Molecular Analysis of the Hud Gene Encoding a Paraneoplastic Encephalomyelitis Antigen in Human Lung Cancer Cell Lines1

ICANCERRESEARCH54, 4988-4992, September 15, 19941 Molecular Analysis of the HuD Gene Encoding a Paraneoplastic Encephalomyelitis Antigen 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

ABSTRACT found in sera from cancer patients may be directed against other as yet unknown oncogene products. Small cell lung cancer (SCLC) is known to express the HuD protein, the A characteristic serum antibody called anti-Hu sera has been dem neuronal antigen homologous to Drosophila Elav and Sxl genes involved in onstrated in patients afflicted with PEM and paraneoplastic sensory neuronal and sex development. HuD is the target of an immune response including high titered antibodies causing paraneoplastic encephalomyeli neuropathy associated with tumors, especially SCLC (10â€”12).These Usand sensory neuropathy. Because thep53 recessive oncogene is mutated sera react with a group of antigens (molecular mass, 32â€”45kilodal and anti-p53 antibodies frequently occur in cancer patients, we wondered tons) that have been found to be specifically expressed in small cell if the development of anti-HuD antibodies signaled the presence of HuD lung cancer and neurons. One hundred % of SCLC patients exhibiting mutations in hmg cancer. TheHUD gene was mapped to chromosome region ip the typical paraneoplastic neurologic syndrome develop anti-Hu an using a hwnan-mouse hybrid cell panel. We confinned that 26 of 46 cancer tibodies and, interestingly, 16% of SCLC patients not showing clinical (43 lung cancer and 3 mesothelioma)celllinesexpressedHuD mRNA and that symptoms of paraneoplastic disease also have anti-Hu antibodies in thisexpre@on,as wellneprotein expre@onby Western blot,com4ated strongly their serum (13). The antigen HuD, detected by anti-Hu sera, was @ththe SCLC neuroendoesine pheno@pe@ Southern Not and singlesthind cloned by screening a human cerebellar A expression library (14); conformation polymorphinn analyses showed that HuD was not mutated in 78 sequence analysis revealed that the deduced amino acid sequence lungcancers, including patients with the severe paraneopiastic syndrome. Nosib contained three potential RNA recognition motifs and was highly em blot analysisshowedthat lung cancer celllinesexpressedtwo major mRNAs homologous to HuC, which was cloned at the same time (14), Hel-Ni (4.3 and 4.0 kilobases) of HuD. We found the three previously described alter native spliced mRNA forms (HuDpro, HuD, and HuDmex). In addition, we also (15), and the Drosophila RNA-binding proteins Elav and Sex-lethal found HuD mRNA had an altemalive spiking foam in its 5'-coding region. This involved in neuronal and sex development and differentiation, respec alternalive spliceintroduced 87 base pairs olsequence and a terminalion codon tively (16â€”19).One possibility is that the HuD gene is mutated in resulting in a predicted small, tmncated protein (ii amino ackis) reminiscent of SCLC patients and the mutant protein elicits an immune response, the male-specific truncated protein in the &r-lethal (Sri) gene of DmsophiIa@ which in turn results in the paraneoplastic syndrome (14, 20). Related However, mRNAs encoding both fill-length and tnancated proteins were ex to this is the possibility that HuD, like p5.3, is a recessive oncogene premed in all SCLCs. These results show that the HuD gene is not mutated in product that may exhibit mutations in SCLC. lung cancer, including tumors from patients producing anti-HuD antibodies, but In this study, we mapped the HuD gene to the chromosome ip HuD expremion is an