for thi s reciprocal hybridization (B) re-iterates that TSP-1 is stil l among a select group of genes that are downregulated by at least 2-fold following UVB irradi ation (i ndicated with an arrow).
Finun 5: Optimizati on of RT-PCR for TSP- 1. Normal human keratinocy tes were treated with 20 mJ/cm2 UVB. RNA was extracted 12 hours post-UVB treatment, and RT-PCR was peiformed to detect expression of G3PDH and TSP-1. Control reactions were performed to demonstrate that pure RT-PCR products for G3PDH and TSP-I were generated when RNA was treated with RNAse-free DNAseI pnor to reverse transcription (RT). PCR products were sire-fractioned in a 1 .O% agarose gel and stained with ethidium bromide. Lane 1 shows a DNA mdecular weight ladder. G3PDH pnmers were used in lanes 2 to 5. Lane 2 shows the G3PDH PCR product (983 bp) when RNA (without DNAseI treatment) is used to make cDNA. Lane 3 shows the G3PDH PCR product when DNAseI-treated RNA i s used to make cDNA. Lane 4 i s the negative control reaction using RNA (without DNAseI treatment) as the PCR template. The band in 1 ane 4 i ndi cates DNA contami nation in the PCR. Lane 5 is the mie negative control for G3PDH using DNAsel-treated RNA as the PCR template. Lanes 6 to 9 exem pli 5 the same order of reactions, however TSP- 1 pri mers were used i nstead. The DNAtontaminated negative control in lane 8 (700 bp) is larger than the TSP-1 cDNA PCR product (lanes 6 and 7; 472 bp), because the TSP-1 primers are intron-spanni ng. Collectively. lanes 3, 5 , 7 and 9 show optimized RT-PCR experiments with proper negative controls for each gene.
Time Cou rse Post-UVB Treitmen t (HOURS)
B 0.7 i
1 0.6 1 (* Std. Dtv.) (n = 4)
T l l t
' C 3 6 9 12 15 18 Time Cou rse Post-UVB Treatmen t (HOURS)
F i m e 6: Confinning downregutation of TSP-1 using semiquantitative RT-PCR. Normal human keratinocytes were treated with 20 ml/cm2 UVB. RNA was extracted every 3 hours fdlowing W B treatment to form an 18 hour time course, and RT-PCR was perfonned to detect expression of G3PDH and TSP-1. PCR products were size- fmctioned in a 1 .O% agarose pl and stained with euiidiurn brornide. A representative gel is shown (A). The negative control is in lane 1. Lane 2 shows the sharn-treated control. Lanes 3 to 8 show the expression l evel s of G3PDH and TSP- 1 for the 18 hour time course. Downregulation of TSP-1 c m be seen starting at the 6 hour time-point. Densi tometer anal ysi s of 4 separate experi ma t s confi rmed the 2-fold downregul ation of TSP-1 (pc0.05) that was aiginally identifteci by cDNA m i c r m y analyPs (B). The average TSP- I/G3PDH densi tometer ratios Std. Dev. (n=4) are plotted in di gnrn ent with the gel shown in (A). A linear regression trend üne is included to illustrate the progressive downregulation of TSP-1 expression.
Nodular BCC - 40X Sclerosiag BCC - 40X
Nodular BCC - 250X Sclerosing BCC - 2SOX
N = 31 Patients N = 19 Patients
Fisure 7: Routine hi stopathology sections for nodular BCC and sclerosing BCC. Sections are stained with hematoxylin and m i n . The dark purple regions in the epidermis and demis represent Nmouf (marked with arrows). Low and hi& magnification views are show. Nuice the different pathological behaviour of these two subhlpes of BCC . For this large-scale mi croamay study , a total of 3 1 nodul ar speci mens and 19 sclerosing specimens (plus normal skin tissue) were exci sed by Mohs' Micrographie Surgery. The surgical sarnples were preserved as described in Materials and Methods.
Finrre 9: Upregulated gene clusters for BCC microarray data. A gene by gene compdson was made between al1 BCC patients and the self vs. self control data, using ANOVA (F-test) anal ysi S. Genes for which differences were detected among the group (p<O.05) were utilized in the clustering process (58 1 total). Hiemrchical cluster M o n s showing gene uprelpilation are shown here. Bright red regions i nd cate at leiist 2-fold upregul ation . Al ong the top are the patient clusters from Fi mre 8 (A-F). Note the consistent M ack shade for the sel f vs. self cluster (A). The upper m q p ifkd image (G) il lustrates genes which appear to have consi stent upregulation across the patient sam ple (pink d uster tree). The genes tiom this cluster region are listed in Table 2A. The lower magnifiecl image (H) illustrates a cluster region with seledve upregulation across the patient sample (inâicated with arrows and brackets dong the bottom). Genes in the lower hslf of panel H are potentially more upregula ted in sclerosi ng BCC (0. Ail genes cl ustered in panel H are 1 isted in Tabl e 2B.
Consistent downregulation across patients (green)
Figure 10: Downregulated gene clusters for BCC microarray data. A gene by gene cornparison was made between ail BCC patients and the self vs. self control data, using ANOVA (F-test) analysis. Genes for which ciifferences were deteaed among the group (pC0.05) were util ized in the cl ustering process (58 1 toel). Hierarchi cal cluster regions showing gene downregulation are shown here. Bright green regions indicate at least 2-fold downregulation. Along the top are the patient clusters h m Figure 8 (A-F). Note the consistent black shade for the self vs. sei f cluster (A). The ma@ fi ed image (G) i ii ustrates genes which appear to have consistent downregulation across the patient sarnple (pink cluster tree). The genes from this cl uster region are l isted in Table 3.
genes which seem interesthg upon first inspection.
IMAGE 1 Gan N a m CIancID 22629 23241
Peptide traasporter involveci in antigen pmcmbg (TU-1) Hypothetid protein KïAAOl21
24652 31382 37462 38551 5 1229 130126 130843
150801 153025 155460 1557 17 155723 158998 173269 178645 178779 182473
1 861 32 196189 200227
204686 240647 241629 245 162 245662 260153 261760 266318 269273 278 195 282023 299251 299724 302042 302190 323326 323777 325 121 341240 342070 343563 3441 16 359630 359898
~eÏnatopoiet-c progeMtor all antigen CD34 AXL oncugene (tyrosine-protein kinase ll~ctptor UFO prcmsor) Protein argininc N-mcthylbransf~~ll~e 2 Coagulation factor Vm @racorsulant componc11t) Restin (CtIP-170) (Rad-Stanbeq intamediate filament-asmciated protein) Sodium-dependent n o m h a h c transporter 0 Platebtderivad endotbelid cell p w t b factar (PD-ECGP) (giiostrrtin) (thymidin4 ~hospm'l-) Von Hi@-Udau discase tumor suppmmr ~~a inbiiitory factor 0 (me.lluiorna~vaii LPL inhibitor) Retinoic acid recqtor, alpha4 (RAR-ALPHA-1) ïmmunoglobuiin-associated B29 proiein (Ig-ka) (CD79B) Vasoprash VIA rcccptor (antidiwtic hormone t'cccptor 1A) Ras-relatai protein RAB- l IB (GTP-bindiag protein YPM) Acetylcholin~.rnanc Mitochondrial stress-70 protein (GRP 75) (mortalin) Guanine nucleotide-b'mding protein G (Om Nuclcar f ~ r NF-KAPPA-B plûû subunit (contains NF- KAPPA-B p52 subunit (oncogene LYT-10) E-aelectin (endothelial leukocyte a8iesion m o l d e 1) (ELAM- 1) CytochromeBS Ubiquitin-conju8ating enzyme E2-32 kD complementing (ubiquitin-protein lig=) Phaspholemman 3-hyclmxyantbranilate 3,4dioxygenase Metallothionein 2 Faritin beavy chah Phma kalikdn precursor (khbogenin) (Fletcher factor) P59 protein (HSP binding immunophilin) (52 kD FKSM binding protein) Hypothetical protein KIM0274 AF-6 protein Hi&-aninity CAMP-sptcific T,S'-cyclic phosphodicsterase Rotein-tyrosine phosphatase, delta Zinc finger p te in 15 1 (MIZ1) DNA-directed RNA polymerases 1, II, and ïü 7.0 Id) polypeptide (ABC10-alpha) Dystrophin Homcobox protein HOX-A5 (HOX-1C) Zinc fiaga protein HRX (ALL-1) TUmr neaosis factor, alpha-inducad protein 1 Discs, iarge homolog 3 (maguk p55 subfamily membcr 3) Alpha crystallin B cbain (R-thal fiber componcnt) Hydroxyacylglutathione hydroiase (glyoxalase II) Roto-oncogene tyrosine-protein kinase ABL (c-abl) MAP kinase-activated protein kinase 2 (MAPKAPK-2) Transformation upreguiated nuclear protein (TüNP) Cathepsin IC (Cathepsins O, X and 02) Diamine aatv lWc~l l se
Table 1A Conbued
c36Q756 361 123 364351 364977 365630 376277 428098 469973 48695 1 491 128 501553
splicinpfaCtatsc3L -
ADPfATP transloasc 3 (daine nucleotide trpnslocator 3) Suinclthreanine procein phcqhtasc 5 matelct factor 4 (ONCOSTATIN A) hmhin, alpha-3 (@grin 170 IED subunit) 26s proteasorne subunit S5B (KIAA0072) Alpba-fetoprotein pmumx (alpha-fet.@obuW cAMP&pcn&nt plottin kinase type 1. beta r~gulatory chain Tumm n~closis factat r#.lleptor 1 (m-R-PSS) Serine/thrto~pmtein kinase 1 1 Lambh, alpha-2 chah
-- - - - - -.. m l B r B n c s which appear to be dowprtgulatcd by at least %fold in nurmai human kcratinocytes (12 hours afta atposurc to 20mllcm2 WB). An '*' is placed next to soxne gents which seem interesting upon fkst inspection.
IMAGE 1 GcncName Clone ID
21815 2256û 3 1 154 39505 1 164 1 1 124526
132602 13659û 14150 1595 12 163209 187 164 19728 1 208059 232390 265328 266438 27 1324
291361 298689 321 758 324239 362798 47OOl8 484578 488534 683334
Dualspecificityproteinpùosphatase6(PYSTl) Extracellulat signal-related kinase 3 @RK3) @97-MAPK) T-lymphocyte mnturation-associatcd protein P p t c i n ( m c l a n ~ s p e c i n c ~ p t c i n ) Corticottopin-releasing factor binding protein (CRH-BP) Probable ubiquitin carboxyl-terminal bydrolase HAUSP (de-ubiquitinating enzyme) (herpes virus associatecl ubiquitin-specific protease) Methionine aminopeptidase 2 (initiation factor 2-8SSOCiated glycoprotein p67) Tissue factor pramsor (mgulation k b r m) (thromboptastin) (CD142 antigcn) Macrophage colony stimulating factor-1 (CSF-I) (MCSF) Integrin, dpha-6 (VLA-6) Zinc hger protein 35 TISI 1B protein (EGF-response factor 1) (ERF-1) Delta-type opioid rcccptor @OR-1) Heaî shock cognate 7 1kD protein Heterogcne~us nuclear r ibonuc l~ te in H (HNRNP-H) Cavcotin-1 Nuclaolin (protein C23) N-ll~etylglucosaminyl-phosphatidylindtol biasyntbetic protein (phosphatidyiinositol giycan complementaîion class A) CytochromeC Probable RNA-depcadent helicase p68 (dead-box protein p68) nibulin, alpha4 chah Thromôospndin 1 Plg-associated splicing factor (PSF) Dipeptidyl-pcptidaschnsf~ I pmmsoc (Cathepsins C and J) T-complex protein 1, gamma subunit (TCP-1-gamma) Monocyte chernatactic protein 1 (MCP-1) Rotcasorne component MECL-1 precursor
_.-_ _ - - 2a:Gamwhichappeer ta k~nnsintGntl)tupreguiatcâ across the BCC patient samp1t. These gmcs corne h m the cluster region of p d G 9). Gsnes listed in bold print wm&m a sub-cluster within panel G which nprestnts a group of the most commonly upregulated gents 8 m s s the patient sampk. An '*' is placed next to some gencs which seem intercsting upon f in t inspection. -
IMAGE h m 347096 2595 12 266838 358449 264297 429361 502990 206391 298825 157828
153025 152469 488548 47 1667 491272 3219û7 305406 346482
265328 195803 5005 1
327676 471 144 264528 22658
471588 296702 195429 324338 487 134 262425 267800 184028 470591 345378 135050 362795 18241 1 502754 361531 266005
Hs.104125 Hs.77326 Hs.7957 Hs.44585 Hs.16003 Hs.89626 Hs.75932 Hs.74598 Hs. 1299 14
Hs.2250 Hs. 169832 Hs.239138 Hs.21486 Hs.1501 Hs.692
Hs. 172216 Hs.95972
Hs.296324 b.89538 Hs. 156346 Hs.70070 Hs.74047 Hs. 194657 Hs.274402 Hs.93 183 Hs.251415 Hs.27990 Hs. 169300 H9.183153 Hs. 1466 Hs. 153961 Hs.999 10 Hs.93649 Hs.388
Hs.22554 Hs.505 H9.738 Hs.1116
Hs.1-0
UniGene Chistcr Hs. 152936
adeayly1 Gy&-- protein insulin-iike growtb f m binding protein 3 &II&DC Acam;"R'E RNA-gPCCific tumor protein pS3-binding protein 2 tctiaoblaatomr-bindiDg~4 parathymid hormone-like hormone Nletbylmaicimide-sensitive factor attachment ptein, alpha polymetase @NA directed), delta 2, rcgulatory subunit (Som) l l ~ ~ ~ t e myeloid leukemirr (ml) 1 oncogene; runt-nlatcd transcription factor 1 leukcmia inhihitory factor (cholinergie dincrcntiation factor) zinc fin- protein 42 (myeioid-spocific ntinoic acid-responsive) pre-B-ce11 colony-cnhancing factor signal teansducer and activator of transcnption (STAT) 1,9 1kD syn&Can 2 (hqmm sulfate proteoglycan 1) ttunor-11880Ciaaed calcium signai trrinsducer 1 chromogranin A (panthymid secretory ptein 1) d m - a s a o c i a t a d ME20 antigen (melanocytc protein PMEL 17/gp100) (Silva (muse homolog)-lilre) cavcoiin 1 caveolae protein, 22kD cholesmyl ester trader protein, plasma t o p o i s o ~ @NA) II alpha (170kD) M 13 electron-trsnsfer-tlavoptein, beta polypeptide epithelial (E)-cadherin, cadherin 1, type 1 k a t shoclc 70kD protein 1B vasadilator-stimulated phosphoprotein ~ddodinase, iodothyronine, type 1 ESTs ?ransfo&g growîb factor, beta 2 ADP-ribosylation factor 4,b glyatol kinase ARP1 (a&-tclated protein 1, yeast) homolog A (ccntractin alpha) phospbofmmWmc, platelet c-fos interacting upstream transcription factor 2 nudix (nucldde diphosphate linltad moiety X)-type motif 1 homco box BS ISL1 tnnsctiption frcîor, LIM/hhomain, (islet-1) carly growth reSpOllSC 1 Lympbotoxin beta rcccptot superfamily, member 3) chlonde channe14
Gene Nune
adaptor-rtlattd p d cornplex 2, mu 1 subunit
Hs. 155530 Hs. 103724 Hs.ll8Sl2 Hs.7547 1 Hs.88778
Hs.182183 Hs, 129953 Hs.9 1747 Hs.111779 Hs.73965 Hs.56845 Hs.181060 Hs.283305 Hs.l56llO Hs. 1 18778
Hs.79914 Hs.75929 Hs. I79735 Hs.75103
Hs.75428
Hs.21537 Hs. 169825 Hs.75243
Hs.75 180 Hs.753 18 Hs.119177 Hs.65114 Hs.211600 Hs. 1334
B.242463 Hs. 180370 Hs. l lIW EIs.159154 Hs. 108014 Hs.75607
Ws.9661 Hs.83450
H9.273385
nS,8068r) Hs.274472
Hs.82689 Hs.162
. ..
