p21(WAF1/Cip1) retards the growth of human squamous cell carcinomas in vivo M. Cardinali a, *, J. Jakus a , S. Shah a , J.F. Ensley b , K.C. Robbins a , W.A. Yeudall a a Oral and Pharyngeal Cancer Branch, National Institute of Dental Research, Bethesda, MD 20892-4330, U.S.A. b Division of Hematology/Oncology, Wayne State University, Detroit, MI 48201, U.S.A. Received 22 September 1997; accepted 13 October 1997 Abstract The excessive proliferation exhibited by cancer cells is frequently a result of their failure to adequately regulate cell cycle pro- gression. In the present study, we developed a xenograft model of oral cancer in athymic mice, using squamous carcinoma cell lines and examined the ability of the cyclin-dependent kinase inhibitor p21 (WAF1/Cip1) to retard tumour growth in vivo, using a ret- roviral delivery system. Human p21 cDNA was cloned by polymerase chain reaction, expressed, and the encoded protein shown to have biological activity in in vitro kinase assays. Amphotropic retrovirus cultures which expressed recombinant p21 were generated and used to treat established squamous cell carcinoma xenografts. Two weeks following onset of treatment, tumours injected with p21 virus producer cells showed a reduction in size between 3- and 10-fold compared with tumours which received control cells which produced control virus alone. The data indicate that recombinant p21 may be of future use for therapeutic intervention in oral cancer. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Oral cancer; Gene therapy; Tumour suppressor; Cell cycle; p21; WAF1; Cip1; sdi1 1. Introduction Normal cellular growth is dependent upon the tightly regulated activation and inactivation of cyclin-depen- dent protein kinases (CDKs) which enable the cell to pass through several regulatory transition points in the cell division cycle [1]. Failure to regulate CDK activity may accelerate cell cycle progression, resulting in unchecked cell proliferation and neoplasia [2]. CDK activity is regulated intrinsically by a series of phos- phorylation and dephosphorylation events [3], and by a number of inhibitory proteins which bind and inactivate CDKs (reviewed in [4,5]). Thus, failure to express CDK inhibitors represents an important mechanism for deregulating cell cycle control in tumour cells. For example, in normal cells the CDK inhibitors p16, p15 and p18 act to suppress the activities of CDK4 and/or CDK6 [6,7], thus preventing phosphorylation of pRb and subsequent release of E2F, thereby blocking cell cycle progression. In many tumour types, it has now been demonstrated that p16 function is compromised through deletion [8,9], point mutation [8,9], or tran- scriptional silencing [10], while re-introduction of p16 into cells lacking this molecule represses growth [11–13]. Expression of p21(WAF1/Cip1), a general inhibitor of CDKs [14–16], is up-regulated by wild-type p53 in response to DNA damage [17], and contributes to G 1 cell cycle arrest under these circumstances. It has also been shown that p21 interacts with proliferating cell nuclear antigen (PCNA) to block DNA synthesis, although PCNA-dependent DNA repair is not aected [18–21]. Mutation of p53 is frequent in human cancers [22], and tumour cells lacking functional p53 fail to arrest in G 1 and repair genetic damage [23]. Cell cycle progression is also modulated by the action of cytokines [2]. Transforming growth factor (TGF)b1 has been characterised as a major negative regulator of epithelial cell proliferation, and blocks cell cycle pro- gression in G 1 in a number of epithelial cell types [24–26]. TGFb-mediated growth arrest may occur through several dierent mechanisms, including ORAL ONCOLOGY Oral Oncology 34 (1998) 211–218 1368-8375/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved PII: S1368-8375(97)00083-3 * Corresponding author: Oncology Branch, Division of Clinical Trials Design & Analysis, Center for Biologics Evaluation & Research, Food & Drug Administration, 1401 Rockville Pike, WOC I HFM-573, Rockville, MD 20852, U.S.A. e-mail: [email protected].
