Small cell lung cancer (SCLC) is known to express the HuD protein, theneuronal antigen homologous to Drosophila Elav and Sxl genes involved inneuronal and sex development. HuD is the target of an immune responseincluding high titered antibodies causing paraneoplastic encephalomyeliUsand sensory neuropathy. Because thep53 recessive oncogene is mutatedand anti-p53 antibodies frequently occur in cancer patients, we wonderedif the development of anti-HuD antibodies signaled the presence of HuDmutations in hmg cancer. TheHUD gene was mapped to chromosome region ipusing a hwnan-mouse hybrid cell panel. We confinned that 26 of 46 cancer(43 lung cancer and 3 mesothelioma)cell linesexpressedHuD mRNA and thatthis expre@on,as wellneprotein expre@onby Western blot,com4ated strongly
@ththe SCLC neuroendoesine pheno@pe@ Southern Not and singlesthindconformation polymorphinn analyses showed that HuD was not mutated in 78lungcancers, including patients with the severe paraneopiastic syndrome. Nosibem blot analysisshowedthat lung cancer celllinesexpressedtwo major mRNAs(4.3 and 4.0 kilobases) of HuD. We found the three previously described alternative spliced mRNA forms (HuDpro, HuD, and HuDmex). In addition, we also
found HuD mRNA had an altemalive spiking foam in its 5'-coding region. Thisalternalive spliceintroduced 87 base pairs olsequence and a terminalion codonresulting in a predicted small, tmncated protein (ii amino ackis) reminiscent ofthe male-specific truncated protein in the &r-lethal (Sri) gene of DmsophiIa@However, mRNAs encoding both fill-length and tnancated proteins were expremed in all SCLCs. These results show that the HuD gene is not mutated in
lung cancer, including tumors from patients producing anti-HuD antibodies, butHuD expremion is an independent maiterordeteeminant ofthe neuroendocdnedifferentiation seen in SCL@
INTRODUCTION
Cancer patients can develop antibodies against tumor-associatedantigens and in some cases oncogene products. Anti-c-myc antibodieswere found in 57% of sera from colon cancer patients (1); anti-c-myb
antibodies were found in 43% of sera from breast cancer patients (2);while anti-p53 antibodies were found in 5—10%of sera from patientswith breast or lung cancer or lymphomas (3—5).We also have analyzed sera from patients against their autologous tumor cell lines andhave shown that 58% of SCLC3 patients develop antibodies againstvarious proteins expressed in their autologous tumor cells (6). p53 is
mutated in nearly all small cell lung cancers and —‘50%of non-smallcell lung cancers (7—9).Using immunoblotting techniques, 13% oflung cancer patients (4 of 40 SCLCs and 2 of 6 non-SCLCs) werefound to have p53-specific antibodies (4). These observations suggestthe hypothesis that antibodies against autologous tumor cell proteins
Received 4/21/94; accepted 7/12194.Thecostsof publicationof thisarticleweredefrayedin partby thepaymentof page
charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Julie Gould Foundation (S. A. B.), the G. Harold and
Leila Y. Mathers Charitable Foundation, and National Cancer Institute Grant P 20CA58220-01.
2 To whom requests for reprints should be addressed, at Simmons Cancer Center,
University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas,TX 75235-8590.
3 The abbreviations used are: SCLC, small cell lung cancer; NSCLC, non-small cell
found in sera from cancer patients may be directed against other as yetunknown oncogene products.
A characteristic serum antibody called anti-Hu sera has been demonstrated in patients afflicted with PEM and paraneoplastic sensoryneuropathy associated with tumors, especially SCLC (10—12).Thesesera react with a group of antigens (molecular mass, 32—45kilodaltons) that have been found to be specifically expressed in small celllung cancer and neurons. One hundred % of SCLC patients exhibitingthe typical paraneoplastic neurologic syndrome develop anti-Hu antibodies and, interestingly, 16% of SCLC patients not showing clinicalsymptoms of paraneoplastic disease also have anti-Hu antibodies intheir serum (13). The antigen HuD, detected by anti-Hu sera, wascloned by screening a human cerebellar A expression library (14);sequence analysis revealed that the deduced amino acid sequencecontained three potential RNA recognition motifs and was highlyhomologous to HuC, which was cloned at the same time (14), Hel-Ni(15), and the Drosophila RNA-binding proteins Elav and Sex-lethalinvolved in neuronal and sex development and differentiation, respectively (16—19).One possibility is that the HuD gene is mutated inSCLC patients and the mutant protein elicits an immune response,which in turn results in the paraneoplastic syndrome (14, 20). Relatedto this is the possibility that HuD, like p5.3, is a recessive oncogeneproduct that may exhibit mutations in SCLC.