Table 2A Continuad
Hs.169900 Hs.82045 Hs.119571
Hs.81915 Hs. 14642% Hs.82985 Hs. 179573 Eh89674 Hs.83727 Hs.83753 Hs.154156 Hs.80905 Hs.740
Hs.289101 Hs.1019 Hs.16340 Hs.82212 Hs. 1422 Hs.79396 Hs.160318
Hs.2076 Hs.2388 1 Hs.265 18 Hs.149609 H9.70434 Hs.77813
Hs.99863 Hs.2484 Hs.198296
mUMiiL (nœrite growth-piomoting Irictor 2) ~ t j p c I f l , i l p b ~ ~ ~ . n l o s s y n d i o m c , aptoiamildomiarnt) Iwikcmlreted pboepbopmtein pl8 (stathmin) c d k p n , t y p e V , * ~ l ~ t p p c V , r l p ~ 2 ~ t s p c 4 s l p ~ 2 &licbylLdiphosphooIig~&-protein glycosyltransfcf~~st clcavage and polyadenylation specific factor 1,160kD subunit d nuclou r i b o n u c l ~ t e i n polypeptides B and B 1 rnyosin, light polypepîi& 4, aikali; atrial, embryonic Ras asso&ion (RaIGDs/AFd) domah family 2 focai adhesion khusc 1 (FAKIl) (procein tyrosine kinase PTK2) giucoge ~tgulated protein, 58kD parathymid hormone receptor 1 sulfite oxidase CDS3 antigea (ieukocyte surface anbigen) v-fgr vira& oncogene homolog, Gardner-Rashad feline sarcoma N-metbylputine-DNA glycosylase PXYD àormwlantaining ion transport replator 1 @hosphol-) zinc figer protein 7 (KOX 4, clone HF.16) keracin7 trrnsmembraae 4 s u p # f d y member 7 integrin, alpha 5 (fibronectin tcccptor, alpha polypeptide) zinc finga protein 133 (clone pHZ-13) spbingomyelin phosphodiestetase 1, acid lysosomal (acid sphiagomyelinase) elastase 2, neutmphil T-cd leukcmiallymphoma 1A protein actin-depewlent rtgulator of chromaiin, SWVSNF rrlattd, marrix
subfamiiy a, nmmber 2
. _ - 2 ~ r Gencs whichappeat to haves&cti.e upngulation~~oss the BCC patient sample. Thesc genes corn h m the clusta ngion of panel H (Figure 9). Genes listexi in bold print appear to have selective upreplation in both nodular and sclerosing BCC. Genes not listed in bold reprcsent subcluster 7' h m panel H (Figure 9) which may potentiaily be upregtdated in sclmsing BCC alone @awd on a few patients). An '*' is plaoed next to some genes which seun intcresting upon first inspection. -
Row Niimbtx
1 2 3 4 5
6
7 8 9 10 11 12 13 14
15 16 17
18 19 20 2 1 22 23 24
25 26 27
28 29 30 31 32 33 34 35 36 -
- IMAGE Ckae II 211771 195925 230370 248679 121479
1878 19
222032 1 10384 259798 291218 202342 324760 223070 3803%
304790 490822 324024
3 1795 347153 504521 155842 162935 163209 156926
309842 129870 505352
39241 324916 3051 20 202665 245474 375923 306175 277932 321607 -
Hs.78881
m. 184050 Hs.278994 Hs.78596 Hs.146409 Hs.78890 Hs.46348 Hs.857
Hs.84298
Hs.3828 Hs.286233 Hs.llS28S
Hs.100016 Hs. 169992 Hs.83%8 Hs. 183857 Hs. 110802 Hs.288658
Hs.77 1
Hs.53 125 Hs.301547 Hs.7 1827
Hs.260U)l Hs. 173714 Hs. l726CB Hs.1012 Hs.47913 Hs.80988 Hs.2393 Hs.2 1227
RNA hdiumrdrrted protdn EST8 v-cric adan mwmr v im CTlO oncogenc homohg giycopborin B (ioduâes Sa blood p o p ) Bci-2-mmâakd 8-1 (BAG1)hqmtoalhiirir cudaomr ampbting h a n o c h m r r ~ protein mgdatory lrctor X-moc&teà uilrgrtaantaining protein
box -paon c n h n a ~ factor 2 (myocgte - -r tniidomhg pmtda p21/IC-m~-2A (RmK2) rheriis blood group,ccEe ~lttigens p- @rosoait, a b a t , beta tgpc, 5 wiugb-type MMTV integmtion site family, member 4 n-m-whw- bdpttnln reaptor BI fetlnd-biMUiig pmtein 3, htcrrtltw CD74 ~ 3 - (iü~uiurt pdgpcpptidt of MEC ClPBB II
mevaionate (diphospho) decarboxylase sperm autoantigeaic protein 17 dihydrolipoamide S-acetyltransfuase (E2 component of pyruvate &hyâmgeme cornplex) protoporphyrhogen oxidase hypothetical43.2 Kd p t c i n LFA- 1 (iymphoeyte function-associateci antigen- 1) acctyl-Canzyme A carboxylase beta von Willebraud factor zinc figer pobsii, 33fclons HF.10) glycogen phosphoylase (Hers dise-, glycogcn storagc discase WM) s m d nuclcar nbanucleoprotein D2 polypepti& (16.5kD) ribosomrirpn,tein S7 (RPS7) KTAA0112 protein; homolog of yeast ribosome biogenesis fegulrtoryproteinRRS1 ESTs MORF-related gent X nucleobindin 1 comp1ement component 4-binding protein, alpha coagulation factor X = ~ t y p e V f , d * 3 phoqhorylase kinase, alpha 1 (muscle) ESTs
Hpl7Ol33 Ifodrbcd box 01A (rhabdomyomrcoma)
,- $
Table 2B Continucd
Hs.8073 1 autocrine mtility factor nceptor fis.250773 signal mpna mqtor, alpha (transiocon-associated protein
alpha) Hs.256290 SlOO calcium-binding protein A l 1 (ca ighdn) Hs.5398 guaniile monophosphate syntbetase Hs. 12 1529 ELK3, ETSdomain protéin (Sm acasmy protein 2) Hs.75589 acid phosphatase 2, lysommai Hs.36992 ATPase, H+/K+ exchanging, alpha polypeptide Hs.900 1 1 adenylo~uccinate synttiasc Hs. 165215 or bypotùetical protein MGC5395lAHNAK nucleopmtwl Hs.301417 (desmoyokia) Hs.76800 alcohol dchydrogcnase 6 (dass V) Hs.823 14 hypoxaathine phosphoribosyltraasftra~e 1 (Lesch-Nyhan
syndrome) Hs. 1721 interlesin 1 1 Hs.1305 serine (or cysteine) proteinase inhi'bitor, clade A (alpha4
antipteinase, antitrypsin), mernbcr 6 Hs.3786 glutamate receptor, mmimttopic 3 Hs.93223 glycapborin E
da ESTs Hs.79372 mtinoid X nccptor, beta Hs. 156016 KIAAOl4û gene product Hs.173894 macrophage colony stimulating factor 1 (MCSF-1) Hs.37109 mcianoma antigea. family A, 8 Hs.9667 butyrobetaint (gamma), 2 ~ 0 ~ ~ ~ dioxygenase Hs.3548 mahm T-celi prolifdon 1 protein Efs.287358 cytochrome P450, subfamily ïID (debrisquine, spartcine, etc., -
metaboIizing), plypepti& 6 Hs.3426 era (E. coli G-protein homolog)-üke 1 Hs.82 193 esterase DJformylglutathione h y b b Hs.7636 f&e SatCOma (Snyder-Tùeilctl) viral (v-fes)/Fujinami avian
sarcoma (PRQI) viral (v-fps) oncogene homolog Hs. 18 1128 EtKl , member of ETS oncogene family Hs.8 1 170 pim- 1 oncogene W49û94 mitrrhnnlfrinl-inihnhnn , .. . fàctor2 Hs.159161 Rba GDP dissociation inhiitor (GD0 alpha Hs.75280 gfy~yl-@NA spthetase Hs.201626 Homo sapiens clone 25015 mRNA sequence Hs. 12592 period @rosophiia) homolog 3 tIs.287912 latin, mmmanc-binding, 1
_ _ . ._-... , _ - A - Table 3: -9eai whiclxappwr to bc.consistentlyY&wnrcOulafed m s s the BCC parisnt sample. These gencs corn fnw the cluster region of panel G (Figure 10). An '*' is placcd next to some genes which seem interesthg upon f h t inspection.