Transcript
p21(WAF1/Cip1) retards the growth of human squamous cellcarcinomas in vivo
M. Cardinali a,*, J. Jakus a, S. Shaha, J.F. Ensleyb, K.C. Robbins a, W.A. Yeudall a
aOral and Pharyngeal Cancer Branch, National Institute of Dental Research, Bethesda, MD 20892-4330, U.S.A.bDivision of Hematology/Oncology, Wayne State University, Detroit, MI 48201, U.S.A.
Received 22 September 1997; accepted 13 October 1997
Abstract
The excessive proliferation exhibited by cancer cells is frequently a result of their failure to adequately regulate cell cycle pro-
gression. In the present study, we developed a xenograft model of oral cancer in athymic mice, using squamous carcinoma cell linesand examined the ability of the cyclin-dependent kinase inhibitor p21 (WAF1/Cip1) to retard tumour growth in vivo, using a ret-roviral delivery system. Human p21 cDNA was cloned by polymerase chain reaction, expressed, and the encoded protein shown tohave biological activity in in vitro kinase assays. Amphotropic retrovirus cultures which expressed recombinant p21 were generated
and used to treat established squamous cell carcinoma xenografts. Two weeks following onset of treatment, tumours injected withp21 virus producer cells showed a reduction in size between 3- and 10-fold compared with tumours which received control cellswhich produced control virus alone. The data indicate that recombinant p21 may be of future use for therapeutic intervention in
oral cancer. # 1998 Elsevier Science Ltd. All rights reserved.
Normal cellular growth is dependent upon the tightlyregulated activation and inactivation of cyclin-depen-dent protein kinases (CDKs) which enable the cell topass through several regulatory transition points in thecell division cycle [1]. Failure to regulate CDK activitymay accelerate cell cycle progression, resulting inunchecked cell proliferation and neoplasia [2]. CDKactivity is regulated intrinsically by a series of phos-phorylation and dephosphorylation events [3], and by anumber of inhibitory proteins which bind and inactivateCDKs (reviewed in [4,5]). Thus, failure to express CDKinhibitors represents an important mechanism forderegulating cell cycle control in tumour cells. Forexample, in normal cells the CDK inhibitors p16, p15and p18 act to suppress the activities of CDK4 and/orCDK6 [6,7], thus preventing phosphorylation of pRb
and subsequent release of E2F, thereby blocking cellcycle progression. In many tumour types, it has nowbeen demonstrated that p16 function is compromisedthrough deletion [8,9], point mutation [8,9], or tran-scriptional silencing [10], while re-introduction of p16into cells lacking this molecule represses growth [11±13].
Expression of p21(WAF1/Cip1), a general inhibitorof CDKs [14±16], is up-regulated by wild-type p53 inresponse to DNA damage [17], and contributes to G1
cell cycle arrest under these circumstances. It has alsobeen shown that p21 interacts with proliferating cellnuclear antigen (PCNA) to block DNA synthesis,although PCNA-dependent DNA repair is not a�ected[18±21]. Mutation of p53 is frequent in human cancers[22], and tumour cells lacking functional p53 fail toarrest in G1 and repair genetic damage [23].
Cell cycle progression is also modulated by the actionof cytokines [2]. Transforming growth factor (TGF)b1has been characterised as a major negative regulator ofepithelial cell proliferation, and blocks cell cycle pro-gression in G1 in a number of epithelial cell types[24±26]. TGFb-mediated growth arrest may occurthrough several di�erent mechanisms, including
ORAL
ONCOLOGY
Oral Oncology 34 (1998) 211±218
1368-8375/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S1368-8375(97)00083-3
* Corresponding author: Oncology Branch, Division of Clinical
Trials Design & Analysis, Center for Biologics Evaluation & Research,
Food & Drug Administration, 1401 Rockville Pike, WOC I HFM-573,
decreasing expression of CDK2, CDK4 and cyclin E[27], blocking CDK2 activation [28] and throughincreasing expression of either or both p15INK4B [29]and p21(WAF1/Cip1) [30±32], and through p27(Kip1)[33,34].