In this study, we mapped the HuD gene to the chromosome ipregion (a site of allele loss in tumors) using human-rodent cell hybridpanels and analyzed the HuD gene for mutations in 78 lung cancer celllines. RT-PCR and Northern blot analyses confirmed that expressionwas highly correlated with neuroendocrine phenotype in lung cancer(20, 21). Southern blot analysis did not show any genomic rearrangements or amplification. Using SSCP analysis covering the entire openreading frame, no somatic mutations were detected in the codingregion. However, we found a new alternative spliced mRNA formoccurring just downstream of the translation initiation site and onesilent nucleotide polymorphism.
MATERIALS AND METHODS
Cell Lines. The tumor cell lines used in this study have been describedpreviously and are deposited at the American Type Culture Collection (Rockville, MD) (22—24).Among 78 cell lines used in this study, 46 cell lines wereavailable for both DNA and RNA analysis: 18 small cell lung carcinomas; 2extrapulmonary small cell carcinomas; 1 atypical carcinoid; 22 non-small cellcarcinomas; and 3 mesotheliomas (Table 1).
Chromosomal Mapping. A human-rodent somatic cell hybrid cell mapping panel 1 was purchased from the National Institute of General MedicalScience (Bethesda, MD). The chromosome 1 hybrid-mapping cell lines are asdescribed by Bader et a!. (25), with the additional mouse cell linesMCH6M2B24and MCH9O6.15(26) containing intact human chromosomes 1and 3, respectively. Other lines from which DNA was extracted for mappingwere: GM1604, human lung fibroblast cell line; A9, mouse cell line; microcellhybrid MCH6M2B2, A9 plus human chromosome 1; microcell hybridMCH5O3c1, A9 plus human t(X;lp); microcell hybrid MCH2O6c1, A9plus human t(X;lq); and microcell hybrid MCH9O6.15, A9 plus humanchromosome 3.
Molecular Analysis of the HuD Gene Encoding a Paraneoplastic EncephalomyelitisAntigen in Human Lung Cancer Cell Lines1
Yoshitaka Sekido, Scott A. Bader, David P. Carbone, Bruce E. Johnson, and John D. Minna2
Departments of Internal Medicine and Pharmacology, Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75235 (1'. S.. S. A. B., D. P. C..J. D. M.J, and Navy Medical Oncology Branch, National Cancer Institute, NIH, Bethesda. Maryland 20889 [B. E. J.J
Table 1 The relationship between HuD expression (RT-PCR) and neuroendocrine phenotype in46 lung cancer celllinesHuDNEexpression°phenotype―HistologyNo.NU
a@ expression of HuD in RT-PCR assay; —, lack of expression.
b ,@ NE, neuroendocrine phenotype (40—42); —, lack of expression.
Southern and Northern Blot Analyses. DNA and RNA were preparedfrom cell lines by standard techniques (27). For Southern blot analysis, 10 @gof high molecular DNA from cell lines were digested with EcoRI or Bglll,electrophoresed, and transferred to nitrocellulose membranes. Hybridizationand washing conditions were performed by standard techniques (28). ForNorthern blot analysis, 10 p.g of total RNA from cell lines were electrophoresod in formaldehyde-1% agarose gel and transferred to Hybond N+ (Amersham). Hybridization and washing were performed by standard techniques.The DNA probe used was the insert of pHBl.5 (14) provided by Dr. J. B.Posner (Memorial Sloan-Kettering Cancer Center, New York, NY).
Western Blot Analysis. Preparation of total cell lysates and Westernblotting were performed as described previously (4). In brief, after homogenization of subconfluently growing cells in lysis buffer, 20 p.g of total celllysate protein were subjected to sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and transferred to nitrocellulose membranes. Following blocking with 1% bovine serum albumin and 5% nonfat dry milk, the filters wereincubated for 2 h at room temperature with a 1:250 dilution of high titer
anti-HuD sera [obtained from a patient (NCI-H1155) with the typical paraneoplastic neurologic syndrome], washed 3 times with phosphate-bufferedsaline, and then reacted with tasI@proteinA (ICN, Costa Mesa, CA) for 1 h atroom temperature.