Hs.73722 ns.77904 Hs. 184776 Hs.5101
Hs. 169793 Hs.184108 Hs.2017 Hs.77039 Hs.153 177 Hs.79123 Hs.394
Hs.155212 Hs.131255 Hs.75573 Hs.180714 Xs.37427 Hs.901
B.28785 Hs. 1735% Hs25960
Hs.81634
Hs.74823
Hs.3709 Hs.57 16
Hs.81097 Hs.80986
Hs.28935 Hs.77393
Hs.75616 Hs.43627 Hs.429
Hs.83834 Hs.78888
Hs.250641 Hs.89862 Hs.695 Hs.790
APEX nuclcase (rnultifuirtond DNA repair enzyme) ribosomai protein S26 ri- protein K3A protein regulatcu of cytokinesis 1 n'bosomal protein L32 n i m a l protein L21 (gene or pseudogene) ribosomai protein L38 n i d protein S3A n i m a l protein S28 KIAAûû84 protein adrenomedullin methylmalonyl Coenzyme A mutase ubiquinol-cytochme c ductase biiding pmtein centromme protein E (312kD) cytochme c oxidase subunit VIa polypeptide 1 q h c y t e membrane protein band 4 J CD48 antigen (Be l l membrane protein) microfîbdlar-associated protein 3 ubiquin01lcytocbrome c reductase core protein II v-myc avian myelocytomatosis viral related oncogenc. neuroblastoma M v c d ATP synthasc, H+ îmnsjmhg, mitochondrial Fû complex, subunit b, isofonn 1 NADH dcb ydrogenase (ubiquinone) 1 alpha subcomplex, 1 (7*51tD, low moldar mass ubiquinonc-binding protein (9.SirD) -03 10 gene product cytochrome c oxidase subunit Vm ATP synthase, H+ trailsporting, mitochondrial Fü complex, subunit c (subunit 9). isofm 1 'transducin-Ue eahancer of split 1. homolog of Dmsopbila E(sp1) fmesyl diphosphate synthase (farncsyl pyrophosphate synthetsse, dimethyMyItrsnsüaasfetase, gcranyItmstrsnsferase) KIM001 8 gent product SRY (se% detemihg region Y)-ôox 22 ATP synthase, H+ transporting, mitochoadrial Fû complex, subunit c (subunit 9) isoform 3 cytochmme b-5 dhqam b i g inbi'bitar (GABA reccptor modulatot, acyl- Coenzyme A binding protein) tropomyosia4 TNFRSFlA-associWd via Aratb domain cystatin B (stenn B) microsorna1 glutatbione S-tmnsferase 1
-
Table 3 Continuai
Ha.621 Hs.155017 Hs.77868 Hs.166160 Hs.80828 Hs.99936 Hs.78943 Hs. 194625 Hs.37034 Hs.171695 Hs.70359 Hs.79070 Eh.169919 Hs.268571 Hs.82028 Hs,ll93Ol
Hs. 180878 rn.8364 Hs.78867 Hs.2465
Hs.83213 Hs.4
Hs.26670 Hs.171862 Hs.278608 Hs.75244
Hs. lO3O42 HA408 Hs.241257 Hs.66151 Hs.76206 Hs.49881
lectin, galac&d&-binding, soluble, 3 (galectin 3) nuciear receptor interacting protein 1 unknown protein LOCSlO35 acetyl-Coenzyme A acyltransf- 1 (pwxisomal) keratin 1 (epidcrmolytic hyperlreratosis) kcraiin 10 (@damolytic hypericetatosis; -sis palmaris bleomycin hydrolase dynein, cytoplasmic, light intcrmediate polypcptick 2 home0 box AS dual specificity phosphatase 1 KIAA0136 protein v-myc avian myelocytomatosis virai oncogene homolog electton W a flavoprotein, alpha subunit apoiipoprotein C-I transfarming p w t h factor, bcta tcccptor ïï (70-80kD) S 100 calcium-bnding protein A10 (annexin II ligand, dpactin 1, ligbt polypeptide (pl 1)) lipoprotein lipase pymvate dehydrogenase kinase, isoeaymt 4 protein tyrosine phosphataso, reccptor-type, Z polypeptide 1 KlAAoOOl gene ptoduct; putative G-protein-couplcd rcccptor; G protein coupleci ncqtor fm UDP-giucose fatty acid b i g protein 4, adipocytc alcohol dehyârogenase 2 (class 1). beta polypeptide H m PAC CI OU^ RP3-515N1 intetfaon-induable guanyiate biiâing protein 2 KIAA0052 protein BCL2-lilte 2 microtubule-associaiai protein 1B endothelin 3 latent üansformiag growth factor beta binding protein 1 mitogcn-activatcâ protein kinasc 1 cadheria 5, type 2, VEcadberin (vascular epithelium)
---- -.._ .. -46: .Germ h m Table 2A thatappearfo be upregulated in at least 25% of the BCC patient sample (Le. at least 13 out of 50 patients).
Row Nomber
1 2 3 4 5 6
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
27 28 29 30 3 1 32 33 34 35 36 37 38 39 40' 41
42 43
- IMAGE clont ID 488548 47 1 667 49 1272 321907 305406 W 8 2
5005 1 327676 195429 487134 262425 267800 362795 18241 1 502754 266005 273 146 344490 358350 381205 360756 489707 488538 163131 155 164 147574
194038 161359 30131 1 36435 1 321758. 4879 16 487868 327080 243549 203999 269 162 147862 184151 180858 291409
683334 365630
8.21486 Id@ trsnsducct and r t i v a t m of transcription (STAT) 1,91kD
Cluntex Hs.239138 p B - c e l î colony.enhancing factor
Hs.1501 Hs.692
Hs.172216 Hs.95972
Hs.156346 Hs.74û70 Hs.27990 Hs. 183 153 Hs. 1466
Hs.153%1 Hs.505 Hs.738 Hs. 1 116 Hs.620
Hs.88778 Hs.182183 Hs.129953 Hs.111779 Hs.73965 Hs.56845 Hs. 18 1060 Hs.283305 Ha. 1 56 1 10 k. 1 18778
W.79914 Hs.75929 Hs. 169825 Hs.75180 Hs.753 18 Hs. 1 19 177 Hs.65114 Hs.211600 Hs.1334
Hs.242463 Els. 180370 Hs.111334 tIs.159154 ~.108014 Hs.75607
syndecan 2 (heparan sulfate protmglycan 1) tumor-agsociated calcium signal transducer 1 chromogranin A (pat'athyroid secrctory protein 1) melanoma-associated ME20 antigen (melanocyte protein FMEL 17/gp100) (silver (musc homolo&ik) topoisomcrase @NA) II alpha (170kD) M 13 ESTs ADP-n'bosylation factor 4lkc glyccro1 kinase ARP1 (actin-relaied protein 1, yeast) homolog A (centtactin alpha) ISLl transcfiption factor, LIMmomeodomriin, (islet-1) earlygrowthrcspow1 Lymphotoxin beta reaptor (JNFR supcrfamily, member 3) bullous pemphigoid antigen 1 (2301240kD) carbonyl ductase 1 caldeamon 1 Ewing -ma bmkpoht region 1 secnhed protein, rcidic, cysteinc-nch (osteonectin) splicing factor, ~elserine-rich 2 (S-2) GDP dissociation inhibitor 2 apelin; peptide ligand fm APJ noeptor Hom sapiens SNC73 protein (SNC73) mRNA, complete cds immuno@obuiin lcappa constant chah KDEL (Lys-AspG1u-Leu) cndoplasmic rcticulum protein retention nceptor 2 Lumican caâherin 1 1, type 2, OBlczidherin (osteoblast) collagen, type IV, alpha 5 (Alport syndrome) p r o t e i n p i q h a s c S , c a & î ~ ~ tubulin, alpha 1 ADP-ribosylation factor 3 keratin 18 n ~ o r d faetor, alpha-indu& protein 3 v-myb avian myeIoblastosis Wal oncogene homolog keratin 8 c o f i 1 (non-muscle) f d t h , light polypeptide tubulin, beta4 tubulin, beta5 myristoylated alanine-rich ptcin kinase C substrruc
Table 4A Continued - Hs.273385
Hs.80684 Hs.82689 Hs.78409 Hs.119129 Hs. l699oO Hs.119571
Hs.81915 Hs.146428 Hs.82985 Hs.179573 Hs.83753 Hs.80905 Hs.740 Hs.1019 Hs.1422 Hs.793% Hs.100000
guaaint-nucleotide deindingptotein CG protein), alpha stimulatiog. aetivity polypepide 1 bigh-mobility group (noahistone chromosom81) protein 2 tuxnor rejection antigen (gp96) 1 ahgea, type m. alpha 1 coliagen, type IV, alpha 1 poly(A)-binding pmtein, cytopiasmic 4 (inducible fom) Icollagcn, type III, alpha 1 (Ehlcrs-Danlos syndrome, autosomal Idominant) leukcmia-awxbd phosphoprotein pl8 (stathmin) collagen, type V, alpha 1 coiiagen, type V, alpha 2 cohgcn, type 1, alpha 2 smrùi nuclear ninucleoprotein polypeptides B and B 1 Rm ~ 0 1 1 (RalODS/AF-6) doinain family 2 focal adhesion kinase 1 (FAK-1) (protein tyrosine kinase PIX2) paratbyroid homone rcccptor I v-fgr viral oncogcne homolog, Gardner-Rashad febe aircoma N-~ylpurine-DNA glycaylase calgmulin A, SlOO caicium-binding protein A8
. -. ... .. ~ ~ W T a h l c 2 A t h a t ~ b - b e u p l e g u l s t e d i n a t l e a s t 3 0 9 b o f t h e BCC patient samp1e (i.e. at least 15 out of 50 patients). -
IMAGE Clone ID 488548 49 1272 321907 346482
50051 327676 195429 487 134 267800 362795 502754 26600s 273 146 344490 38 1205 489707 488538 163131 155164 147574
194038 161359 364% 1 321758 487916 487868 327080 243549 207999 269162 147862 184151 180858 291409
683334 365630
326463
341045 502323 486832 300089
Cliia# Hs.239 138 Hs.1501
prc-Ba11 coloay-cnhancing factor syndealn 2 (heparan sulfw ptotcoglycan 1)
Hs.692 Hs.95972
Hs. 156346 H9.74070 Hs.27990 Hs. 183 153 Hs.153961
Hs.505 Hs. 1 1 16 Hs.620
Hs.88778 Hs.182183 Hs. 1 1 1779 Hs.56845 Hs. 18 1060 W.283305 Hs.156110 Hs. 1 18778
Hs.79914 Hs.75929 Hs.75 180 Hs.75318 Hs. 1 19177 Hs.65114 ~.211600 Hs.1334
Hs.242463 Hs, 180370 Hs. 1 1 1334 Hs,lS9154 Hs.108014 Hs.75607
Hs.9661 Hs.83450
Hs.273385
Hs.82689 Hs.78409 Hs. 1 19 129 Hs. 1 1957 1
bimm-associated calcium signai transducer 1 melanom-annneiatcd MEZO antigcn (melanocytc protein PMEL 17/gp100) (Silva (mouse homolog)-lilre) topoisomerase @NA) II alpha (170kD) keratin 13 ESTs ADP-ribosylatio~~ factor 4-lilrt ARP1 (actin-related protein 1, yeast) homolog A (centractin alpha) ISLl trpnscription factor, LIWhomcOdomain, (islet-f ) Lymphotoxin beta receptor (TNFR superfamily, member 3) builous pemphigoid antigen 1 (230n40kD) carbonyl reductase 1 caldesmon 1 8ccrcted protein, acidic, cysteine-rich (osteonectin) GDP dhociation inbi'bitor 2 apelin; peptide ligand for APJ receptor Homo sapiens SN03 ptoteia (SNC73) mRNA, complete cds immunoglobulin kappa constant chah KDEL (Lys-AspGlu-Leu) endoplasmic nticulum protein retention rcecptor 2 Lumican cadherin 1 1, type 2, OBaâhaia (ostcoblast) protein phosphatase 5, catalytic subwiit tubulin, alpha 1 ADP-ribasylation factor 3 kcraîin 18 tumot necrosis factor, alpha-induced protein 3 v-myb avian myeloblastosis viral oncogeac homolog keratin 8 cofilii, 1- (MIIMWBC~~)
faritin, light polypeptide tubulin, beta 4 nibulin, beta 5 mydstoylated danine-rich protein kinase C substrate (MARCKS, 80K-L) proterisorne @osorne, mecropain) subunit, beta type, 10 iam;nin, alpha 3 (nicein (150kD), kaMn (IdSkD), BM600 (150kD), epilegrin) guaniae nucleotide binding protein (G protein), alpha stimuiating activity polypeptide 1 aimer rejection a n t i p (gp96) 1 cohgen, type XMII, alpha 1 coliqen, type IV, alpha 1 colhgcn, type Iiï, alpha 1 (Ehlers-Daalos syndrome, autosoma1
aaochmi phoqhqmkhpl&(stnthmin) cdagcn. type V, alpha 1 =wen, type v* alpha 2 collap, rypc L alpha 2 Ras assocMon (RaiODS/AFa) domain family 2 'focal adhesion kioase 1 (FAK-1) (protein tyrosine b a s e PTK2) parathymid homo= receptor 1 v-fgr Wal oncogene homolog, Gardner-Rashecd feline sarcoma ca lpul in A, SI00 calcium-biiding protein A8
L-z- - - - - 4c Gencs from Table 2A that a p p - to be upregulated in at least 40% of the BCC patient sample (i.e. at least 20 out of 50 patients).