independent maiterordeteeminant ofthe neuroendocdne region (a site of allele loss in tumors) using human-rodent cell hybrid differentiation seen in SCL@ panels and analyzed the HuD gene for mutations in 78 lung cancer cell lines. RT-PCR and Northern blot analyses confirmed that expression INTRODUCTION was highly correlated with neuroendocrine phenotype in lung cancer (20, 21). Southern blot analysis did not show any genomic rearrange Cancer patients can develop antibodies against tumor-associated ments or amplification. Using SSCP analysis covering the entire open antigens and in some cases oncogene products. Anti-c-myc antibodies reading frame, no somatic mutations were detected in the coding were found in 57% of sera from colon cancer patients (1); anti-c-myb region. However, we found a new alternative spliced mRNA form antibodies were found in 43% of sera from breast cancer patients (2); occurring just downstream of the translation initiation site and one while anti-p53 antibodies were found in 5â€”10%of sera from patients silent nucleotide polymorphism. with breast or lung cancer or lymphomas (3â€”5).We also have ana lyzed sera from patients against their autologous tumor cell lines and MATERIALS AND METHODS have shown that 58% of SCLC3 patients develop antibodies against various proteins expressed in their autologous tumor cells (6). p53 is Cell Lines. The tumor cell lines used in this study have been described mutated in nearly all small cell lung cancers and â€”â€˜50%ofnon-small previously and are deposited at the American Type Culture Collection (Rock cell lung cancers (7â€”9).Using immunoblotting techniques, 13% of ville, MD) (22â€”24).Among78 cell lines used in this study, 46 cell lines were lung cancer patients (4 of 40 SCLCs and 2 of 6 non-SCLCs) were available for both DNA and RNA analysis: 18 small cell lung carcinomas; 2 extrapulmonary small cell carcinomas; 1 atypical carcinoid; 22 non-small cell found to have p53-specific antibodies (4). These observations suggest carcinomas; and 3 mesotheliomas (Table 1). the hypothesis that antibodies against autologous tumor cell proteins Chromosomal Mapping. A human-rodent somatic cell hybrid cell map ping panel 1 was purchased from the National Institute of General Medical Received 4/21/94; accepted 7/12194. Science (Bethesda, MD). The chromosome 1 hybrid-mapping cell lines are as Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage described by Bader et a!. (25), with the additional mouse cell lines charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. MCH6M2B24and MCH9O6.15(26) containing intact human chromosomes 1 1 This work was supported by the Julie Gould Foundation (S. A. B.), the G. Harold and and 3, respectively. Other lines from which DNA was extracted for mapping Leila Y. Mathers Charitable Foundation, and National Cancer Institute Grant P 20 were: GM1604, human lung fibroblast cell line; A9, mouse cell line; microcell CA58220-01. hybrid MCH6M2B2, A9 plus human chromosome 1; microcell hybrid 2 To whom requests for reprints should be addressed, at Simmons Cancer Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, MCH5O3c1, A9 plus human t(X;lp); microcell hybrid MCH2O6c1, A9 TX 75235-8590. plus human t(X;lq); and microcell hybrid MCH9O6.15, A9 plus human 3 The abbreviations used are: SCLC, small cell lung cancer; NSCLC, non-small cell chromosome 3. lung cancer; RT-PCR, reverse transcriptase-polymerase chain reaction; SSCP, single strand conformation polymorphism; PEM, paraneoplastic encephalomyelitis; PBS, phos phate-buffered saline; nt, nucleotide; cDNA, complementary DNA. 4S.A. BaderandE.Stanbridge,unpublishedresults. 4988