PCR/SSCP and Sequencing Analysis. Random primed cDNA wasprepared by standard methods using total RNA (28). Four overlappingHuD mRNA regions were amplified with primers is and 2as, 3s and 4 as,55 and 6as, and 7s and 8as (Fig. 2C). The sense primers used were: is(nt 65-85), 5'-AGCAAOCITFCfGCGAGACCCAATA1TFGC-3'; 3s (nt348-368), 5'-AGCAAOCVFGAGmAGGGTATOGA1TfGT-3'; 5s (nt591-611), 5'-AGCAAGC1TFCAAGTCACAGGAGTGTCCAG-3'; and 7s(nt 930-950), 5'-AGCAAGCrFATGGAATGACAAGCcTFGTGG-3'. Theantisense primers used were: 2as (nt 410-430), 5'-AGCl@CFAGAGAGTC1@-GAGTCCAmAAAGT-3'; 4as (nt 666-686), 5'-AGCl@CfAGAGCflCrGGCCATfCAGCCCfT-3'; 6as (nt 983-1003), 5'-AGCFCTAGAGlTGTAGACAAAGATGCACCA-3'; and 8as (nt 1355-1375), 5'-AGCFCFAGAACTGGCTTATAAAGTCCATGG-3'. Nucleotide coordinates and sequencewere obtained from the data of Szabo ci aL (14). All sense and antisenseprimers had extraneous nucleotides comprising the HindIll and XbaI sites attheir 5' ends, respectively. PCR products were cloned into the HindIII-XbaIsite of pBluescript KS (Stratagene, La Jolla, CA), and the plasmid DNASwereprepared from pooled clones were sequenced using T3 and Ti primers.
Each PCR amplification for SSCP was carried out using 0.5 @gof randomprimed cDNA in a final volume of 10 @d,which was labeled during thereaction with 0.5 pi (5 gtCi) of[a-32P]dCTP (>3000 Ci/mmol; Amersham); 50ng of each primer were combined with buffer. PCR conditions consisted of 1cycle at 95°Cfor 5 mm followed by 35 cycles at 95°Cfor 1 min, 52°Cfor 1mm, and 72°Cfor 2 min. The PCR products were digested with appropriaterestriction enzymes to yield a higher sensitivity due to their smaller size.Restriction enzymes used in this study were EcoRI, SstI, Bc!!,AluI, Dde!, andKpnI. The PCR products were electrophoretically separated both on a 6%polyacrylamide nondenaturing gel at 4°Cand a 6% polyacrylamide nondenaturing gel containing 5% glycerol at room temperature.
RESULTS
TheHuDGeneMapsto ChromosomalRegionip. Todeterminechromosomal localization ofHuD, we used two methods. First, a filter
containing DNAS of a human-rodent somatic cell hybrid mappingpanel was hybridized with the HuD probe, which gave results completely consistent with assignment to chromosome 1 and discordantwith assignment to any other chromosome (data not shown). Second,a filter of the chromosome 1 hybrid mapping panel was hybridized
with the HuD probe. The HuD probe detects strong —‘30-kilobaseandweak 2.1-kilobase bands in EcoRI digested human genomic DNA notseen in mouse A9 cell DNA (Fig. 1). This —‘30-kilobaseband wasalso seen in mouse cell line MCH6M2B2, which was derived frommouse A9 cells containing only human chromosome 1 prepared as a
monochromosome transfer hybrid (25). This unequivocally confirmsthe assignment to chromosome 1. This probe also detects a band inhybrid MCH5O3c1, which contains a t(X;lp) chromosome, but not
o@QooQ
kb0 @-‘ ‘0-
23.1—@ S9.4 —6.6—4.4 —
.@,
2.3 —2.0 —
Fig. I. The HuD gene maps to chromosome ip. DNAS were digested with EcoRI,separated in 1.0% agarose gel, transferred onto filter, and hybridized with HuD cDNAprobe. The DNAS used were: GM1604, human lung fibroblast cell line; A9, mousefibrosarcoma cell line; MCH6M2B2, A9 plus human chromosome 1; MCH5O3c1,A9 plushuman t(X;lp); MCH2O6c1,A9 plus human t(X;lq); and MCH9O6.15,A9 plus humanchromosome 3. Arrow, —30-kilobase(kb) EcoRI fragment of the human HulL)gene. The2.1-kilobase band of MCH6M2B2 and MCH5O3c1was visible but very weak.