Row NPmbu
1 2
3 4 5 6 7 8 9 10 11
12 13 14 15 16 17 18 19 20 21 22 23
24 2s
26
27 28 29
30 3 1 32 33
- IMAGE aOnelE 488548 346482
327676 195429 267800 344490 38 1205 489707 163131 155 164 147574
194038 f 61359 36435 1 321758 487916 487868 327080 243549 207999 269 162 147862 291409
683334 365630
326463
502323 486832 300089
299106 270672 264960 346628 -
UniGene cluBter
Hs.239138 Hs.95972
Hs.74070 Hs.27990 Hs. 153961 Hs.182183 Hs. 11 1779 Hs.56845 b.283305 Hs.156110 Hs. 1 18778
Hs.79914 Hs.75929 Hs.75 180 Hs.753 18 Hs. 1 19177 Hs.65114 Hs.211600 Hs.1334
Hs.242463 Hs.180370 Hs.111334 Hs.75607
Hs.9661 B.83450
Hs.273385
Hs.78409 KS. 1 191 29 Hs. 1 1957 1
Hs.8 19 15 Hs.82985 Hs. 179573 Hs. 100000
Gene Name
p B 4 l c o t o n y ~ c i n g factor melanoma-associated ME20 antigen (meiaaocyte protein PMEL 17/gp100) (silver (muse homolog)-lte)
13 B T S ARP1 (actin-related protein 1, yeast) homolog A (centractin alpha) caldesmon 1 m t e d protein, acidic, cysteine-nch (osteoncctin) GDP dissociation inbi'bitor 2 Homo sapiens SNC73 protein (SNC73) -A, cornpletc cds immunoglobuiin kappa constant chain KDEL (Lys- AspGlu-Leu) endoplasmic reticulum protein retention receptor 2 Lumiam cadherh 11, type 2, OB-cadberin (osteoblast) protein phosphatase 5, caîaiytic mbunit tubuiin, alpha 1 ADP-n'bosylation factor 3 kcratin 18 tumor necrosis factor, alpha-induad protein 3 v-myb avian myeloblasbsis viral oncogene homolog kcmh 8 cofilin 1 (non-muscle) ferritin, ligbt polypeptide myristoylated danine-nch protein kinase C substrate (MARCKS, 80K-L) proteasorne @rosomc, macropain) subunit, beta type, 10 iamin;n, alpha 3 (nicein (150kD), kalinin (16SkD). BM600 (150kD), epiiegrin) guanine nucleotide binding protein (G protein), alpba stimulating activity polypeptide 1 collagen, type XVm, alpha 1 coiiagen, type IV, aipha 1 coiiagen, type III, alpha 1 (Ehlers-Dados syndrome, autosornai dominant) leukcmia-associated phosphoprotein p 18 (stathmin) coiiagen, type V, alpha 2 coilagen, type 1, alpba 2 calgranulin A, S 100 calcium-binding protein A8
_----. _ =- 1D*Omes fromTable 2A-that appçat to kupfcgulateâ inat least 50% of the BCC patient sampie (i.e. at kast 25 out of 50 patients). -
IMAGE CIone ID 195429 38 1205 489707 155164 194û38 161359 36435 1 487868 327080 207999 269 162 147862 683334 326463
299 106 270672 264960 346628
Hs. 1 1 1779 axcted protein, acidic, cysteint-rich (osteonectin) Hs.56845 GDP dissociation inhibitor 2 Hs. 1561 10 immunagiobulin kappa collstsnt chairi Hs.79914 Lumican Hs.75929 ramicrin 11, type 2, OB-cadhorin (osteablast) Hs.75180 protein phosphaîasc 5, caîaiyîic subunit Hs.65114 lceratin 18 Hs.211600 Tumor necrosis factor, alpha-induad protein 3 Hs.242463 keratin 8 Hs.180370 cofilin 1 (non-muscle) Hs, 1 1 1334 ferritin, light polypeptide Hs.9661 proteasorne @morne, macn,pain) subunit, beta type, 10
Hs.273385 guaaiac nucleotide binding protein (G protein), alpha stimulRting activity polypeptide 1
Hs.8 19 15 Leulrernia-associateci phosphoprotein pl8 (stathmin) Hs.82985 CoIlagtn, type V, alpha 2 m. 179573 CO-eIl, type 1, alpha 2 Hs.100000 calpulin A, S 100 calcimb'iding protein A8
--- A=,-- ~ h T ~ ~ t h o t s p p c s s r t o k & w i u e g u l o t e d i o a t l e a s t 2 5 % o f the BCC patimt semple (ir. at least 13 out of 50 patients). - Row
Nuber - 1 2 3 4 S 6 7 8 9 10 11 12 13 14
15
16
17 18
19 20
21 22 23
24 25
26 27 28 29 30 31 32 33 34 35 36
37 38 39 -
- IMAGE aancm 193474 491 155 310074 176650 489907 3 IO908 321334 346462 233938 193348 430083 125134 307310 415656
415278
300104
247992 242033
1%112 203773
415303 298001 272575
196189 345809
310673 321541 299 183 490086 221092 173228 323428 340709 341732 364607 346620
22483 302042 149421
Chistu Hs. 184776 Hs.5101 Hs. 169793 Hs.153177 Hs.79123 Hs.394 Hs. 155212 Hs. 13 l Z 5 Hs.75573 Hs.180714 Hs.37427 Hs.901 Hs. 1735% Hs.25960
Hs.81634
Hs.74823
Hs.3709 Hs.80986
Hs.28935 Hs.77393
Hs.75616 H9.43627 Hs.429
Hs.83834 Hs.78888
Hs.2so64r Hs.89862 Hs.695 Hs.790
Hs.7 8915 Hs. 15 1413 Hs.621 Hs.166160 Hs.1408 Hs.80828 Hs.99936
Hs.78943 Hs.37034 Hs.171695
ribosomai protein L23A proteia iegulator of cytokinesis 1 ribosomal protein L32 ribosonmi protein S28 KIM0084 protein Adrenomedullin methylmaionyl Canzyine A mutase ubiquinolcytocbme c ductase binding protein centromere protein E (312kD) cyiocbme c oxidase subunit VIa polypeptide 1 q t h q t e membraue protein band 4.1 CD48 antigon ( B a i l menhane protein) ubiquinol-cytochromc c reductase core protein II v-myc avian myelocytomais virai rclattd oncogene, neumblastoma duivcd ATP s y n h , H+ transportiag, mitochonâtial Fû complwt, subunit b, isoform L NADH de&ydn,gaasc (ubiquinone) 1 alpha subcomplex, 1 CI*=, m low molecular m a s ubiquinone-b'mding protein (9.5kûj ATP synthrse, H+ transporting, mitocbondrial Fû complex, subunit c (subunit 9). isoform 1 transducin-iîke enhancer of split 1, homolog of Drosophila E(sp1) farnesyl diphosphate synthasc (fmcsyl pymphosphate synthetase, dirilethylallyltranstransferese, geranyl transtransferase) KlAA0018geneproduct SRY (sex deteminhg region Y)-box 22 ATP syntthase, H+ transportiag, mitochonbial Fû cornplex, subunit c (subunit 9) isoform 3 cytochrome b-5 diazcpam biding inhibitor (GABA receptor modulator, acyl- Coenzyme A binding protein) tropomyosin J TNFRSFl A-associateci via Arath domain cystatin B (stefh 0) rnicrosomal glutathione S - d e r a s e 1 GA-binding pmtcin &ption factor, beta subunit 1 (53kD) glia maturation factor, beta (GMF-beta) lectin, galactoside-binding, mlublc, 3 (galcctin 3) acetyl-Cocnzyme A acyltransf- 1 (peroxisomal) endothclin 3 keratib 1 (epidennolytic h-) kemtin 10 (epidennoIytic hyperlreratosis; kmtosis pairnaris et plantaris) blcomycia hydrok homea box AS duai specincity phosphatant 1
Table SA Continucd
- - = - ,KTAAQ136 pto_ain v-myc a h myelocytomatOgis viral oncogene homolog elecmn transfct !lavopmtein, alpha subunit apolipopmtein C-I mammacyderivd growth inhibitor (fatty acid binding protein 3)
- 40 41 42 43 44
T
-14,7031 417226 415747 205499 298149
. Hs+70359 Hs.79070 Hs. 16991 9 Hs.26857 1 Hs.49881
- - -WC Gcncs h T a b l e 3 tbatappearîa k - d o ~ g u l a t e d i n at least 30% of the BCG patient sample (Le. at least 15 out of 50 patients).
UniGene ClMter
Hs. 184776 Hs.5101 Hs. 169793 Hs. 153177 Hs.79123 Hg. 155212 Hs. 13 1255 Hs.75573 Hs.37427
Hs.173554 Hs.25960
Hs.81634
Hs.74823
Hs.3709 Hs.77393
Hs.75616 Hs.429
Hs.83834 Hs.78888
Hs.250641 Hs.695 Hs.790
Hs.78915 Hs.621
Hs.166160 Hs.1408 H9.80828 Hs.99936
Hs.37034 Hs. 17 169s Hs.70359 tIs.79070 Hs. 169919 Hs.26857 1 Hs.49881
Gent Name
ribosomal protein L23A protein regulator of cytol)cinesis 1 ribosomal protein W2 ribosomal protein S28 KIAA0084 protein methylmalonyl Coenzyme A mutase ubiquinol-cytochtome c teductase binding protein centromcrt protein E (3 12kD) wybmytc xmnbme protein band 4.1 u b i q u i n o l ~ h r o m e c teductase core protein II v-myc avian myeldcytamatosis viral relatai oncogene, neuroblastoma derivtd ATP synthase, H+ transporting, mitochondnal M complex, subunit b, isofonn 1 NADH dehyârogemsc (ubiquinone) 1 alpha subcomplex, 1 CI-m, MWFE) low molccuiar mass ubiquinone-binding protein (9.SkD) fmesyl diphosphate synthase (famesyl pyrophosphate synthetase, dimetbylallyltmustransferase, geranyltranstransferase) KZAAûû18 gene product ATP synîhat~~, H+ transparting, mitocbondnal Fû complex, subunit c (subunit 9) isoform 3 cytochrom~ b-5 diazepam binding inhibitor (GABA tcceptor modulator, acyl- Coenzyme A binding protein) tropomyosin4 cystatin 8 (st& B) micmomal giutatbione S-transfttitsc 1 GA-binding protein transcription factor, beta subunit 1 (53kD) lectin, gaiactosjde-binding, soluble, 3 (galectin 3) acetyl-Canzyme A acyliransferasc 1 @eroniwmai) endothclin 3 k a t h 1 ~ e p i ~ I y t i c byperkcfatosis) lreratin 10 (epidermolc hypcrkmtosis; kcratosis pairnaris et plantaris) homm box AS dual speciticity phmphatase 1 KfAA0136 protein v-myc avian r n y e l o c y t o ~ viral oncogene homdog electron înmsfkr flavoprotein, alpha subunit apolipopmtein C-1 mammary-derived growth iohibitor (fw acid binding protein 3)
-- ---- - - . - T~SCr~fromTsble3thatippearUrk&~inotlesrt40%ofthe BCC patient sample (i.e. at least 20 out of 50 patients). -
IMAGE Cbacm 310074 346462 233938 430083 415656
415278
203773
415303 272575
1%189 345809
3 10673 299 183 323428 340709 364607 346620
14942 1 14703 1 417226 415747 205499 298 149 -
ribosomai protein L32 ub'iquinol-cytochrome c rductasc binding protein ccntromtrt protein E (312kD)
kentin 1 (epidamolytic b m s ) keratin 10 (epidermolytic b-; kcratosis pairnaris et phtaris) dual spedicity phosphatase 1 KIM0136 protein v-myc avian myelocytamatosis viral oncogenc homolog cltctron transfer flavoprotem, alpha subunit apolipopmtein C-1 mammuy-àuived growth inhibitor (fatty acid binding protein 3)
Hs.37427 Hs.25960
Hs.8 1634
Hs.77393
Hs.75616 Hs.429
Hs.83834 Hs.78888
Hs.25û641 Hs.695 Hs.621
Hs.166160
eryihrocyte membrane protein band 4.1 v - y a y viral rciated oncogene, neumbiastom8 ddved ATP synthase, H+ transportin& mitochondrial Fû cornplex, subunit b, isoform 1 fainesyl diphosphate synthaso (frrniesyl pyrophosphate synhetase, dimethylaIlyltrsnstrensfcrase, g~ra~~yltranstrahsferase) KïAAûûl8 Bene product ATP synthase, H+ ûanqmhg, mitochonbial FO cornplex, subunit c (subunit 9) isoform 3 cytociuome b-5 d b p m binding inbiiitor (GABA receptor modulatort acyl- Coenzyme A binding protein) tropomyasin4 cystatin B (stefin B) l& galactoside-binding, soluble, 3 (8alcctin 3) acetyl-coenqm A a c y l t r a n s f ~ 1 (peroxisornal)
----..- - T.blc!iD: Gcna~aOmTable3 thatappearioba d o ~ i a a t l e a s t 50% of the BCC patient sample (ir. at least 25 out of 50 patients).