linesHuDNEexpressionÂ°phenotypeâ€•HistologyNo.NUTable 1 The relationship between HuD expression (RT-PCR) and neuroendocrine phenotype in46 lung cancer cell

lines++Small lungcancercell cell lung cancer18H82, H128, H146, H182, H209, H249, H526, H711, H862, H1045, H1092, H1105, H1284, H1618, H1770, H2107, H2195++Carcinoid1H720++Extra H2171,

H1870++Large pulmonary small cell cancer2H1341, H1385+â€”Adenocarcinoma2H23, cell carcinoma with NE properties3H460, H1155, H1792â€”â€”Adenocarcinoma13H322, H358, H522, H838. H920, H1355, H1395, H1466, H1573, H1651, H2077--Squamous H1648,H1666, 11226--Adenosquamous cell carcinoma2H157, carcinoma1H647â€”â€”Large cell carcinoma1H1334--Mesothelioma31128, cell 11290, H2052

@ a expression of HuD in RT-PCR assay; â€”, lack of expression. b ,@ NE, neuroendocrine phenotype (40â€”42); â€”, lack of expression.

Southern and Northern Blot Analyses. DNA and RNA were prepared RESULTS from cell lines by standard techniques (27). For Southern blot analysis, 10 @g of high molecular DNA from cell lines were digested with EcoRI or Bglll, TheHuDGeneMapsto ChromosomalRegionip. Todetermine electrophoresed, and transferred to nitrocellulose membranes. Hybridization chromosomal localization ofHuD, we used two methods. First, a filter and washing conditions were performed by standard techniques (28). For containing DNAS of a human-rodent somatic cell hybrid mapping Northern blot analysis, 10 p.g of total RNA from cell lines were electrophore panel was hybridized with the HuD probe, which gave results com sod in formaldehyde-1% agarose gel and transferred to Hybond N+ (Amer pletely consistent with assignment to chromosome 1 and discordant sham). Hybridization and washing were performed by standard techniques. with assignment to any other chromosome (data not shown). Second, The DNA probe used was the insert of pHBl.5 (14) provided by Dr. J. B. a filter of the chromosome 1 hybrid mapping panel was hybridized Posner (Memorial Sloan-Kettering Cancer Center, New York, NY). with the HuD probe. The HuD probe detects strong â€”â€˜30-kilobaseand Western Blot Analysis. Preparation of total cell lysates and Western weak 2.1-kilobase bands in EcoRI digested human genomic DNA not blotting were performed as described previously (4). In brief, after homoge nization of subconfluently growing cells in lysis buffer, 20 p.g of total cell seen in mouse A9 cell DNA (Fig. 1). This â€”â€˜30-kilobasebandwas lysate protein were subjected to sodium dodecyl sulfate-polyacrylamide gel also seen in mouse cell line MCH6M2B2, which was derived from electrophoresis and transferred to nitrocellulose membranes. Following block mouse A9 cells containing only human chromosome 1 prepared as a ing with 1% bovine serum albumin and 5% nonfat dry milk, the filters were monochromosome transfer hybrid (25). This unequivocally confirms incubated for 2 h at room temperature with a 1:250 dilution of high titer the assignment to chromosome 1. This probe also detects a band in anti-HuD sera [obtained from a patient (NCI-H1155) with the typical para hybrid MCH5O3c1, which contains a t(X;lp) chromosome, but not neoplastic neurologic syndrome], washed 3 times with phosphate-buffered saline, and then reacted with tasI@proteinA(ICN, Costa Mesa, CA) for 1 h at room temperature. PCR/SSCP and Sequencing Analysis. Random primed cDNA was prepared by standard methods using total RNA (28). Four overlapping HuD 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 o@QooQ (nt 65-85), 5'-AGCAAOCITFCfGCGAGACCCAATA1TFGC-3'; 3s (nt 348-368), 5'-AGCAAOCVFGAGmAGGGTATOGA1TfGT-3'; 5s (nt 591-611), 5'-AGCAAGC1TFCAAGTCACAGGAGTGTCCAG-3'; and 7s (nt 930-950), 5'-AGCAAGCrFATGGAATGACAAGCcTFGTGG-3'. The kb antisense primers used were: 2as (nt 410-430), 5'-AGCl@CFAGAGAGTC1@- 0 @-â€˜ â€˜0- @ GAGTCCAmAAAGT-3'; 4as (nt 666-686), 5'-AGCl@CfAGAGCflCr 23.1â€” S GGCCATfCAGCCCfT-3'; 6as (nt 983-1003), 5'-AGCFCTAGAGlTGTA 9.4 â€” GACAAAGATGCACCA-3'; and 8as (nt 1355-1375), 5'-AGCFCFAG 6.6â€” AACTGGCTTATAAAGTCCATGG-3'. Nucleotide coordinates and sequence were obtained from the data of Szabo ci aL (14). All sense and antisense 4.4 â€” primers had extraneous nucleotides comprising the HindIll and XbaI sites at