Squamous cell carcinoma of the head and neck region(HNSCC) is the sixth most common cancer in devel-oped countries [35]. In spite of its high incidence, themolecular pathogenesis of the disease is poorly under-stood, although inactivation of p53 by various mechan-isms is a common feature of these tumours [36±38], andmay contribute to loss of cell cycle control. In addition,loss of responsiveness to TGFb is a feature of some celllines derived from squamous cell carcinomas, and mayrepresent a fundamental mechanism for evading growtharrest during tumour development. This may occurthrough loss of receptor expression [39,40], as a result ofreceptor mutation [41], or by inactivation of distal ele-ments in the signalling pathway. As both loss of wild-type p53 function and TGFb-mediated inhibition arelikely to compromise normal cell cycle control via fail-ure to upregulate p21, this molecule is a potentiallyattractive therapeutic for gene replacement strategies tocombat HNSCC. Therefore, in the present study wedeveloped an in vivo model of HNSCC, engineeredviruses which express recombinant human p21, andexamined their e�ect on tumour growth.
2. Materials and methods
2.1. Cell lines
The squamous carcinoma cell line HN8, derived froma lymph node metastasis of a human squamous cellcarcinoma of the epiglottis, has been described pre-viously [42,43]. HN8 cells were cultured in Dulbecco'smodi®cation of Eagle's medium (DMEM) supple-mented with 10% fetal bovine serum (FBS) and 0.4 mg/ml hydrocortisone on a feeder layer of lethally-irra-diated Swiss 3T3 ®broblasts. The feeder cells wereremoved prior to subculturing by standard techniques[44]. A431 human epidermoid carcinoma cells andSCC15 squamous carcinoma cells were obtained fromthe American Type Culture Collection (ATCC, Rock-ville, Maryland, U.S.A.) and grown in DMEM supple-mented with 10% FBS. The retroviral packaging cellline c2 [45] and the amphotropic producer cell linePA317 [46] were obtained from the ATCC and grown inDMEM supplemented with 10% calf serum (CS).
2.2. Recombinant plasmids and virus production
Human p21 cDNA was prepared by reverse transcrip-tion polymerase chain reaction (PCR) as previously des-cribed [43], using 1mg of total RNA from HeLa cells as a
template and the following oligonucleotides as primers:P21S 50-ACTCGAGATCTCCTATGTCAGAACCGG-CTGGGGATG-30; and P21AS 50-ATCTAGAATTCG-GATTAGGGCTTCCTCTTGG-30. PCR consisted of35 cycles of 95�C±55�C±72�C, 1min each. The ampli®-cation products were puri®ed (Wizard DNA Clean-up,Promega, Madison, Wisconsin, U.S.A.), restrictiondigested with BglII and EcoRI, gel-puri®ed and ligatedinto the prokaryotic expression vector pGEX4T3(Pharmacia, Piscataway, New Jersey, U.S.A.) whichhad been modi®ed to encode an in¯uenza virus hae-magglutinin (HA) epitope tag (pGEX4T3-HA). Theplasmid DNA was isolated and sequenced (Sequenasev2, USB, Madison, Wisconsin, U.S.A.) to con®rm thepresence of human p21 cDNA sequences. Clones wereanalysed for expression of glutathione-S-transferase-HA-p21 in Escherichia coli, and the HA-p21 cDNA wasexcised as a BamHI-EcoRI fragment and subclonedinto the retroviral vector pBABEneo [47] previouslylinearised with BamHI and EcoRI, to generate pBA-BEHAp21neo. pBABEHAp21neo plasmid (100 ng), orpBABEneo (100 ng) as a control, were used to transfectc2 cells by the calcium phosphate method [48]. The cellswere selected in 750 mg/ml G418. The supernatantobtained from pooled G418-resistant colonies was ®l-tered through a 0.45 mm ®lter and titred on NIH3T3®broblasts. To generate amphotropic virus, c2 cellswere cocultured with PA317 cells at a ratio of 5:1 in thepresence of 10 mg/ml polybrene.