A B0) (0 0 1'-. CD,@. ‘@1'C%JU) U,oJ ,- N. @-. C')I I I I I
KD
200-
97-
and random primed SCLC cDNA, and the PCR products wereseparated on a 6% nondenaturing gel. These experiments with 26lung cancer lines expressing HuD mRNA showed that the HuDtype is the predominantly expressed form, with HuDmex the nextmost abundant, while HuDpro was only expressed at very lowlevels (data not shown).
Searching for Mutations in the HuD Gene. To examine whetherexpression of HuD in SCLC is associated with gene rearrangement oramplification and whether the HuD gene had point mutations or smallmutations, we performed Southern blot and SSCP analyses. DNASfrom 78 cell lines digested by EcoRI or BglII were tested using
@E the 1.5-kilobase insert of pHB1.5 as a probe. This probe detects —30-and 2.1-kilobase EcoRI fragments and 8.2-, 5.8-, and 4.0-kilobaseBglII fragments (data not shown). The genomic region of HuD wasnormal by this analysis in all 78 lung cancer genomic DNAS (data notshown).
Using cDNA from the 26 cell lines positive for expression of HuDamong 46 cell lines tested, SSCP analysis was performed to testwhether point mutations exist in the coding region. Since the codingregion of HuD was 1140 base pairs, it was divided into four overlapping domains which were separately amplified with four sets ofprimers between is and 2as, 3s and 4as, 5s and 6as, and is and 8as
IIIIIIIIIIIIIII
28S- :: :@@
18S-
29-
a
Fig. 2. Expression of HuD mRNA and protein in lung cancer cell lines. (A) Northernblot analysis of HuD in lung cancer cell lines. Armwheads, two major HuD transcripts of4.3 and 4.0 kilobases. (B) Western blot analysis of HuD in lung cancer cell lines usinganti-HuD sera from patient Hi 155. Arrows, HuD antigens from 35 to 38 kiodaltons (KD).The histologicaltypeswere:SCLC,H249and H146;carcinoid,H720;squamouscellcarcinoma, Hi57; and adenocarcinoma, H358.
in hybrid MCH2O6c1, which contains a t(X;lq) chromosome. Thissublocalizes the HuD gene to chromosome arm ip. These data areconsistent with the assignment of HuD to lp34 by in situ hybridization by Muresu et al. (29).
TheHuDGeneIs PreferentiallyExpressedin LungCancerCellLines with Neuroendocrine Properties The HuD protein has beenshown to be expressed in SCLC by immunological techniques (20).To study the expression of HuD mRNA in lung cancer cell lines, weperformed RT-PCR and Northern blot analyses. First, cDNA wassynthesized from total RNA obtained from 46 tumor cell lines (22—24). Then, RT-PCR amplification was performed on two differentregions of HuD between primers is and 4as (from nt 65 to 686) andprimers 5s and 6as (from nt 591 to 1003). PCR products were loadedon 3.0% NuSieve agarose gel and the expected sizes were confirmedby ethidium bromide staining (data not shown). Among the cell lineswith neuroendocrine phenotype, expression of HuD was detected inall 18 SCLCS, 2 extrapulmonary small cell carcinomas, 1 atypicalcarcinoid, and 3 large cell carcinomas with neuroendocrine properties(Table 1). On the other hand, HuD expression was detected in only 2adenocarcinoma cell lines of the 19 non-small cell lung cancerswithout neuroendocrine properties and 0 of 3 mesotheliomas. Northem blot analysis confirmed the result of RT-PCR analysis and determined that all cell lines expressing HuD exhibited two major sizes ofmRNA of 4.3 and 4.0 kilobases (Fig. 14). Finally, Western blotanalysis was performed using cell lysates from 20 cell lines and theexpression pattern of the HuD protein was identical to the results ofRT-PCR and Northern blot analyses (Fig. 2B).
Three forms of HuD which differ by alternative mRNA splicinghave been described: HuDpro (unspliced); HuD (spliced between nt868 and 909); and HuDmex (spliced between nt 829 and 909)@(14).PCR amplification using primers 5s and 6as confirmed that the tumorcell lines express these three different alternative spliced forms.The three different amplified bands representing HuDpro, HuD, andHuDmex were subcloned and confirmed by DNA sequencing. Theexpression experiments (with [32P]dCTP) were then repeated usingthe subcloned HuDpro, HuD, and HuDmex constructs as controls
BHi28
3, ::
TIC-@CC
5,@
GA T C
H2i07
—
GATC
Hi46
—S —T
GA TC
C5'
lOObp
@1
is EcoRl 2as 5s AluiDdel 6a5@ I@@ II3s sstiecu4as@@ ‘1 7s KpnI 8as@ I
Fig. 3. Identification ofdistinct electrophoretic mobilities ofthe HuD cDNAs in SCLCby PCR-SSCP method. (A) PCR-SSCP analysis of the fragments amplified with primers55 and 6as followed by AluI restriction digestion yielded a different electrophoretic pattern(arrow). (B) Sequence analysis of the HuD cDNAs of the SCLC cell lines. Polymorphismwith silent nucleotide substitution (CCC to CCI', proline) is observed, which correspondsclearly to the distinct PCR-SSCP pattern. (C) schematic diagram of the strategy forPCR-SSCP analysis of HuD cDNAs. 0, coding region. bp, base pairs.