1 UniGe~e cltM4m Hs.75573 Hs.37427 Hs.77393
Hs.75616 Hs.429
Hs.83834 Hs.78888
Hs.99936
Hs. 169919 Hs.4988 1
G t n t N m ~
centromere protein E (312kD) a y t h q t e mcmbi9nt protein band 4.1 famesyl diphosphate synihasc (famesyl pyrophosphate synthctase, dimethylallyltranstiansfcrast, getanylbanstransfctasc) KïAA0018 gent product ATP synthasc, H+ tmsporting, mitochondrial FO complex, subunit c (subunit 9) i s o f m 3 cytochrome b-5 diazepam biiding inhibitor (GABA naptor moduiator, acyl- Coenzyme A binding protein) kentia 10 (cpidermolytic b y p d m a s h ; kcratosis palmais et phtaris) electran transfer fiavopmtein, alpha subunit mammaryderived growth inhibitor (fafîy acid binding protein 3)
- =C-Ge~ whichappeand ta be-upgdatcd inasmsUlllltnbCT of pathts; some gens of whidi did not pass the statisticai ANOVA (F-test) criteria to be included in the hicrarchicai cluster (Figure 9). Ciosa inspection wili be rcquired to discem whether these gens should be included in the upregulated gene list for BCC. -
Row NPmkr
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
IMAGE ( GcncNunt
molanoma adhesion mo1ccule matrU metalloproteinase 2 (MvlP-2) mimgen-activateci protein kinase-activated protein base 2 (MAPKAPK2) intcrfmn (alpha, beta aud omega) rtaptdr 2 v-jun avian sarcoma Virus 17 oncogcne homolog transcription factor AP-2 alpha (activating cnhancc+ bindiag protein-2) îumor necrosis factar, alpha-induced protein 1 (eudothelid) danoma-associated antigen IO (MAGE-10) üanscription f~ AP-1 @rr,to-oncogene c-jun) TIMP-1, metalloproteinase inhibitor 1 e+B3 receptor tyrosine itinasc endothelin-1 fibroblast growth factor raqm 3 (FGFR-3) rasdateci protein RAB-27A NF-hppaB pl05 subunit melanoma-associateci antigen 1 (MAGE4 ) heat shock cognate 7 1 melanoma-associated antigen ME491 (-3) colorcctd mutant cancer protein MSH3, DNA misinatrh Tcpair pmtein mehoma-associated antigen MuCl 8 MSH6, DNA mimatch tcpair protein mat& metalloproteinase 9 (MMP-9) transforming growth factor, alpha (TGF-alpha) metastasis suppressor kangai-1 (CD82) ~vascular endothelia1 mwth factor NEGF)
. . - - ~ ~ : - ~ w h i c h a p p e a a d t a b e . & w m e g u l a t e d i n a ~ n i i m k e i ofpatiCrits;some genes of which did not pass the statistical ANOVA (F-test) criteria to be included in the kcrarchical chiatm ( ~ i b 10). Cioset iaspsction will be required to discern whether these geiies should be hcluded in the dowmguiated gene list for BCC. -
Row Niimki -
1 2 3 4 5 6 7 8 9 10 11 12 13 14 1s 16 17 18 19 20 -
IMAGE 1 Gent Nemc
.Fm06 b i g pmtciri 12 (npamycin-8S80CiBttd protein 1) RAD23 homalog B epidermal p w t h factor reaptor pathway substrate 15 multidrug mistance protein 1 annexin A8 -phase-protein4(MIP-4) inf#fen,n-inducad guanyhte binding protein 1 interferon r e m fadc#.4 giowth arriest spacitic p t e h @AS-1) cyclin G1 caspase-8 ( F A D I E b ICE) thrombospandin-2 (TSP-2) cbemolcint hccptor CXC-R4 nimor necrosis factor, dpha naptm 2 (TiW-Rp75) OM-CSF nceptor, alpha prostqlandin E2 q t o r ïL-1 receptor, beta (type 2) niinot necrosiS factot, aipha L16 (lymphocyte chemoataactant factor) IL6
It is first important to discuss the preparative experhents that were performeà
prîor to utilizing the cDNA microarray assay (for examining UVB-inducible genes and
BCC gene regdation). The UVB dose optimization by RT-PCR (Figure 1) yielded
nsuits as expecteà. M o u s studics have shown that TNF-a is a prototypical UVB-
induceà cytokine 1913196-201. Because TNF-a plays a role in meny of the dccumented
iaimunosupprcssivc and apoptotic effects of WVB ""Oo, we hypothesizeà that whatever
dose of W B induced the highest expression of TNF-a in our in vitro keratinocyte (KC)
mode1 would likely yield other interesthg changes in gene expression that could be
revealed by microarray analysis. This proved to be tme, and the nsults of the mimarray
enalysis of WB-inducible genes are discussed m e r below.
The miaoarray technique is dependent upon hybridization conditions that permit
stable interaction betwem two DNA species: a) fîuorescently labelled cDNA probes (test
sample), and b) spotted cDNA clones (organizcd on the microarray glass surface). An
important part of establishg the use of mi-ys for determinhg UVB-inducible
genes was to perform the assay whik monitoring for specific DNA binding interactions.
TBis was done ushg positive control cDNA (Mved bmm o ~ o p s i s thdiana) in the
mi~r081~8y hybridization reaction (Figures 2 and 3). Arobadopsis is a plant species
whose cDNA sequences have no sequence homology to human genes. Therefore, by
placing arabadopsis RNA into the reverse transcription (RT) labelling reaction of both
test samples (Le. matment and control), it fields a cDNA proâuct that binds specifically
to the arabu&psfs cDNA fmtures on the microanay. This provides a positive conrrol in
two different ways. Firstly, the arubu&psis faturcs iadicate that specifîc cDNA
Q- . . . are occiuria~. Sec&&, orobodoyHis CDNA is labclled with botb
fluorescent dyes (Q5 and Q3). therefore when these fmtures yield an equivalent
fluorescent signal h m each dye (ye11ow; see Figure 3) it indicates that the differentiaiiy
labelid test samples (e.g. UVB-CyS and sham-Cy3) have likely hybndized to the
microarray with qua1 efficiency. This equivalency in hybridization behaviour is
something that is important in tenns of &ta nomakation, and it is one reascin for
pedorming miprocal labeliing expaiments in a micn>anay study (see below). The
rabbit globin negative control features also provide a good indication that specific
interactions arc occurring between the cDNA probes and the microarray fatum. because
if no binding occurs at thwe sites it is lürely that specific human-human cDNA
hybridization is occurring ( s a Figure 3).
Reciprocal labelhg expaiments are not cornmonplace in the microenay
literature. However, arguments can be made in support of using recipracal labels to
c o h positive hybridization nsults in a microarray expriment (as was done for these
studies). Some investigators claim the possibility that CyS- and Cy3-conjugated dCTP
fiuonscent dyes are incorporated into cDNA with Merent efficiency by reverse
transcriptase enzymes. It is possible that these clnims have ken made due to the
different rates of photobleaching that are experienced by these two fluorophores. thereby
suggesting that Cy5 and Cy3 are incorporated di&nntly when really they are not.
Regardless methods have ban developcd to control for the possibility of different steric
hindrances by Cy5- and Cy3conjugated dCTP d h g nvase transcription. One option
is to utilize an indirect fluorescent labelhg approach when generating cDNA probes.
For example. one could incorparate the s a w biotinylated d m into each cDNA probe
(Lek treatmcnt eid conttol) to creatc quai dncicncy of dCTP incoqmation. Secondary
reactions wouid then be pafomcd us@ CyS and Cy3 conjugateâ to streptavidin, which
wodd bind imversibly to the biotin motif on dCTP nsidues. A n o h option is the
miprocal label approach that was used in these studies. In this approach, a high fidelity
reverse transcriptase enzyme was used (SuperscxiptTï) makiag it unlürely that there are
Merences in incorporation efficiency for dinct labebg with Cy5- and Cy3-dCTP.
Two microamy hybridizations w e n thai p & o d for each analysis with the Cy dyes
nverseà in the second bybridization (reciptocal). Gaies of intenst were identifid when
comparable results were obtainad even when the dyes were reversed. An example of this
is shown in Figure 4. A slightly modifiai approach was utüizcd in the large-de BCC
study to accommodate the clustcring process. Reciprocal labeuiag experiments were stiii
perfomied in this case, howeva the normelizcd net fluorescence intensity ratios for each
gene were configureâ with the same dinctionality using the OCI AMAD database. For
example, each normaïized gene expression ratio was a representation of net fluorescence
h m tumour cDNA over top of net fluorescence h m contml cDNA (ir.
nimout/contro). The quadruplicate data from the four features of each gene was then
averaged to give one npnscntative gene expression ratio per gene per patient.
Thenfore, the reciprocal label control data was containeci w i t b these averaged ratio
values.
Data wrmaiization is another crucial step to analyzing microarray data. This
stems h m the thwry that global hybnâization (across the en th microarray surface)
should k comprised of @valent fluorescence intensity h m both Cy5 and Cy3. In the
microamiy scanning process attempts are made to balance these signais by adjusting the
-1- powa andlol.pioîaxd@k tube (PbîT) semioga for collection of data h m each
fluorophon. However, these adjustments are cru& approximations based on combined
signals of the cDNA f e a ~ n s and the background fiuorcscence h m the glass surface
surroundhg ai i of the features. Data nomukation is thercfore requireà to create a
Cy5/Cy3 baiance for gene-specific fiuorescence intensities within each m i m y . This
is because the interesthg data cornes h m the micro-vicinity of the cDNA fatures, and
not from the empty glass surfaces that surround these fatum. For these studics, gene
expression ratios were calculated based on a ratio of background-subtracted median pixel
intensities. in other words, the image of each faturc on a microarray is compnsed of a
numkr of pixels that contain data h m both CyS and Cy3, and there is also a sxnali
background signal from within the micro-vicinity smunding each feature. To calculate
a CyS/Cy3 gene exps ion ratio, the median of the CyS intensities h m every pixel
(minus the median Cy5 signal of the local background pixels) gets divided by the median
of the Cy3 htensities h m every pixel (minus the median Cy3 signal of the local
background pixels). Median intensities an often prefed to arithmetic mean intensities
because they are not as senously affectcd by extreme values at either end of a normal
distribution. TheorcticaUy, a perfect micmamy feature should have the same gene
expression ratio whether calculateci h m means or medians. This is most ofken the case,
however medians w m used for the featurc ratios in these studies keeping in mind that
outiier pixel events can potentialiy skew the information obtained h m a microarray
image. Normalization factors, to balance the global CySICy3 feature signals to a value of
1 .O, were then calculated based on the ratios of net median pixel intensity.
-&- -a .---- @an- exists as to how
ratios that corn out of microamy
one aould i n e t me nomaihaî gene expression
experiments. Difffxent approaches were used in the
two phases of this thesis (Le. UVB-KC vs. %CC experiments). Both approaches are
cqually valid, however it was possible (and necessary) to @orm a more intricate
aiiaiysis on the BCC data bccause of the large 50 patient sarnple size obtained (discussed
later in this chapta). ûne item of generai importance is that microarrays are best utilized
for determintog 'ons' and 'offs' of gene expression, as opposed to quantitative fold-
changes ktwem the genes analyzed. For example, it is possible to say that a p u p of
genes are tumed on (upregulated) by at least two-fold, because within each microarray
featurc the CyS and Cy3 intensities an calculatd based on hybridization of a particular
transcript from the treatment W o r conîrol specimens. It becornes difncult, however, to
malte cornparisons between genes and statc that e particular gene is precisely two-fold
more upttgulated tbsn another gene in the array. In otder to make these conclusions, one
rcquires more sensitive analyses such as northem blots or semi-quantitative RT-PCR.
This quantitation caveat of the micromy technique stems from a few item. First of aii,
detection of gene expression on a microerray relies on incorporation of fluorescent labels
conjugated to a nucleotide in the cDNA (Cy5- and Cy3-dCTP in this case). Comparing
the CySlCy3 ratios between two genes can then be problematic if the mRNA transcripts
of those two genes have substantial ciifferences in their biochemical content. For
instance, two gencs may have extreme ciifferences in th& guanine and cytosine (GC)
content, Gens with high OC content will have more label incorporateci into their cDNA
than gcnes with low GC content, and a problem then &ses due to confidence intervals of
fiuoresccnce intcnsity data (i.e. consistent CyS/Cy3 gene expression ratios corne h m
- strong fl~rcsctnce emission sipals, and an therefm associateci
confidence). High GC content could potentidy give rise to
intensity, and more consistent CyS/Cy3 ratios, which therefm
with higher levels of
greater fluorescence
malces it difficult to
consistently compare CySiCy3 ratio amplitudes betwwn features of variable GC content.
For a similar m o n , a second cause for this quantitation caveat of microarrays arises, and
it cm be deshbed as follows. In biological systems, certain mRNA transcxipts an more
abundantly expresseci thm others20) (coincd by the phrase 'copy numôer'). Thus,
difficulty can aise from rnicroamy data when cornparhg fold gene expression changes
between cDNA probes representing abundantly expressed genes (high copy numba), and
genes whose expression produces an eff'ct h m lower levels of transcription (low copy
number). Additionaiiy, celis ofaii require a hi@ copy number to translate an eff- h m
mRNA tmscripts that possess instabîiity or a short helf-life. How this affects micmarray
analysis a g a . relates to the confidence intervals for fiuonscence detedon. cDNA
probes arising h m a high mRNA copy number may collectively incorporate more
fluorescent label than a probe with low copy number. The CySlCy3 gene expression
ratios may then be more consistent for abundantly expressed transcripts, which hstigates
a similar probkrn to the GC content of genes, where feanues having a higher amphtude
of net fluorescence intensity may not be completely comparable to features of lower
amplitude. These arguments are not stating that faturcs with low GC content md/or low
copy number should be excluded, but ratha the possibiiity that it can be inaccurate to
compare the fold changes in gene expression between two genes on a microarray. For
this nason, microarray data is usualiy displaycd as lists of genes which appear to be
upreguiated or dowmgdated (as is done in this thesis). Certain elements of microarray
an&sis arise fFom these concepts: a) @xisions must be made ngarding -- A
ratios define a gene as behg turned on or off (usually two-fold up or
what Cy51Cy3
down), and b)
thresholds of fluorescence detection must be estabüshed to define feahire signais which
are too close to the surrounding background noise (based on signal-to-noise ratio).
For the analysis of W-inducible genes, we used arbitrary two-fold cut-offs for
the CySICy3 gene expression ratios to deüneate Lists of genes that appear to be tumed on
(ratio >= 2; base two logarithm >c l), or off (ratio <= 0.5; base two logarithm <= -1).