their 5' ends, respectively. PCR products were cloned into the HindIII-XbaI .@, site of pBluescript KS (Stratagene, La Jolla, CA), and the plasmid DNASwere prepared from pooled clones were sequenced using T3 and Ti primers. 2.3 â€” Each PCR amplification for SSCP was carried out using 0.5 @gofrandom 2.0 â€” primed cDNA in a final volume of 10 @d,which was labeled during the reaction with 0.5 pi (5 gtCi) of[a-32P]dCTP (>3000 Ci/mmol; Amersham); 50 ng of each primer were combined with buffer. PCR conditions consisted of 1 cycle at 95Â°Cfor5 mm followed by 35 cycles at 95Â°Cfor1 min, 52Â°Cfor1 mm, and 72Â°Cfor2 min. The PCR products were digested with appropriate Fig. I. The HuD gene maps to chromosome ip. DNAS were digested with EcoRI, restriction enzymes to yield a higher sensitivity due to their smaller size. separated in 1.0% agarose gel, transferred onto filter, and hybridized with HuD cDNA Restriction enzymes used in this study were EcoRI, SstI, Bc!!,AluI, Dde!, and probe. The DNAS used were: GM1604, human lung fibroblast cell line; A9, mouse KpnI. The PCR products were electrophoretically separated both on a 6% fibrosarcoma cell line; MCH6M2B2, A9 plus human chromosome 1; MCH5O3c1,A9 plus human t(X;lp); MCH2O6c1,A9 plus human t(X;lq); and MCH9O6.15,A9 plus human polyacrylamide nondenaturing gel at 4Â°Cand a 6% polyacrylamide nondena chromosome 3. Arrow, â€”30-kilobase(kb)EcoRI fragment of the human HulL)gene.The turing gel containing 5% glycerol at room temperature. 2.1-kilobase band of MCH6M2B2 and MCH5O3c1was visible but very weak. 4989

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. MOLECULAR ANALYSIS OF THE HuD GENE A B and random primed SCLC cDNA, and the PCR products were ,@.0) â€˜@1'C%JU)(0 0 1'-. U,CD separated on a 6% nondenaturing gel. These experiments with 26 oJ ,- N. @-. C') lung cancer lines expressing HuD mRNA showed that the HuD IIIII 11111 KD type is the predominantly expressed form, with HuDmex the next 200- most abundant, while HuDpro was only expressed at very low levels (data not shown). 97- Searching for Mutations in the HuD Gene. To examine whether expression of HuD in SCLC is associated with gene rearrangement or @ 28S- :: :@ amplification and whether the HuD gene had point mutations or small mutations, we performed Southern blot and SSCP analyses. DNAS from 78 cell lines digested by EcoRI or BglII were tested using

18S- @E the 1.5-kilobase insert of pHB1.5 as a probe. This probe detects â€”30- and 2.1-kilobase EcoRI fragments and 8.2-, 5.8-, and 4.0-kilobase BglII fragments (data not shown). The genomic region of HuD was 29- normal by this analysis in all 78 lung cancer genomic DNAS (data not a shown). Using cDNA from the 26 cell lines positive for expression of HuD Fig. 2. Expression of HuD mRNA and protein in lung cancer cell lines. (A) Northern among 46 cell lines tested, SSCP analysis was performed to test blot analysis of HuD in lung cancer cell lines. Armwheads, two major HuD transcripts of whether point mutations exist in the coding region. Since the coding 4.3 and 4.0 kilobases. (B) Western blot analysis of HuD in lung cancer cell lines using region of HuD was 1140 base pairs, it was divided into four overlap anti-HuD sera from patient Hi 155. Arrows, HuD antigens from 35 to 38 kiodaltons (KD). The histologicaltypeswere:SCLC,H249and H146;carcinoid,H720;squamouscell ping domains which were separately amplified with four sets of carcinoma, Hi57; and adenocarcinoma, H358. primers between is and 2as, 3s and 4as, 5s and 6as, and is and 8as

in hybrid MCH2O6c1, which contains a t(X;lq) chromosome. This sublocalizes the HuD gene to chromosome arm ip. These data are