2.3. Western blot analysis
To analyse the expression of recombinant HAp21,c2/PA317 cocultures were washed with ice-cold phos-phate bu�ered saline (PBS) and lysed in NP40 lysisbu�er (50mM Tris±HCl pH 7.4, 150mM NaCl, 20mMethylene diamine tetra acetic acid (EDTA), 0.5% Non-idet P-40, 1mM phenyl methylsulphonyl ¯uoride(PMSF), 5 mg/ml aprotinin, 5 mg/ml leupeptin; 600 mlbu�er per 100mm dish) on ice for 10min. Cellular deb-ris was removed by low speed centrifugation and theprotein content of the supernatant determined by a col-orimetric assay method (Biorad, Hercules, California,U.S.A.). Immunoprecipitation was carried out on 1mgaliquots of cleared cell lysates, using 2mg of anti-HAmonoclonal antibody (12CA5, Babco, Berkeley, Cali-fornia, U.S.A.). Immune complexes were captured onGammabind Sepharose, washed three times in lysisbu�er, electrophoresed in denaturing 12% poly-acrylamide gels and western blotted on to Immobilonmembrane (Millipore, Bedford, Massachusetts, U.S.A.).The membranes were blocked in Tris-bu�ered saline/0.05% Tween 20 (TBST) containing 5% skimmed milkat ambient temperature for 1 h, washed twice in TBSTand incubated with a 1:1000 dilution of anti-HA anti-body, or a 1:500 dilution of a monoclonal antibody
212 M. Cardinali et al./Oral Oncology 34 (1998) 211±218
which recognises human p21 (Ab-1, Oncogene Science,Manhasset, New York, U.S.A.) for 1 h. The membraneswere washed three times in TBST, incubated withhorseradish peroxidase-conjugated goat-anti-mouse orgoat anti-rabbit secondary antibodies, as appropriate,and detected with ECL (Amersham, Arlington Heights,Illinois, U.S.A.).
2.4. Immune complex kinase assays
To assay cdk2 kinase activity, cdk2 immune com-plexes were immunoprecipitated from HN8 cell lysatesas described above, using an a�nity-puri®ed rabbitanti-cdk2 polyclonal antibody (sc-163; Santa Cruz Bio-technology Inc.). The immunopreciptates were washedthree times in lysis bu�er, once in 50mM Hepes pH 7.5,and resuspended in 20 ml of kinase assay bu�er (50mMHepes pH 7.5, 10mM MgCl2, 1mM dithiothreitol(DTT), 25mM adenosine triphosphate (ATP)) togetherwith 10 mCi of g-[32P]ATP (3000Ci/mmol) and 0.2mg/mlhistone H1 as substrate. The reactions were incubated at37�C for 30min, terminated by addition of sodiumdodecylsulphate (SDS)-gel loading bu�er, and resolvedin denaturing 12% polyacrylamide gels, as describedabove. The gels were dried and autoradiographed.
2.5. Animals
Immunode®cient athymic nu-nu mice were obtainedfrom Harland-Sprague-Dawley (Madison, Wisconsin,U.S.A.) and housed according to National Institute ofHealth guidelines for the care and use of laboratoryanimals. The animals were maintained in laminar ¯owhoods under pathogen-free conditions and fed a stan-dard laboratory diet.
2.6. Establishment of tumour xenografts in athymic mice
SCC15 and HN8 cells were cultured to 80% con-¯uency on lethally irradiated ®broblast support. Thefeeder cells were removed by vigorous agitation with0.02% EDTA in PBS, and keratinocytes released fromthe culture dish by trypsinisation. Cells were washedonce in DMEM/FBS, resuspended in DMEM at a con-centration of 1.25�107 cells/ml and 400 ml of cell sus-pension was injected subcutaneously in the ¯ank regionof 4-week-old athymic mice. When tumours reached15mm in diameter, the animals were killed by CO2 suf-focation and the tumours aseptically removed. The®brous tissue surrounding the tumours was removedand fragments of viable tumour tissue measuring 3mm3
were dissected for transplantation. The recipient animalswere anaesthetised by inhalation with methoxy¯uorane,and 8mm incisions practiced bilaterally in the ¯ankregion. The tumour fragments were inserted in the sub-cutaneous space, and the wounds closed with stainless
steel wound clips (Becton Dickinson, Sparks, Maryland,U.S.A.). Clips were removed 3 days postoperatively.