Our fmt hypothesis for these shdies stated that microamay anaiysis would eluciàate
severai new genes modulateci by UVB, and this was satisfied upon examination of the
results (Table 1A and Table 1B). From the WB-upreguiated gene list (Table lA),
certain gens were consistent with previous studies of UVB-induced gene expression in
KCs. For example, the cytokine leukemia inhibitory factor 0 (Table 1A; row Il),
and nuclear factor NF-kappa (Table 1A; row 19) were both upregulated. Members of
the TNF-a signahg pathway w m a h uprcgulated, including TNF-a-induced protein 1
(Table 1A; row 38) and TNF reccptor p55 (Table 1A; row 55). These hdings strengthen
the rest of the microarray data, because they estabiish that the microamy assay is
effective for detecting hown WB-upngulated genes (sa Chapter 2 - Literature
Review). The appearance of LIF signified that the assay could detect previously nported
cytokine regulation in KCs. Finding upregdation of the TNF-related genes coincides
with W B regulation of TNEa 1). and previous sbidies on UV-induced
apoptosis mediated by TNF-R-p5S. Detection of NIF-kappaB ais0 coincides with the role
of WB as a tumour initiator, for its overexpttssion has k e n previously shown to cause
the apoptotiisresistant phenotype of KCs. Some of the newly detccted WB-upregulated
- - -gemsinTsblclAaloofPlLintocategnriPs~~
carcinogentsis, which satisfies our second hypothesis
m ~ y k nl9ied t~ W - W
(see Chapter 3). These include
oncogenes (Le. AXL oncogene, row4; pmtwncogme c-abl, row 42), angiogenesis
activators (i+. platdet-derivai endothelid cell growth factor (PD-ECGF)/thymidine
phosphorylase, row 9). cancer-related genes (i.e. transformation uprcgtdated nuciear
protein TüNP, row 44; Reed-Sternberg intermediate fîiament-associated restin, row 7).
proliferative signalling molecules ( i r ras-related RAB- 11B. row 15; MAPKAPK-2, row
43). immuuology-relateci and accessory molecules (i.e. peptide transporter TAP-1, mw 1;
immunoglobulin-associateci B29 protein, row 13; E-selectin, row 20; p59 HSP-binding
immunophilin, r0~28), and cytokines (i.e. platelet fador 4/oncostatin A. row 50).
Interestingiy, oncostath A is potentially a new addition to the KC cytokine pronle
induced by W. 0 t h genes appear to k part of the UVB-induced stress response (i.e*
mitochondnaî stress-70 protein, row 17; ntinoic acid nceptor alpha-1 , row 12; ubiquitin-
pmtein ligase, row 22; proteasorne subunit SSB, row 52). Numemus obier UVB-induced
signal transduction candidates as well as transcription factors may also play bona fide
roks in the UV-induccd immune suppression &or carcinogenic process by mediating
the transaiption of their target genes. However, this is only specdation, and the role of
these molecules would require m e r investigation due to the network-like interactions
of signal transduction processes. This group of upregdated genes included signalling
molecules (i.e. high-aninity CAMP-specific cyclic phosphodicsterase, row 3 1 ; protein-
tyrosine phosphatase delta, row 32; discs large homolog 3, row 39; adenine nucleotide
traiislocator 3, row 48; serindthreonine protein phosphatase 5, row 49; CAMP-dependent
protein kinase 1 beta regdatory ch&, row 54; strindthrconine pmtein kinase 11, mw
PL - 56),and transaiption factors 0.e. zinc protein I51,row 33; Pnc pmtein
HRX, row 37; homeobox pmtein HOX-AS, row 36). From a downregulation standpoint,
astain genes üsted in Table 1B may aiso be relevant to W-induced carcinogenesis
a d o r immune suppression. This includcs downreguiation of certain cytokines (Le.
macrophage colony-stimulahg factor- 1 (CSF-1), row 9; monocyte chernotactic protein- 1
(MB-1). row 26). irnmunology-rclated and accessory molecules (i.e. T-lymphocyte
maturation-associatd pfotein, mw 3; integrin alpha-6 (VU-6), row IO), stress-rslated
and apoptosis molecuîcs (i.e. ubiquitia-specific protease HAUSP, mw 6; heat shock
cognate 71, row 14; dead-box pmtein p68, row 20; proteasorne component MECL-1, row
27). and endogenous angiogenesis inhibitors (i.e. thrombospondin-1 (TSP-l), row 22).
Similar to the UVB-upregulated gaie Est, the downregulated list also contains numemus
signalîing molccult~ and üanscription factors which may be important, but would require
detailai investigation to eluciôate the consequcnccs of th& domgdation. These
inciude signalling moiecules (i.e. dual specificity protein phosphatase 6, row 1;
extracellular signal-related kinase 3 (ERIC-3). mw 2; EGF-response factor-1 (ERF-l),
row 12; FTB-associated splicing fpctor (PSF), row 23). and transcription factors (ie. zinc
h g e r protein 35, row 11).
To elaborate on the second hypothcsis that certain UVB-regulateâ genes
discovercd by mi~108ff8y may be relevant to the pathogenesis of non-melanoma skin
cancer, we decideci to focus on the downregdatcâ genc TSP-1 (Figure 4; Table 1B, row
22). TSP-1 downregdation was confïmd by RT-PCR (Figure 6). which also indicated
that the mimarray technique is capable of detecting two-fold changes in gene
txpression. As descxibed in Chapta 2, TSP-1 is a potent endogenous inhibitor of tumour
m g @ d khOPbOcOmcbelpailiorrceoty~to.thioloftheswitchtoththimn~~
angiogenic phenotype as a net balance betwan positive and negative replators of blood
vesse1 growth. The extent to which the negative regdators are dtcreased during this
switch may dictate whether a primary tumour grows rapidly or slowly and whether
metastases grow at ell ? The finding that W B cm stimulate domgdat ion of TSP-1
could be an early event in promoting the angiogenic phenotype of BCC.
The anti-angiogenic importance of TSP-1 cm be b t l y linked to rnalignant
pâth010g~. Reports have shown that endogenous TSP-1 expression is transcriptionally
rcguiatcd by wildtype p53, and domguiated upon loss of the p53 aiiele Reports
that TSP-1 gent expression can also be repssed by oncogenic signals of c-jun/AP-1
mm, v-SC and v-myc ( and silenced by DNA methylation imply that
dow~vcgulation of TSP-1 coiitributcs to oncogcncsis 210. The rolc of TSP-I in tumour
angiogenesis is also well established 'sR211413. For example, TSP-1 dow~ltegdation bas
an inverse correlation with ovenxpression of VEGF and bFGF in lung carcinoma, and
this is somehow mediated by p53 214.
An established role for p53 in BCC was mentioned in Chaptu 2, because BCCs
commonly possess mutations in p53. Additionally, it was described how p53 mediates
W-induced cell cycle amst and apoptosis. Thereforc, a strong possibility exists that
p53 regdation of TSP-I gene expression could k affecteci by W exposure, and the
effet of UV on p53 status could cause the two-fold dowmgulation of TSP-1 observed in
these studies. Coinciding with this downrcgulation in TSP-1, our microarray analysis of
UV-inducible genes showed uprcgulation of PD-ECGF. &O known as thymidine
phosphorylast (TP) (Table 1A, row 9). TP is an angiogenesis activator in certain forms
angiogenesis for head and nedr SCC '17. Studies have aiso suggested that angiogenesis
in SCC aises fiom combination of TP upgulation and TSP-1 downreguiation, and that
this could be related to lm-of-function mutations in p53 *18. Taken togetha. the UVB-
induced upregulation of TP and dowmcgulatioa of TSP-I (dctermined by our microarray
anelysis) could be an important carly event in the onset of malignant growth in the skin.
To mon dircctly examine gene regulation in BCC. we utilized microamy
technology for an in vivo study examining tissue biopsies h m 50 BCC patients.
Tumour and normal skin specimens w n obtained via Mohs' Micrographie Surgay. The
benefit of Mohs' surgery was that ali twnow and normal skin specixneas were
histologicaily confimeci at the timc of excision. Therefore, site-matched n d tissue
sainples wae obtained with confiàence that no nsiduai tumour celîs remained. Care was
also taken by the surgeon to match the skin-depth of the himour and normal biopsies so
as to evoid stroma1 Merences between the two specirnens. Microamys were utilized to
compare tumour tissue to normal skin for each patient, using duplicate reciprocal label
analysis (sa Chapter 4 - Materials and Methods, and Chapter 5 - Results).
For d y s i s of gene regulation in BCC, a different more intuitive approach was
required for analyzing the microarrtly gene expression ratio data as compared to the
investigation of W-inducible genes in KCs. This was for two misons: a) the number of
microarrays used in these BCC experiments was mach greater, which yieided a rnuch
largcr data set, and b) each BCC patient rcprcsented a potentially distinct data set (unlüre
the UVB-KC expcrimcnts which were consistently conaolled in a cell culture settiiig).
methods -.wae.- aequllrdto dco. ccqa&msbetwtto th BCC @en& A
first step to adyzing the BCC data was to pafonn a set of what are called seif vs. self
miaoanay hybridizations. These seWseîf hybriàizations comprisxi of pooled BCC and
n o d sicin RNA specimens labelied with both Cy5 and Cy3 (see Chapter 4), and they
functioned as a controiied data set for defining upregulation and domgdation
Cy5iCy3 ratio cut-offs for the BCC patient data. To bnefiy explain, aii sewself
miaosrray data normakd to a mean CySlCy3 ratio of 1.00, because the cDNA probes
were labeiied with both C y S and Cy3. This type of balancd Cy5 and Cy3 data provides
useful information, because the âata for evay fatute theoretically represents 'no change'
in gene expression. Using the mean of the n o d z e d Cy5iCy3 ratios (equal to 1.00;
taken h m a combination of 12 separate seWseîf hybridizations), plus-minus (f) 1.96
times the standard deviation (SD) of the seWseif data set (Le. 1.00 f 1.96SD). one can
defîne a 95% confidence interval whae the CyS/Cy3 ratios within that range are
statisticaily considend to be part of the same data popdation as the 1.00 mean value
@<O.OS). For the sewself hybridizations in these studies, this 95% confidence range for
the nomislized Cy51Cy3 ratios was h m 0.61 up to 1.65. This demonstrateci that using
CWO-fold CySlCy3 ratio cut-offs for ciasyfying genes as upngulated or downrepuiat#i
(as was done for the UVB mimarray data) may be too stringent for the large-scale BCC
study where we want to obsme trends in the BCC data That is, we want to delineate
genes that an potentially upregdated or downreguiated across the patient sarnple, but
avoid excluding them h m individual patients due to variation in CyS/Cy3 ratios mund
a set two-fold c u t d . For example, if four patients showed upregdation of a gene with
CySiCy3 ratios of 1.7, 1.85, 2 and 2.1, then two-fold cut-offs wouid exclude the data
interval cut-off of 1.65 (defined by the sewsclf hybridizations), the upregulation trend
would be observed for that gene in a i i four patients, becme ail four CySICy3 ratios an
pater than 1.65. Using this epproach it prcvents the 1.7 and 1.85 ratio data h m
bccoming fdse negatives. In this manncr, sewself hybridizations aiiowed us to
potentiaüy avoid false negatives for the entire BCC patient sample. Additionally,
seWstIf hybiàizations provided an excellait control for idcntifyiag statistical variance
within the patient data set. Using the OC1 AMAD database, ANOVA (F-test) analysis
was pedormed to identify genes with significant diffaences in gene expression ratios
(pcû.05) when compand to the sewself experiments (which exhibited no change in gene
expression). Importantiy, a high Ievel of statistical powa couid be associatcd with this
aaalysis of variance due to the large data set. To visualize the trends in gene expression,
the colour d e of the cluster diagrams (Figure 9 and Figure 10) was calibrated to show
green or rcd bcy*nd the iiomaiized CySICy3 ratio 95% confidence intervals of 0.61 and
1.65, respectively. These 0.61 and 1.65 cut-offs then assisted in delineating the various
upngulated and downregulated gene lists for BCC (Tables 2-5). In terms of the
hierarchical clustering process, the seWseif experiments dso functioned as a positive
control, and this is discussed below.
Micmmy experiments generate hu11dreds of thousands of data points, and such
volumes of data are tw large to analyze by simple sorting in spreadsheets, or plothg on
a single graph. For sense to k made of large-scale expression data, systematic
organizational methods and dgorithms must be used. Agglomerative hierarchical
clusierirg was the process utilized to try and malce sense of the BCC data for these
studies (sec Figures 8-10). 'Ibis technique has rectived encumous attention in die
literatwc due to its capability for identikying trends within gene expression data, and
iiîustmting these patterns in a cw&ated f a o n that is casily discaned by the eye 219.
This technique was first employed by Michael Eisen et al. to display genome-wide
expression patterns in yeast PD. 'Ibere have since been hi@-impact publications using
the clustering technique to andyzc differtnt forms of cancer including human breast
carcinoma "lm, and diffuse large B-ceU lymphoma =. A nahiral basis for organizing
gene expression data is to p u p toge- genes with similar pattern of expression. The
first step to this end, and thaefon to cluster maiysis, is to adopt a mathematical
description of similarity (correlation). Different options are available for use as a
8imilarity metnc depending on the experimcntai design and type of gene expression data
that npuires clustering. For these studits, uncentreci Pwson pmduct-moment correlation
was utilized. nie Pearson comlation coefficient was chosen because the gene
expression ratio data is nonnaDy distributed on an intervaücontinuous number scaie
(linearlled by base two logarithm conversion), and these criteria are best analyzed using a
paramctric statistical method The uncentrcd modification of this coefficient was used
because cornparisons wae king made between distinct patient experiments with separate
patient-matched controis. In technical terms, this meaiis that comlation between the
gew expnssion ratios was dependent on the amplitude of the ratios and the vector
dircctionality of those ratios (unliLe centreci Pearson comlation which is independent of
data amplitude) For example, if two data vectors X and Y exist with identicai shape
(directionality), but are offset relative to each other by a nxed value, they wiU have a
standatd Pearson comlation (centreâ comlation) of 1. However, an uncentnd
-- ~ w o u k L m & b c s q u s l - t u l-k.?ui..thcvectamueoffset fram each&@.e.
have di&ant amplitude). Centrecl comlation would potentially be better for examining
paaans of gene expression across multiple expuiments in a time course, where cach
individual micmamy hybridizaîion contaias the same control cDNA and one waats to
comlate expression pattern for diffcrcnt genes m i s a series of time points. However,
uncentnd conelation was chosen for the BCC patients bacause the amplitude of the gene
expression ratios for an individuai geae mss the entire patient sample may possess
relevant information to be camed through the clustaing process (Le. düf'erences between
the tumoG and patient-matcbed control may be importPnt witbin the individuel patients).