consistent with the assignment of HuD to lp34 by in situ hybridiza IIIIIIIIIIIIIII tion by Muresu et al. (29). TheHuDGeneIs PreferentiallyExpressedinLungCancerCell Lines with Neuroendocrine Properties The HuD protein has been shown to be expressed in SCLC by immunological techniques (20). To study the expression of HuD mRNA in lung cancer cell lines, we performed RT-PCR and Northern blot analyses. First, cDNA was synthesized from total RNA obtained from 46 tumor cell lines (22â€” 24). Then, RT-PCR amplification was performed on two different regions of HuD between primers is and 4as (from nt 65 to 686) and primers 5s and 6as (from nt 591 to 1003). PCR products were loaded on 3.0% NuSieve agarose gel and the expected sizes were confirmed by ethidium bromide staining (data not shown). Among the cell lines with neuroendocrine phenotype, expression of HuD was detected in all 18 SCLCS, 2 extrapulmonary small cell carcinomas, 1 atypical B Hi28 H2i07 Hi46 carcinoid, and 3 large cell carcinomas with neuroendocrine properties 3, :: (Table 1). On the other hand, HuD expression was detected in only 2 â€” adenocarcinoma cell lines of the 19 non-small cell lung cancers @ TIC- â€”S â€”T without neuroendocrine properties and 0 of 3 mesotheliomas. North C C em blot analysis confirmed the result of RT-PCR analysis and deter mined that all cell lines expressing HuD exhibited two major sizes of mRNA of 4.3 and 4.0 kilobases (Fig. 14). Finally, Western blot @ 5, analysis was performed using cell lysates from 20 cell lines and the expression pattern of the HuD protein was identical to the results of GA T C GATC GA TC RT-PCR and Northern blot analyses (Fig. 2B). Three forms of HuD which differ by alternative mRNA splicing have been described: HuDpro (unspliced); HuD (spliced between nt C lOObp 868 and 909); and HuDmex (spliced between nt 829 and 909)@(14). 5' @1 PCR amplification using primers 5s and 6as confirmed that the tumor @@@ cell lines express these three different alternative spliced forms. is EcoRlI 2as 5s AluiDdelII 6a5 The three different amplified bands representing HuDpro, HuD, and 3s sstiecu4as 7s KpnI 8as @@@ HuDmex were subcloned and confirmed by DNA sequencing. The â€˜1 I expression experiments (with [32P]dCTP) were then repeated using Fig. 3. Identification ofdistinct electrophoretic mobilities ofthe HuD cDNAs in SCLC the subcloned HuDpro, HuD, and HuDmex constructs as controls by PCR-SSCP method. (A) PCR-SSCP analysis of the fragments amplified with primers 55 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. Polymorphism 5 J. Liu, J. Dalmau, A. Szabo, M. Rosenfeld, J. Huber, and H. Furneaux. HuD: the with silent nucleotide substitution (CCC to CCI', proline) is observed, which corresponds paraneoplastic encephalomyelitis antigen binds to the ARE element of c-fos mRNA, clearly to the distinct PCR-SSCP pattern. (C) schematic diagram of the strategy for submitted for publication. PCR-SSCP analysis of HuD cDNAs. 0, coding region. bp, base pairs. 4990