2.7. In vivo tumour therapy
c2/PA317 cocultures expressing recombinant humanp21, or cells containing empty vector as control, wereharvested by trypsinisation, washed once in DMEM/CS, resuspended in DMEM at a concentration of2.5�106 cells/ml, and sublethally irradiated with6000 rads from a 137Cs source. The tumours on the left-hand side of each animal received 1�106 virus producercells containing empty vector, while the tumours on theright-hand side received 1�106 producer cells expressingrecombinant p21. The injection of producer cells wascarried out 4 days after initial tumour transplantation,and repeated weekly for 2 weeks. Tumour growth wasassessed twice weekly by measuring the tumour withcalipers. Prior to the second round of treatment, aphotographic record of tumour size was made. Oneweek following the second round of injection, animalswere killed by CO2 su�ocation and the tumours excisedand weighed.
3. Results
3.1. Establishment of tumour xenografts for in vivotherapy
In order to be able to test the e�ect of recombinantproteins on tumour growth, we needed to establish asuitable in vivo model system. Therefore, we assessed theability of the HNSCC-derived cell lines SCC15, HN22and HN8 to grow as xenografts in athymic mice. Cells(5�106) were injected subcutaneously into the ¯ankregion, and tumour development assessed over a12-week period. As shown in Table 1, both SCC15 andHN8 cell lines formed tumours in vivo, while HN22 wasnon-tumourigenic. Marked di�erences were apparent inHN8 and SCC15 tumours. Histological examinationrevealed that HN8 formed poorly di�erentiated, highlyvascular tumours which grew as a solid mass in thesubcutaneous space (Fig. 1A). In contrast, SCC15tumours were well di�erentiated, with numerous keratinpearls (Fig. 1B) while, at a gross level, they underwentcolliquation, with copious exudation of keratin. As wewished to use established lesions for gene replacement
Table 1
Tumorigenicity of SCC Cell Lines in Athymic Mice
Cell Line Tumorigenicity in vivo (nu/nu) Histological features
HN8 + poorly differentiated
HN22 ÿ ±
SCC15 + well differentiated
M. Cardinali et al./Oral Oncology 34 (1998) 211±218 213
experiments, SCC15 tumours were found to be unsui-table because of their failure to retain injected materialsduring mock treatments (data not shown). HN8 formedsolid lesions which grew uniformly when transplantedbilaterally in athymic mice (M.C., unpublished data).Thus, bilateral transplantation of HN8 was deemed tobe a suitable in vivo model for gene therapy experiments.
3.2. Recombinant p21 is biochemically active
As p21(WAF1) is a downstream e�ector of wild-typep53 and TGFb signalling, both of which may be com-promised during the development of HNSCC, we chosethis molecule as a candidate for HNSCC therapyexperiments. The entire coding region of human p21was obtained by PCR, and the cDNA cloned intopGEX4T3-HA, which facilitated the addition of anN-terminal HA epitope tag to the p21 protein, and theconstruct sequenced to ensure ®delity of ampli®cation.
Fig. 1. Propagation of head and neck squamous cell carcinoma
xenografts in vivo. Cells were cultured to 80% con¯uence, trypsinised,
washed, counted, and 5�106 cells were injected subcutaneously in the
¯anks of athymic mice. The tumours which developed were excised,
processed for histology, and stained with haematoxylin and eosin.
(A) HN8 tumour. (B) SCC15 tumour. Original magni®cation 20�(inset is 4�).
Fig. 2. Recombinant p21 inhibits CDK2 kinase activity. Human p21
cDNA was cloned into pGEX4T3-HA, and expressed as a haem-
fusion protein in Escherichia coli, as described in Materials and meth-
ods. Cyclin-dependent protein kinase 2 (CDK2) immunoprecipitates
prepared from HN8 cells were used to phosphorylate histone H1, in
the absence or presence of GST-HA-p21, or GST-HA as control, as
indicated. Reaction products were resolved in 10% polyacrylamide
gels, dried and autoradiographed.