With the Pearson uncentreci simil@ metxic dehed, agglomerative hienuchical
clustmng then uses a bottom up approach, whereby single expression profiles are
successively joiDed (corrclaâd) to form nodcs, which in tiirn branch apart furthcr and
furthcr until a l l individual profles and nodes have b a n joined to fonn a single
hiemhical tree '19. The mon simiiar a set of gene expression ratios are, the shorter the
hierarchicaî branches WU be between those genes, and the closer they will appear
togcther in the cluster diagram. This is why we desaibe the condensed regions of rd
and green in Figures 9 and 10. The individuai experiments or patients can also be
clustercd to mate a two dimensional cluster to detect patient similarity profiles in
addition to sirniiar changes in gene expression within the microanay gene list (see Figure
8). Within the patient cluster we also utilized the sewself experiments as a positive
control, whereby the distinct cluster region of sewself experiments indicated that the
similarity metcic used for clustering workcd propcrly (sec Figures 8-10).
-Lx- -- - ï n c o n j h w i h the abouemciirionedclusta anrilysis, and the mvel seWseif
hybridization appmach, we chose to include another type of analysis showing the
percentage of BCC patients that exhibited the gene expression changes identfied by
clustering ( d d b e d in Chapter 5). This additional type of aaalysis once again relates to
the debate in this field of nsearch as to what is the k t way of intcrprcting micromy
data. Our initial cluster analysis, and this other patient percentage method, each have
there own set of advantagcs and disadvantages. For example, one advantage of the
cluster proass is that groups of simüarly rcguîateà genes are identifiai without
depending on the numbet of patients that exhibited those gene expression changes. ûne
potential disadvantage of this, howeva, is that certain genes which cluster together may
have only b a n upreguiated or domgulateâ in a few patients. if we condense the
clustercd gene iists d o m to those genes which appar to be upregulateâ or
downreguîated in at least 25% of the patients (and w a k up to bigher paccntages), the
advaatage is that we have the ability to identify more commonly regulated genes aaoss
the patient saniple. It is for this m o n that we performed the patient percentage analysis.
Unfortunately, a major disadvantage of this approach is that we may exclude certain
isolated gene expression defects, which are potentially relevant to BCC, but were only
observed in a handful of patients due to sensitivity limitations of the microarray assay.
At any rate, we included this percentage-wise analysis to suggest that there are different
ways of interpreting our mimairay data.
The gene and patient clusters which were gentrateci fkom the BCC data provideci
some intertsting information. Gents which appearcâ to be upreguiatecl (by cluster
anaiysis) were listed in Ta& 2A and Table 2.B (and patient percentags-Wise in Tables 4
&DL hile-ihooe appeaad to &do- ~ ~ r l i s t e d io Tsblc 3 (Pod
patient perccntage-wise in Tables 5 A-D). These gene lists h m the BCC study may
appcar larga than the lis& from analyzing WB-indutible genes due to the fact that these
BCC nsults mise f h n clustering a much larga data set. Upon close inspection, the
largest list Uable 2A) oniy contains 116 genes, wbich is not unmanageable. în addition,
many interesthg genes (marked with a '*' in Table 2 and Table 3) can be deciphend
h m these lists. From the consistmtly uprcgthted k t (Table 2 4 , the genes f d into
several categories that may be relevant to the pathogenesis of BCC. These inclub
onwgenes and relatai molecules (i.e. AML-1 oncogene, row 10; c-fos interacting
upstream transcription factor 2, mw 34; ras homolog gene member C, row 60; v-myb
oncogene homolog, row 71; ras-associateci RalGDS, row 98; v-fgr oncogene homolog,
row 104). c k r - or melanoma-related molecules (i.e. tmour-associatecl caicium signal
transducer 1, row 16; me1anoma-associated ME20 antigen, row 18; Ewing sarcoma
bmkpoimt region 1, row 49; tumour rejection antigen 1, row 83; leukemia-associated
p18, row 90; T-cell leukemiallyrnphoma 1A protein, row 114), proliferative signaiiing
molecules (Le. insulin-lüre gmwth factor binding protein 3, row 3; STAT-1, row 14;
insulin-like growth factor binding protein 2, mw 84), ceîl cycle-nlated molecules (i.e.
tumour protein p53-binding protein 2, row 5; rehoblastoma-binding protein 4, row 6).
cytokines and their receptors (Le. LIF, mw 11; TGF-beta 2, row 29; lymphotoxin k t a
receptormùFR superfamiiy member 3, row 39; TNF-a-induced protein 3, row 70),
interferon-rclated m o l d e s (Le. interferon gamma-inducible protein 16, row 43),
chemokines (i.e. midkhc, row 88), accessory molecules (i.e. epithelial (E)-cadherin, row
24; integrin alpha V, row 45; cadherin 11. row 59; focal adhesion kinase 1, row 99),
stress-nlated mo1ccuit~ (Le. heaî shock ZOkD protein iB, row 251, cxüacllular matrix
and matriceL1Utat gencs (ia. syndecan 2, mw 15; lumican, row 58; laminin alpha 3, row
79; coilagens type I d , IIIal, Nul, IVaS, Val, Va2, XVmal, in rows 93,89,86,64,
91, 92 and 85, nspectively), and structural/cytoskeletal molecules and keratins (i.e.
bullous pemphigoid antigcn 1, row 41; tubulins al, a4, a5, in rows 67, 75 and 76,
nspectively; myosh light polypeptide 4, row 97; keratins 7, 8, 13, 18, in rows 108,72,
22, and 69, respcctively).
From the list of selectively upregulated genes (Table 2B). many of which may be
upreguiated in sclemsing BCC, there also appears to bc some interesting genes that fit
into the categones discussed above. These include oncogenes and nlated molecules (i.e.
v-crk oncogene homolog, mw 3; tmmforming protein pZlK-ras-2A, row 7; vwfes/v-Qs
oncogene homolog, row 64; ETS onwgenc ELK-1, row 65; pim-1 oncogene, row 66),
cancer- or melanoma-related molecules (i.e. melanoma antigen family A8, row 58),
signaMg molecules (ie. wingless-type MMTV integration site family member 4, row
10; numb homolog, row 11; rethol-binding protein 3, row 13; retinoid X receptor k a ,
row 55; tmslocon signal sequence receptor alpha, row 40; Rho GDP dissociation
inhibitor, row 68), cell cycle related (Le. Bcl-2-associated athanogene-1 (BAG
1)hcpatocelîular carcinoma complicating hemochmnatosis protein, row S), cytokines
(ic. interleukin-11, row 50; MCSF-1, row 57), accessory molecules (ia. CD74, row 14;
LFA-1, row 20; mannose-binding lectin 1, row 72). stress-related molecules (Le. HSP-
10lchaptronin 10, row 38), and extraceUuiar math genes (Le. collagen type V W , row
33). Several intaesthg upregulated genes an &O present in the Est of those that werc
&tccted in a SIMU number of patients ( s a Table 6). This small list of genes in Table 6
- - --- - - - -incliUtp hirrhct-additions @the list of a m g o 1 ~ 8 6 , cciaca- stid mdm. . r t1a tad ge-
cytokines, interferon-related genes, ma& remoàclling molscules (e.g. MMP-2, row 3;
MMP-9, row 24). angiogenesis activators (eg. endothelin-1, row 13; VEGF, row 27), and
a suppmsor of metastasis (e.g. kangai- 11CD82, row 26).
From the consistentiy do~ll~egulated list (Table 3), thm were not as many
striking genes of interest, howeva certain genes in this list may also be important to the
pathogcpesis of BCC. These include cytokines and bis receptars (Le. TNFRSFIA-
associateci via &th domain, row 36; glia maturation factor (GMF)-beta, row 40; TGF-
beta nceptcm 2, row 55; latent TGF-beta binding protein 1, row 69). interferon-related
gencs (Le. interferon-inducible guanylate bhding protein 2, row 64), cell cycle-related
molecules (i.e. BU-2-likt 2, row 66; ~ d r r i v s d growth inhibitor, row 72),
accessory molecules (i.e. gaiactosiâe-binûing lectin 3, row 41; cadherin 5, row 71),
angiogcnesis-rclated molecules (Le. cndo thelin-3, row 68), and keratins (i.e. keratin 1,
row 45; keratin 10, row 46). Several interesthg downreguiated genes are also present in
the list of those that were detectcd in a small numkr of patients (see Table 7). This srnail
list of genes in Table 7 includes further additions to the list of cytokines (e.g. ma, row
la), chemokines (e.g. interleulcin-16, row 19). interferon-related molecules (e.g.
interferon ngulatory factor 4, row 8). celî cycle-related molecules (e.g. cyclin G1, row
10; GAS-1, row 9), apoptosis-related molecules (e.g. caspase-8/FADDDlike ICE, row 1 l),
and angiogenesis inhibitors (e.g. TSP-2, row 12).
Collectively, ail of the gene lists h m Tables 2-7 represcnt a very large amount of
data to absorb. It is difficult at this timc to speculate rolcs for aii of the genes which
appear to k reguîated in BCC by this microarray analysis. However, when certain genes
a t e p l a c t d i n t o ~ ~ g ~ a p . w a s ~ u p a b o v e , s o m e s c n s e c a n b e m a d e â o m
the data. For example, numaous oncogenes and me1anoma-mlated gems appear to be
upguiatad in BCC, while p w t h inhibitors and apoptosis-nlated molecules appear
downnguiated. There also appears to be interesting regdation of cytokines and
interfmn-rclated genes, as weiî as extraceiluiar matrix and matncelîular genes. Much
work will be nquired to pinpoint the key playas withia these gene families.
One may ask why thae wac no obsmed changes in the ShhlPTCH signaiiing
pathway considering the estabrished rolc for these genes in BCC pathogenesis.
Unfortuastely, the ody member of this pathway that was pnsent on the microarrays used
was Gli-3, and regdation of this gene is irrelevant to BCC (sec Chapter 2). nimfore, in
one respect it is good that we did not observe any abnormal regdation of Gli-3. Since we
codd not look for involvement of PTCH-related genes, one way to validate that the
microarray technique worked propcrly, is to identQ other obseweâ gene expression
changes that were previously reported in the literature. This cm be difncult due to the
numkr of gent expression changes king analyzed. However, one good example of this
for these studies is the rcgulation of certain keratins that was obsmed Keratins are the
major structural proteins of aU epidermai celis including KCs. Previous
immunohistochemical d y s i s of 15 cases of BCC revealed that keratin 7 is upregulated
in some BCCs, and Lemtins 1 and 10 an 10st in BCC tissue 38a We obsemd similar
regdation of these 3 kcratin genes, in addition to upreplation of some other keratins.
A few interesthg cornments cm be made at this t h e regardhg c e m i n patterns of
gene regulation that wae observeci and how thesc relate to concepts described in Chapter
2. Additionally. certain aspects of BCC gene regulation that wcre obsewed may help to
provide insight into the biological devance of candidate genes in cutaneous malignancy,
and this satisfies the third hypothesis stated in Chapter 3. The following are some
examples of this. Activation of latent TGF-p was discussed in Chapter 2 for its abiiity to
inhibit tumour angiogenesis and growth via signaihg thugh Smad4. In contrast,
Indignant epithelial ~imours o k n develop mchanisms of escaping TGF-p signaliing,
and develop a TGF-p-resistant phenotype =ln . Studies have shown that the growth
inhibitory effects of TGF-p are mediated through the TGF-&receptor II (TGF-p-RU),
and mutations in this receptor have been reported in numemus cell lines 2 2 ~ 2 9 ,
primary cancers of the colon Z28*au3, head and neck *, and pancnas In agreement
witb these fiadings, our BCC m i m m y data indicate a domgdat ion d TGF-PRII.
At the same time, we observed upregulation of TGF-@2 and downregdation of latent
TGF-bbindiiig protein 1, which in combination may inmase concentrations of active
TGF-p. However, this would probably have a low p w t h inhibitory effect due to the
TGF-BR11 stahis. When tumour cells lose sensitivity to TGF- p-growth inhibition, the
excess TGF-p that nsults may act on tumour cells and stromal celis (via other TGF-p
receptors) to facilitate invasion and metastasis, hiduce angiogenesis, and suppress
mtitumour immune responses lm. F m our obscnva(i0os it is possible that this
phenornenon is occurring in BCC, albeit not in temu of metastatic potentid.
Anotha obsewation of interest is the upregulation of intaferon gamma-inducible
protein 16, interferon (alpha, beta and omega) receptor 2, and the downregulation of
interferon guanylate binding proteins 1 and 2, and interferon ngulatory factor 4.