5 J. Liu, J. Dalmau, A. Szabo, M. Rosenfeld, J. Huber, and H. Furneaux. HuD: the
paraneoplastic encephalomyelitis antigen binds to the ARE element of c-fos mRNA,submitted for publication.
this expression strongly correlated with the neuroendocrine phenotype. In addition, we found a newly identified RNA-splicing forminserting 87 base pairs between nt 103 and 104, as well as thepreviously reported forms HuDpro (unspliced), and alternative splicedforms between nt 868 and 909 (HuD), and between nt 829 and 909(HuDmex) (14). If the previously predicted translation initiationcodon is used in this new spliced form mRNA, the entire HuDdownstream from this start site is not translated because a stop codonappears at amino acid ii. However, this insertion, which occurs justbefore the coding region, has two other ATG codons in frame, ofwhich the second one satisfied the Kozak consensus sequence (35). Ifthis initiation codon was used during translation, it would make aproduct with an altered amino terminus lacking two amino acids.These findings suggest that HuD expression might be regulated byalternative splicing like the Sex-lethal gene of Drosophila melanogaster, which has striking homology (42% identity) to HuD. TheSex-lethal gene plays a key role and is regulated by sex-specificalternative splicing (17, 36, 37), such that the female-specific formencodes a functional product while the male-specific form containsanother exon introducing a stop codon, truncating the open readingframe, and resulting in nonfunctional transcripts. While an analogousdifference could occur in lung cancer, we found both the truncated(“male―)and full-length (“female―)forms to be expressed in lungcancers. HuD has been shown to bind to the AU-rich segment of c-fosmRNA.5 Thus, it will be important to study how the proteins encodedby each alternative spliced product of the HuD gene participates inbinding mRNAs.
One interesting question that arises from this and previous studies(14, 20) is why only a small number of SCLC patients make aprofound immune response to the HuD product, while almost allSCLCs express HuD. One hypothesis is that a mutated form of theHuD gene induces these immune reactions. Dalmau et al. (38) reported on an “aberrantlyspliced―HuD mRNA (lacking 13 aminoacids) in tumor cells in a patient with PEM/paraneoplastic sensoryneuropathy. The purpose of the present study was to determinewhether HuD was aberrantly spliced or mutated in a more extensiveset of lung cancer cell lines. Southern blot analysis did not detect anyrearrangement or amplification among 78 cell lines and SSCP analysisusing the 26 lung cell lines expressing HuD did not detect any somaticmutation. Among the 26 cell lines expressing HuD, 13 sera corresponding to the same patients were available to test for the existence
of HuD antibody. Three sera had anti-HuD antibody (data not shown)and 1 of the patients (corresponding to cell line Hi 155) also had aclinically severe paraneoplastic syndrome. However, these lung cancer cell lines did not have any mutation in the HuD-coding region, nordid the 10 lung cancer cell lines whose corresponding patients had nodetectable anti-Hu antibodies. Even though we found no point mutations in the three lung cancer cell lines whose corresponding patientsexhibited anti-HuD antibodies, it still remains possible that the HuDgene is mutated in some patients with high titer anti-Hu sera. Onepossible explanation why these three cell lines have no mutations ofHuD is that the populations of mutated tumor cells which trigger theimmune response were eliminated by the host immune response. It
will be necessary to screen a large panel of fresh SCLC tumor samplesfrom patients with PEM and paraneoplastic sensory neuropathy toformally rule out this possibility. It is also possible that other familymembers, such as the HuC and Hel-Ni genes, might have mutations
which may induce the immune responses that would cross-react withHuD. In this regard it is notable that HuC was initially cloned asanother protein reacting with anti-Hu sera (14) and that Hel-Ni wasalso shown to be reactive with autoantibodies from patients withPEM (i5).