103 this expression strongly correlated with the neuroendocrine pheno type. In addition, we found a newly identified RNA-splicing form I ATGGTTATG - inserting 87 base pairs between nt 103 and 104, as well as the 2 ATG GTr ATG CCT TCT AGA ATC CTA AAG previously reported forms HuDpro (unspliced), and alternative spliced forms between nt 868 and 909 (HuD), and between nt 829 and 909 TFGACCIG&AGCCAAGAAG.MAATTCT (HuDmex) (14). If the previously predicted translation initiation codon is used in this new spliced form mRNA, the entire HuD downstream from this start site is not translated because a stop codon GGT GAT GGG AGA AGT GGA GCC ACT TAA appears at amino acid ii. However, this insertion, which occurs just before the coding region, has two other ATG codons in frame, of 104 which the second one satisfied the Kozak consensus sequence (35). If this initiation codon was used during translation, it would make a . ATA AU AGC product with an altered amino terminus lacking two amino acids. ATl@ACTTAC ATG ATG ATA AU AGC These findings suggest that HuD expression might be regulated by A alternative splicing like the Sex-lethal gene of Drosophila melano Fig. 4. Sequence analysis of the 5' end of the HuD cDNA. The upper sequence (1) is gaster, which has striking homology (42% identity) to HuD. The reportedby Szaboet al. (14) and lowersequence(2) representsa newlyidentified alternative splicing form containing the 87-base pair insertion. The ATG codon indicated Sex-lethal gene plays a key role and is regulated by sex-specific byboldtypeisthepredictedintiationcodonandtheATG(arrowhead)isa newpredicted alternative splicing (17, 36, 37), such that the female-specific form initiation codon. Underline, inframe stop codon. The number on the letter is the nt number. encodes a functional product while the male-specific form contains another exon introducing a stop codon, truncating the open reading frame, and resulting in nonfunctional transcripts. While an analogous (Fig. 3C). After PCR amplification, the product was digested with difference could occur in lung cancer, we found both the truncated appropriate restriction enzymes yielding two smaller fragments. Al (â€œmaleâ€•)andfull-length (â€œfemaleâ€•)formsto be expressed in lung though no somatic mutation was found within 26 samples expressing cancers. HuD has been shown to bind to the AU-rich segment of c-fos the HuD gene, 3 cell lines had an identical aberrant migration in the mRNA.5 Thus, it will be important to study how the proteins encoded region between primers 5s and 6as (Fig. 3A). The amplified band was by each alternative spliced product of the HuD gene participates in subcloned into pBluescript KS plasmid and the mixture of transfor binding mRNAs. mants was sequenced (Fig. 3B). SCLCs H128 and H2107, which One interesting question that arises from this and previous studies express both forms, had a single base substitution of cytosine for (14, 20) is why only a small number of SCLC patients make a thymine at nucleotide 781, which is the dominant type shown in profound immune response to the HuD product, while almost all H146. This change did not alter the encoded amino acid and was SCLCs express HuD. One hypothesis is that a mutated form of the therefore scored as a nucleotide sequence polymorphism. HuD gene induces these immune reactions. Dalmau et al. (38) re In addition, the PCR amplification between primers is and 2as showed an extra, larger band in all tumor lines expressing HuD cDNA ported on an â€œaberrantlysplicedâ€•HuD mRNA (lacking 13 amino in addition to the expected size band (data not shown). Since this extra acids) in tumor cells in a patient with PEM/paraneoplastic sensory band, as well as the predicted band, were able to be amplified by PCR neuropathy. The purpose of the present study was to determine using only cDNAs as templates but not using genomic DNAS as whether HuD was aberrantly spliced or mutated in a more extensive templates, we concluded that this extra band represents an alternative set of lung cancer cell lines. Southern blot analysis did not detect any splicing form. The extra cDNA has an additional 87 nucleotides rearrangement or amplification among 78 cell lines and SSCP analysis inserted between nt i03 and 104, which is on the 3' side of the using the 26 lung cell lines expressing HuD did not detect any somatic predicted initiation codon. This results in the generation of an mRNA mutation. Among the 26 cell lines expressing HuD, 13 sera corre encoding both a predicted truncated 11-amino acid peptide and a new sponding to the same patients were available to test for the existence downstream potential initiation codon (Fig. 4). 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 a DISCUSSION clinically severe paraneoplastic syndrome. However, these lung can cer cell lines did not have any mutation in the HuD-coding region, nor The data presented here are the first examination of the HuD gene did the 10 lung cancer cell lines whose corresponding patients had no for mutations in a large number of lung cancer cell lines by molecular detectable anti-Hu antibodies. Even though we found no point muta genetic techniques. First, we demonstrated that HuD is located on tions in the three lung cancer cell lines whose corresponding patients chromosomal region ip using Southern blot hybridization of two exhibited anti-HuD antibodies, it still remains possible that the HuD independent somatic cell hybrid panels. This result is in complete gene is mutated in some patients with high titer anti-Hu sera. One agreement with the recent results of Muresu et a!. (29), who used possible explanation why these three cell lines have no mutations of fluorescence in situ hybridization to map HuD to chromosome site HuD is that the populations of mutated tumor cells which trigger the lp34. Small cell lung cancer is frequently associated with chromo immune response were eliminated by the host immune response. It somal deletions at 3p, 9p, 13q, and l7p, all of which are now will be necessary to screen a large panel of fresh SCLC tumor samples recognized to contain recessive oncogenes (8, 30â€”32). Chromosome from patients with PEM and paraneoplastic sensory neuropathy to region ip is also deleted in lung and other cancers and harbors the formally rule out this possibility. It is also possible that other family L-myc gene (lp32), which is also frequently overexpressed in small members, such as the HuC and Hel-Ni genes, might have mutations cell lung cancer (33, 34). Therefore, it will be interesting to determine which may induce the immune responses that would cross-react with the correlation between expression of HuD and other genes located at HuD. In this regard it is notable that HuC was initially cloned as ip, including the L-myc gene. another protein reacting with anti-Hu sera (14) and that Hel-Ni was HuD mRNA expression was tested by RT-PCR and Northern blot also shown to be reactive with autoantibodies from patients with analyses. Among 46 cell lines, 26 were shown to express HuD and PEM (i5). 4991