Modulation of tâese interferon-nlated genes implicates them as molecular causes for
pmious reports on intecferon signaüing defats in BCC. Spontaneously regressing
----- - - BCCs have b n nported to show elevatcd -gamma activiîy ? Numerous
BCC patients also nspond to intcrferon tùerapy using alpha-interferon, or a topical
intcrfmn-inducing agent known as imiquimod p7wa9* The action of these
pharmacological tnatments couid involve compensatory mcchanisms to overcome the
downreguiated interferon-related moleculcc we have observed. The upreguiation of
intcrfmn reccptor 2 could also be nsponsible for the potent action of these forms of
thcrapy in BCC. Also rclated to the interferon signallhg pathway is the observed
upreguiation of STAT-1. STAT-1 is a cytoplasmic transcription factor that is
phosphorylated by Janus kinases (Jak) in nsponse to intaferon-gamma Phosphorylated
STAT-1 translocates to the nucleus, where it interacts witb promota interferon response
elements to tum on specific sets of interferon-gamma-inducible genes The elevated
levels of STAT-1 we have observed mey also enhance the activation of interferon-
inducible genes during BCC interféron thcrapy, by pviding a geater substrate
concentration for Jaic phosphorylation (i.e. by enhancing the,emyme kinetics).
The observed uprcgulation of ECM compontnts and matricellular proteins is also
potentidy important to the pathogenesis of BCC. Molecules which appeerad to be
upngulated included 8 fonns of collagen, laminia alpha 3, lumicari, and syndecan-2.
ECM deposition is important in BCC as shown by the pattems of tumour growth within
the suwmding strom (see Figure 7). Pahaps this deposition of ECM is involved in the
non-invasive phenotype of BCC, and the few patients that showed upreguîation of MMP-
2 anâ MMP-9 (sec Table 6) possess a more invasive phenotype due to elevated
rcrnodelling of ECM deposits. Upftgulation of syndccan-2 is cspecially interesthg due
to its ability to regdate matrix deposition "12. In addition, ment snidics have also
revr_cilrd thet syndeceiis medi& sigmii& pathways inrîigated by c d ndhrsinn
(integins) and p w t h factors lm. From these signalhg pathways, syndemn-2 can
control microfilament bundle formation, and cell morphology %'. Our otha finding that
integrin aipha 5 was upgulated in BCC could k related to the elevateà expression of
syndecan-2. because syndecan-2 and aSB1 integrin interactions arc required for the
cytoskeletal organization effect W. and tumorigenesis is often associated with abnord
ceii mo~phology. Overexprcssion of syndecan-2 may also c o m p e ~ for
domgdat ion of thrombospondins, because maaicellular proteins possess overlapping
functions in ECM assembly and cellular adhesion '? WC obsaved a dowmeguiation of
TSP-2 in some patients, which may create a requhrnent for upregulated syndecan-2.
Domgdation of TSP-2 would also support increased tumour angiogenesis, for the
s a m arguments as the observd domgdation of TSP-1 in the analysis of UVB-
inducible gaies.
DNA mismatch npair was mentioned in Chapter 2 regarding the documenteci
uprcguiation of MSH-2 in BCC, and how this may contribute to the inherent genomic
stability of BCC. It has been suggested that ihis may also be related to the non-invasive
phenotype of BCC. Numemus investigators are interesteci in identifjhg endogenous
moleculcs which give BCC this phenotype, and thcrefore prevent it h m metastasizing
(udike otha more aggressive carcinomas of the breast and colon). Intenstingly, some of
the BCC patients exhibited upreguiation of MSH-3 and MSH-6, which couid enhance the
mismatch repair activity of MSH-2. Therc also appeared to be some instances of
upteguîation for a metastasis suppressor gene known as kangai-11CD82, which may
warrant fuithcf investigations.
Our fourth hypothesjs fot rhese studics (see Chaptu 3) stated that hict~~zchical
clustcr analysis of the BCC microarray data wodd illustrate common trends in gaie
expression that exist for different histological subtypes of BCC. Indeed, this was the case
for our exmination of nodular and sclerosing BCC as indicatcd by the patient and gene
clusters (sec Figures 8-10). One goal that we had in mind, however, was to elucidate a
more clear-cut diffmnce between the gene expression profiles of nodular and sclerosing
BCC. Recall h m the patient clusta in Figure 8 that these two subtypes of BCC
clustercd together, suggesting that this was not the case. There are several possibilities
for this observation. The first possibility is that h m the genes spotted on the
micmamys we used, and the patient specixnens examine& the two BCC subtypes cannot
be differcntiated. A second possibility is that different clustering algorithm could be
required to ansnge the data in such a way that a clear-cut difftttnce between nodular and
sclmsing BCC cm be noticed. For instance, hiairchicai clustering can lead to artifacts.
With the agglomerative method, as clusters become larger the expression profile that
represents that cluster, which is the average of all profiles that belong to the cluster, may
not accurately reflect any of the containtd gene profiles. Hence, the higher up in the
hierarchical tree une look, the less nievant the genes within a cluster may k to each
other, Additionally, if a 'bad' mathematical decision is made early on during tree
construction, it c m o t be comcted later, b u s e agglomerative clustering is a bottom up
approach 219. niese problems may be avoided by first partitioning the data into
nasonably homogeneous groups. These groups can then be individually clustered, in two
dimensions (i.e. patient and gene clwtc~g) . Data partitionhg cm be perfonneû using
self-organizing maps, or K-muuis ciuste~g, before inputthg the gene expression data
inîathe -ve ciuster algorithm 219, Perhsps if we pedition our BCC deta. we
msy be able to discm the subtle gene expression differcnces between nodular and
sclerosing BCC. Quanâary also exists as to how one should normalw between
miaoerray txperiments. For these studies, we normalizcd within arrays (discussed
above), and then compared the resuiting data betwcen the patients without normaliziag
the data betwœn the patients. n ie mason for this is that we did not have a common
expaimcntai control cDNA probe that could k utilized to normalize between arrays.
Our expesimental design was good in that within each mimarray we obtained 'rd' &ta
about each patient because the control cDNA probe was patient-matched normal slM.
Howevcr, one potentiai cavcat relates to the specimens we utilized to m a k our tumour
and normal cDNA. It is possible that the tumour specimens h m the nduiar patients
containcd a higher penxntage of tumour tissue than did the sclmsing spccimens, due to
the scattend nature of sclemsing nimours (sec F i p 7). For example, a nodular tumeur
spccimen may contain 80% tumour tissue, while a sclerosing specimen may contain 40-
50% tumour tissue. Because we did not perform micro-dissection procedures to purify
the tumour tissue within these specimens, and just compared them directly to normal
tissue, it is possible that the amplitude of the gene enpression ratios for the nodular
patients was grcater (due to the higher percentage of tumour tissue) and that the nodular
BCC deta biased the agglomerative clustering process. In this manner, the data for
sclerosing patients could have becn mathematidy 'dragged' in to the hierarchicai trees
of the nodular patients. Nonnakation ktwcen arrays could potentially have prevented
this issue. This provides a strong argument for using a 3-fluof~~hoie microarray, which
is a cumntly âebated topic in the field of miaoamay technology. Ushg a 3-fluorophole
anqy~wccouldhaive ~ e a c A ~ s tiimniir~DUwithCy5~ the pah&ma!chcd
normal cDNA with Cy3, and then a common cell-line contd cDNA (hybridized io every
microarray in the study) with a dye such es Cy2 which hap distinct excitation and
emission sptctra h m the other two dyes. Normalization within arrays could then be
p&ormed using the Cy5lCy3 data, and normalization between arrays could be performed
using the Cy2 data as a commm reference h m every micromy experiment. This 3-
fiuorophorc microarrsy technique is something that wiii iikely appear over the next few
years for the exact rasons being discussed hem. Cumntly, we can only hypothesize that
the eXpmmental design we used is valid considering Ws limitation of the technique.
One way of better n m g betwan arrays is to avoid using patient-matched controls,
and WC a pool of c d lines to mdce a common cDNA conml for al1 mimarray
hybridizations. This was the expcrimental design used by Paou et al. in their ''Molecdar
portraits of human bmst tumours" publication of August 2000 The main caveat of
this experimental design is that the patient-mtcheà control c D N A reference is Iost.
Which method is ktter remains a topic of debate in this field of rcsearch. For now we
must be confident in the experimental approach we have taken for andyzing our BCC
patients, and even though we hopeà to observe the diagnostic potential of microarrays for
disceming between noduiar and sclerosing BCC, the conclusion may be that this is not
possible using the humaii 1.7k microarrays.
This brings us to our final hypothesis ( s a Chapter 3) that the discovery of
candidate genes in BCC will provide a basis for future rrsearch, and aid in delineating
potential therapeutic targets for non-melanoma slM cancer. Indced, this hypothesis may
be truc, and future directions of this nsearch are discussed in Chapter 7. In closing, the
undoubtcdly aid in bettcr undastanding the genetic processt~ of cutaneous neoplasia.
Hopefiilly these snidics will improve upon the curent understanding of BCC
pathogenesis, and lcad to new ideas for matment of non-melanoma skin cancer, and
other forms of cancer in generaï.
To m e r ûevelop our data on W-inducible genes, we would like to examine
whether the genes we delineated in vitro are biologicdly relevant to W-induced
carcinogenesis in vivo. The first gene of interest in this regard wouid be the W-
dow~veguiated gene thrombospondin-1. To investigate the angiogenic rde that this
dowmguiation may play in the omet of W-induced carcinogenesis we would obtain
TSP-1 lmockout mice and skin-targeted TSF1 transgenic mice (both of which have ban
reporteà in the fiterature), and utüize them for a UV-induced carcinogenesis mode1 to
examine changes in non-melanoma tumongenesis. This approach could also be talcen for
assessing the in vivo devance of some of the othcr W-inducible genes that were
elucidated, depending on whether or not there are viable biockout andor transgenic mice
available for those genes. Some of the other genes of interest m y be the AXL oncogene,
PD-ECGFIthymidine phosphorylase, LIF, c-ab1 proto-oncogene, oncostath A, HSP-71,
MCSF- 1 and MCP-1 (see Table 1A and Table 1B).
The d e of the above-wntioned genes in W-induced apoptosis may ais0 be of
interest. This could be investigateâ with M e r in vitro analyses. For example, it may
k of interest to transfect keratinocytes with some of the WB-upreguiateâ genes, or
knock-in dominant-negative mutants for some of the WB-downreguiaied genes, and
detennine the behaviour of these celis following acute or chronic W B exposure. This
may help to assess whether any of these genes are involved in apoptosis, the apoptosis-
resistant phenotype of E s , or the immunosuppressive profiie of gene expression
exhibitcd by KCs folIowing UVB exposun.
. Severai future dirocdons dm-apply to our analysis of gene reguiation in BCC.
One item of interest wiîi be to detemine relationships between certain upregulated (or
- dowmgulated) molecules, and icientiry the potentid hmction that their upreguiation may
have fm BCCw Che way to do this wiii be to further calibrate the agglomerative
clustering techique using data partitionhg methods (im. selfsrgauizing maps or K-
means clustcIing), and then utilize the proximity of gene clusters in relationship to each
other to try and assign related functions in gene expression levels. This application of
clustering has show to be effective for studies of genome-wide expression patterns in
yeast by Eisen et ai. Once we establish how to partition our data we also hope to
detect subtle Merences in the gene expression phenotypes of nodular and sclerosing
BCCw
It wiîî be of interest to examine the BCC diagnostic capabiiities of microarray
analysis as well. The nason king that there is a lot of discussion in the field of
microarray technology regardhg fiinire uses for microamys in differentially diagnosing
various human diseases. To assess the diagnostic potential for BCC, we would like to
obtaia microarray data for other fomis of cancer (either from a database web-îink cited in
other publications, or h m future studies of our own) and perform cluster anaîysis to
depict which clusters of BCC gene replation act as a gene expression fingerprint for this
fonn of cancer.
With our cumnt BCC data we plan to investigate each patient in more detail to
determine whethcr the= are sub-ciustas of gene expression that are associated with a
patient's d c a l history. For example, when we examine with more detail our
established gene expiession profiles for BCC, we may find that certain genes en
cammbnly rcgulated in rtcment BCCg or even BCCs with a mon ap~ressive
phenotype. In addition, we may also delincate molecular candidates which give BCC its
non-invasive phenotype. These genes couid then be investigated for matment of other
forms of aggressive (and fkequently metastatic) cancer, which couid potentialiy be
thempeutidy altercd to possess a BCC-like phenotype, thereby improving the survival
and Mestyle of cancer patients.
Lastly, once we have decided on somc candidate genes which may have
therapeutic potential for the mamient of BCC (confirmeci by protein studies), it may be
appropriate to set up pre-chical ai& in mice (and eventualiy clinical trials in humaris)
using recombinant proteins, immunotherapy, or antistnse technology to target these
candidate genes. Tben is a possibiiity that treatmcnts targeting certain candidate genes
detennined by mimamy may be beneficial for augmenthg the existing topicai
treatmcnts for BCC (i.e. ndwids and/or imiquimod and other interféron therapies).
These forms of matment for BCC are desirabte for üeating aggressive or recumnt forms
of BCC that either do not respond to invasive surgery, or cannot be excised surgically due
to their cosmetic location in s u exposai areas of the body. It wouid therefore be usefbl
in the future to offer patients other f o m of non-invasive therapy for BCC.
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