Fig. 3. Expression of recombinant HAp21 by c2/PA317 cocultures.c2 cells were stably transfected with pBABEneoHAp21, or pBABEneo as control,
cocultured with PA317 cells and cell lysates prepared from subcon¯uent cultures, as described in Materials and methods. (A) Aliquots (1mg) of cell
lysates, as indicated, were incubated with anti-haemagglutinin (HA) antibody and immune precipitates denatured, resolved in 12% polyacrylamide,
western blotted, incubated with anti-p21 antibody and detected with an horseradish peroxidase-conjugated secondary antibody and enhanced chem-
iluminescence. (B) Aliquots (50mg) of total cell lysate, as indicated, were western blotted, incubated with anti-HA antibody and detected as in (A), above.
214 M. Cardinali et al./Oral Oncology 34 (1998) 211±218
To test biochemical function, the recombinant HAp21was expressed in E. coli as a fusion protein with glu-tathione-S-transferase (GST), puri®ed, and tested for itsability to inhibit CDK2 activity in an in vitro kinaseassay. As shown in Fig. 2, GST-HAp21 inhibited CDK2histone H1 kinase activity in vitro, and in a dose-dependent manner, whereas a control reaction incu-bated with GST-HA alone produced no reduction inCDK2 activity. These data demonstrate that therecombinant HAp21 protein retains the CDK2-inhibi-tory function associated with cellular p21, and is likelyto be useful for CDK2 inhibition in vivo with the aid ofa suitable delivery system.
3.3. Expression of recombinant p21 by 2/PA317 cells
To facilitate delivery of recombinant HAp21 tohuman tumour cells in vivo, we subcloned the HAp21cDNA into the retroviral vector pBABEneo, and stablytransfected the resultant pBABEneoHAp21 into theecotropic packaging cell line c2. As a control, c2 cellswere transfected with pBABEneo lacking HAp21sequences. c2/neo and c2/HAp21 cultures of equivalenttitre were cocultured with PA317 amphotropic producercells to generate amphotropic viruses. As shown inFig. 3, recombinant HAp21 was expressed by c2/HAp21 cells, but not by control cells. The data suggestthat HAp21 protein is expressed e�ciently from theretroviral construction.
3.4. In vivo therapeutic e�ect of recombinant HAp21
To test the ability of HAp21 to inhibit tumour growthin vivo, we harvested control and HAp21-expressing c2/PA317 cocultures, sublethally irradiated them, andinjected aliquots of 1�106 cells into the HN8 xeno-grafts. A second injection was performed 1 week later,following assessment of tumour size. Two weeks afterthe initial injection, the animals were killed and the sizeand weight of the tumours determined. Fig. 4A showsthe results of a representative experiment in whichtumour volume was reduced in a group of ®ve experi-mental animals on the side which received HAp21-pro-ducing cells. The volumes of control-treated and p21-treated tumours in individual animals within oneexperimental group are indicated in Fig. 4B. Thereduction in size of tumours treated with HAp21-expressing c2/PA317 cocultures is further emphasisedin Fig. 4C. Tumour volume is minimal on the right-hand side of the animals (treated with p21 viruses) butprominent on the left-hand side, which was treated withcontrol cells. The reduction in tumour weight rangedfrom 3- to 10-fold compared with tumours whichreceived control cells (data not shown). The resultsindicate that HAp21-expressing c2/PA317 cells retardthe growth of HN8 xenografts in vivo.
Fig. 4. In vivo therapeutic e�ect of recombinant HAp21. HN8 xeno-
grafts were established and grown bilaterally in athymic mice and in-
jected with c2/PA317 cocultures, as described in Materials and
methods. Left-hand side tumours received injections of control cells,
while right-hand side lesions were injected with HAp21-expressing
cells. (A) Mean tumour volume recorded at termination of experiment
in one group of ®ve mice. Bars represent 1 standard deviation. Data
are representative of those obtained in four separate experiments.
(B) Tumour volume at termination of experiment in individual mice
within one experimental group. Data are representative of those
obtained in four separate experiments. (C) Comparison of treatment
with control virus (left ¯ank) or p21 virus (right ¯ank) on tumour size,
prior to the second round of producer cell injections.