103
I ATGGTTATG -
2 ATG GTr ATG CCT TCT AGA ATC CTA AAG
TFGACCIG&AGCCAAGAAG.MAATTCT
GGT GAT GGG AGA AGT GGA GCC ACT TAA
104
. ATA AU AGC
ATl@ACT TAC ATG ATG ATA AU AGC
AFig. 4. Sequence analysis of the 5' end of the HuD cDNA. The upper sequence (1) is
reportedby Szaboet al. (14) and lowersequence(2) representsa newly identifiedalternative splicing form containing the 87-base pair insertion. The ATG codon indicatedbyboldtypeis thepredictedintiationcodonandtheATG(arrowhead)isa newpredictedinitiation codon. Underline, inframe stop codon. The number on the letter is the nt number.
(Fig. 3C). After PCR amplification, the product was digested withappropriate restriction enzymes yielding two smaller fragments. Although no somatic mutation was found within 26 samples expressingthe HuD gene, 3 cell lines had an identical aberrant migration in theregion between primers 5s and 6as (Fig. 3A). The amplified band wassubcloned into pBluescript KS plasmid and the mixture of transformants was sequenced (Fig. 3B). SCLCs H128 and H2107, which
express both forms, had a single base substitution of cytosine forthymine at nucleotide 781, which is the dominant type shown inH146. This change did not alter the encoded amino acid and wastherefore scored as a nucleotide sequence polymorphism.
In addition, the PCR amplification between primers is and 2asshowed an extra, larger band in all tumor lines expressing HuD cDNAin addition to the expected size band (data not shown). Since this extraband, as well as the predicted band, were able to be amplified by PCRusing only cDNAs as templates but not using genomic DNAS astemplates, we concluded that this extra band represents an alternativesplicing form. The extra cDNA has an additional 87 nucleotidesinserted between nt i03 and 104, which is on the 3' side of thepredicted initiation codon. This results in the generation of an mRNAencoding both a predicted truncated 11-amino acid peptide and a newdownstream potential initiation codon (Fig. 4).
DISCUSSION
The data presented here are the first examination of the HuD genefor mutations in a large number of lung cancer cell lines by moleculargenetic techniques. First, we demonstrated that HuD is located onchromosomal region ip using Southern blot hybridization of twoindependent somatic cell hybrid panels. This result is in completeagreement with the recent results of Muresu et a!. (29), who usedfluorescence in situ hybridization to map HuD to chromosome sitelp34. Small cell lung cancer is frequently associated with chromosomal deletions at 3p, 9p, 13q, and l7p, all of which are nowrecognized to contain recessive oncogenes (8, 30—32). Chromosome
region ip is also deleted in lung and other cancers and harbors theL-myc gene (lp32), which is also frequently overexpressed in smallcell lung cancer (33, 34). Therefore, it will be interesting to determinethe correlation between expression of HuD and other genes located atip, including the L-myc gene.
HuD mRNA expression was tested by RT-PCR and Northern blotanalyses. Among 46 cell lines, 26 were shown to express HuD and
Our results showed that the HuD gene was not mutated in lungcancer and that its expression correlated highly with tumor cell typesshowing neuroendocrine features. This suggests that HuD is notinvolved in the etiology of lung cancer but may be involved indetermining neuroendocrine cell development and differentiation inthe tumor cell lines that express it. This idea is compatible with thereport that monoclonal antibody MAb 16A1 1, which binds specifically to an epitope present in gene products of all known Hu genes,including HuD, HuC, and Hel-Ni, detects Hutproliferating cells innascent avian sensory and sympathetic ganglia in vivo and in populations of cultured neural crest cells (39). In addition, the study ofHel-Ni showed that this product binds specifically to the 3' untranslated regions of a number of mRNAs, including the transcripts for thecytokine granulocyte macrophage colony-stimulating factor and protooncogene mRNAs such as c-myc and c-los (15). Therefore, it wouldbe interesting to study whether HuD might affect the regulation ofoncogene mRNAs in SCLC, such as myc family members. In addition,transfection assays using an HuD expression construct could also bedone to determine whether expression of HuD would confer neuroendocrine characteristics to non-small cell lung cancers (40—42).
ACKNOWLEDGMENTS
The authors would like to acknowledge Dr. J. B. Posner for the gift of HuDcDNA clone, Dr. H. M. Fumeaux for preprints of his work prior to publication,and Dr. A. Gazdar for the lung cancer cell lines. We thank Shari Cundiff fortechnical assistance.
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