Our results showed that the HuD gene was not mutated in lung regions of growth factor mRNAs. Mol. Cell. Biol., 13: 3494â€”3504,1993. cancer and that its expression correlated highly with tumor cell types 16. Bier, E., Ackerman, L, Barbel, S., Jan, L., and Jan, Y. N. Identification and characterization of a neuronspecific nuclear antigen in Drosophila. Science (Wash showing neuroendocrine features. This suggests that HuD is not ington DC), 240: 913â€”916,1988. involved in the etiology of lung cancer but may be involved in 17. Bell, L. R., Maine, E. M., Schedl, P., and Chine, T. W. Sex-lethal, a Drosophila sex determining neuroendocrine cell development and differentiation in determination swith gene, exhibits sex-specific RNA splicing and sequence similarity to RNA binding proteins. Cell, 55: 1037â€”1046,1988. the tumor cell lines that express it. This idea is compatible with the 18. Robinow, S., Campos, A. R., Yao, K-M., and White, K. The clay gene product of report that monoclonal antibody MAb 16A1 1, which binds specifi Drosophila, required in neurons, has three RNP consensus motifs. Science (Wash cally to an epitope present in gene products of all known Hu genes, ington DC), 242: 1570â€”1572,1988. 19. Robinow, S., and White, K. Characterization and spatial distribution of the EI..AV including HuD, HuC, and Hel-Ni, detects Hutproliferating cells in protein during Drosophila melanogaster development. J. Neurobiol., 22: 443â€”461, nascent avian sensory and sympathetic ganglia in vivo and in popu 1991. 20. Dalmau, J., Furneaux, H. M., Cordon-Cardo, C., and Posner, J. B. The expression of lations of cultured neural crest cells (39). In addition, the study of the Hu (paraneoplastic encephalomyelitis/sensory neuropathy) antigen in human Hel-Ni showed that this product binds specifically to the 3' untrans normal and tumor tissues. Am. J. Pathol., 141: 881â€”886,1992. lated regions of a number of mRNAs, including the transcripts for the 21. Budde-Steffen, C., Anderson, N. E., Rosenblum, M. K., and Posner, J. B. Expression of an antigen in small cell lung carcinoma lines detected by antibodies from patients cytokine granulocyte macrophage colony-stimulating factor and pro with paraneoplastic dorsal root ganglionopathy. Cancer Res., 48: 430â€”434, 1988. tooncogene mRNAs such as c-myc and c-los (15). Therefore, it would 22. Carney, D. N., Gazdar, A. F., Bepler, G., Guccion, J. G., Marangos, P. J., Moody, be interesting to study whether HuD might affect the regulation of T. W., Zweig, M. H., and Minna, J. D. Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res., 45: oncogene mRNAs in SCLC, such as myc family members. In addition, 2913â€”2923,1985. transfection assays using an HuD expression construct could also be 23. Brower, M., Carney, D. N., Oie, H. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. Molecular Analysis of the HuD Gene Encoding a Paraneoplastic Encephalomyelitis Antigen in Human Lung Cancer Cell Lines

Yoshitaka Sekido, Scott A. Bader, David P. Carbone, et al.

Cancer Res 1994;54:4988-4992.

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