M. Cardinali et al./Oral Oncology 34 (1998) 211±218 215
4. Discussion
In the present study, we used recombinant retro-viruses which express the human CDK inhibitor p21(WAF1) as a therapeutic agent to treat HNSCC. Thepractical di�culties in establishing a suitable modelsystem were not inconsiderable, as several HNSCC celllines tested in athymic mice either failed to formtumours or underwent coliquation and ulceration, thusmaking them unsuitable for injection of viral producercell suspensions. This may also be an important issue tobe considered if a similar approach is adopted in futureclinical trials, as HNSCC lesions undergo necrosis andulceration in vivo in greater than 50% of cases [49,50].This likely re¯ects poor tumour vascularity and/or oxy-genation, probably as a result of high cellular prolifera-tion rates. For the purposes of this study, the use ofHN8 cells as a model enabled us to circumvent theproblems encountered initially, as these cells grow as asolid mass upon subcutaneous inoculation into athymicmice. Furthermore, the phenotype of HN8 cellsappeared to be stable upon serial passage in mice, asjudged by morphological analysis of cells reculturedfrom tumour xenografts and uniform tumour growth invivo (unpublished data).
By performing multiple injections of retroviral pro-ducer cells which expressed recombinant p21, we wereable to retard the growth of HN8 tumours when com-pared with tumours which received injections of con-trol cells lacking p21. This is not surprising, as p21 actsas an inhibitor of CDKs [14±17] and PCNA [18±21],and is a critical mediator of growth arrest under anumber of cellular conditions. These include wild-typep53-dependent cell cycle arrest in response to DNAdamage [17], signalling by TGFb [30±32], and di�er-entiation signals [51±54]. Several previous studies havedemonstrated the therapeutic e�ects of wild-type p53on tumour cell growth both in vitro and in vivo [55±59].Indeed, the introduction of wild-type p53 into tumourcells which express dominant-negative mutant p53proteins has been reported to suppress growth [57].Although wild-type p53 is thought to have several bio-logical e�ects such as tumour suppression [60±62] andinduction of apoptosis [63,64], p53-dependent cell cyclearrest is likely mediated through induction of p21expression with subsequent CDK inhibition [14,15].The results of this study, therefore, demonstrate thatp21-mediated cell cycle arrest may be su�cient forinhibition of tumour cell growth in vivo, therebyseparating this function from other p53-dependentbiological responses. This may be in addition to theability of exogenous wild-type p53 to induce pro-grammed cell death, as demonstrated by other recentreports [65±67].
The use of recombinant viruses expressing p21 totreat models of malignant disease has been reported
previously [58,59,68]. Adenoviral vectors containingrecombinant p21 have been shown to suppress growthof p53-de®cient murine prostate cancer cells in vitro to agreater extent than similar constructs carrying wild-typep53, while in vivo experiments to treat establishedtumours have demonstrated that recombinant p21 ade-noviruses are not only more potent than p53 viruses atreducing growth rate and tumour size, but also prolongthe survival of tumour-bearing animals [58]. In a separ-ate study, adenoviral delivery of p21 to p53-null astro-cytoma cells decreased their growth rate in vitro andsuppressed their tumorigenicity in vivo when trans-planted either ectopically or orthotopically [68]. In con-trast to these results and those of the present study,recombinant p21 viruses were unable to inhibit thegrowth of HNSCC cells in vitro, or to reduce the size ofestablished tumours in vivo, while p53 adenoviruseswere e�ective in both situations [59]. This is not sur-prising, as some HNSCC cell lines grow well in vitro andin vivo, in spite of relatively high endogenous levels ofp21 (unpublished data). Taken together, these ®ndingssuggest that some tumour types or, indeed, sometumours within a speci®c class of lesions may responddi�erently to the same molecular therapeutics. Theclinical success of therapy will likely require prior ident-i®cation of markers, such as DNA content parameters[70,71], which are predictive of the biological responseof individual tumours to speci®c treatment regimes.
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