A Gene from Human Chromosome Region 3P21 with Reduced Expression in Small Cell Lung Cancer1

(CANCER RESEARCH 52. 1536-1541, March 15, 1992] A Gene from Human Chromosome Region 3p21 with Reduced Expression in Small Cell Lung Cancer1

Ben Carritt, Klaas Kok, Anke van den Berg, Jan Osinga, Alison Pilz, Robert M. W. Hofstra, Mary B. Davis, Anneke Y. van der Veen, Pamela H. Rabbitts, Kiran Gulati, and Charles H. G. M. Buys MRC Human Biochemical Genetics Unit, University College London, 4 Stephenson Way, London NW1 2HE, United Kingdom [B. C., A. P., K. G.J; Department of Medical Genetics, University of Groningen, Ant. Deusinglaan 4, 9713 AW Groningen, The Netherlands [K. K., A.v.d. B., J. O., R. M. W. H., A. Y. v. d. V.. C. H. C. M. B.J; Department of Clinical Neurology, Institute of Neurology, Queens Square, London WC1N 3BG, United Kingdom [M. B, D.]; and MRC Clinical Oncology & Radiotherapeutics Unit, MRC Centre, Hills Road, Cambridge, United Kingdom [P. H. R.]

ABSTRACT normal lung as compared to SCLC tumors and cell lines. Here we report a gene in 3p21 which is expressed in normal adult A combination of cytogenetic and molecular studies has implicated the lung and in cell lines derived from a wide variety of tumor types p21 region of human chromosome 3 as the probable site of a gene the but which is consistently and specifically not expressed or loss of which contributes to the development of small cell lung cancer. We report here the isolation of a gene from this region which is expressed underexpressed in SCLC cell lines. in normal lung tissue and in cell lines derived from a number of different types of tumor, but the expression of which in small cell lung cancer cell MATERIALS AND METHODS lines is undetectable by RNA blot analysis. Although the more sensitive polymerase chain reaction did detect transcripts, a novel quantitative Library Screening. Cosmid libraries in the vector Lorist B were polymerase chain reaction assay showed that their concentration in small screened with radiolabeled probes as described (17), and the resulting cell lung cancer cell lines is less than 3% of that in normal lung. clones were assembled into contigs by restriction mapping. Regions enriched in the dinucleotide CpG were identified in the contig by digesting individual cosmids with restriction enzymes with a recogni INTRODUCTION tion sequence that includes one or more copies of this dinucleotide. cDNA libraries in XgtlO or Xgtl 1 were screened with single-copy probes Characteristic chromosome deletions are found in a number isolated from the vicinity of Cp( .-enriched regions. of different tumor types and are thought to signal the presence Fluorescent in Situ Hybridization. In situ hybridization was carried of a gene or genes the loss of which contributes to tumorigenesis out essentially as described (18). For use as probe somatic cell hybrid (1-3). Such genes have been termed "tumor suppressor" genes. DNA was amplified using the Alu-IV primer (19). Human chromo Deletions may be homo- or heterozygous and have been found somes were identified in R-banded metaphases (20) using a filter for propidium iodide fluorescence. both in cancers with a strong genetic component and in those Northern Blot Analysis. Polyadenylated RNA was isolated using without. In either case, where the deletion affects only one guanidium HC1 (21), electrophoresed on a 1.4% agarose gel, blot- homologue of a chromosome pair in the tumor, submicroscopic transferred to Hybond N (Amersham), and hybridized to 32P-labeled events appear to have inactivated the tumor suppressor gene on cDNA inserts. the other homologue (4, 5). In cancers with an inherited pre Pulsed Field Gel Electrophoresis. Agarose plugs containing 5 ng disposition, a chromosome carrying a mutant alÃeleor, in rare lymphocyte DNA were made as described (22). The plugs were digested cases, a microscopically visible deletion, is transmitted through for 4 h with 40 units of the indicated restriction enzyme in a buffer the germ line. The mutation is therefore constitutional and recommended by the manufacturers. The DNA plugs were applied to a 1% agarose gel and electrophoresed in a Pulsaphor CHEF apparatus heterozygous but becomes effectively homozygous in the tumor (LKB-Pharmacia). Electrophoresis was for 18 h at 200 V, using ramped through the (functional) loss of the remaining active alÃele.It pulse times. DNA was transferred to Hybond N-plus (Amersham) in has been postulated (1) that the same genes are inactivated or alkaline 1.5 MNaCl. Filters were hybridized in 0.5 Msodium phosphate, lost in both inherited and sporadic forms of the same tumor pH 7.5/1 mM EDTA/7% sodium dodecyl sulfate with probes labeled type. by random hexanucleotide priming. Microscopically visible deletions of the short arm of chro RT-PCR Analysis and Quantitative PCR. First strand cDNA synthe mosome 3 have consistently been found in SCLC2 (6-8) with sis was carried out on 30 MStotal cellular RNA using 32 units of the shortest region of overlap in 3p21. The pattern of alÃele Moloney murine leukemia virus reverse transcriptasÂ«:after priming with 450 ng random 14-nier primers, under conditions recommended loss in tumors at loci for which the patients are heterozygous confirms these findings, which suggest that a likely position for by the manufacturer (Pharmacia). Without further purification, a vol a lung tumor suppressor gene is in 3p21 (8-11). The locus most ume corresponding to 1 ug total cellular RNA was amplified with a set of specific primers. Amplification was for 33 cycles in a total volume consistently involved in alÃeleloss in SCLC is D3F15S2 (for of 30 u\; of this 15 u\ were analyzed on a gel consisting of 2% w/v merly DNF15S2), which has been assigned by somatic cell Nussieve agarose (Research Organics) and 1% NA agarose (Pharmacia). hybrid and in situ hybridization studies to 3p21 (8, 12, 13). A recombinant cDNA was constructed by ligating a 102-base pair Rare cases of SCLC have been found (14-16) in which hetero- restriction fragment of pUC19 into the insert of a cDNA clone. After zygosity is retained at other loci similarly assigned to 3p21, linearization of this construct, recombinant RNA was synthesized in suggesting that a putative SCLC tumor suppressor gene might vitro using T7 polymerase. Mixtures of varying amounts of in vitro- lie close to D3F15S2. The pattern of expression of genes synthesized recombinant RNA and 5 Â¿tgofcellular RNA were subjected to a RT-PCR analysis, using the same primer set in each case; because localized to this region is therefore of interest, particularly in of the insertion, the PCR product from the in v/fro-synthesized RNA Received 9/6/91; accepted 1/7/92. is larger than that from the native mRNA. The amount of specific The costs of publication of this article were defrayed in part by the payment mRNA in the cellular RNA sample is determined by comparing the of page charges. This article must therefore be hereby marked advertisement in intensities of the two bands. Due to competition for primers, there is accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by the Dutch Cancer Society. some reduction in the amount of product from the cellular RNA sample â€¢¿'Theabbreviationsusedare:SCLC,smallcelllungcancer;cDNA,comple when high concentrations of the in v/iro-synthesized RNA are included mentaryDNA;RT-PCR,reversetranscript-polymerasechainreaction. in the PCR. The detailed method will be published elsewhere. 1536

RESULTS

The locus most consistently reduced to hemizygosity in SCLC is D3F15S2. However, in a systematic search for ex pressed sequences in 100 kilobases of normal genomic sequence cloned from around this locus only a single gene was found. This was the previously identified acylpeptide hydrolase, APEH (15, 23, 24), formerly known as D3S48E. No other genes were found within the approximately 100 kilobases around the D3F15S2 locus. Therefore, in order to isolate further sequences from 3p21, a genomic library was constructed from DNA of the Chinese hamster-human hybrid DIS2.6. This hybrid contains the human D3F15S2 locus (25), but not the human RAF1, ERBA2, D3S2, D3S3, or D3S6 loci (not shown), all of which have been regionally assigned on the short arm of chromosome 3 (26). The amount of human chro mosome 3 material present in this hybrid was assessed, on the basis of blot hybridization to these probes, as not more than 3p21-p24. The absence of the human D3S2 locus from this hybrid is of particular interest inasmuch as it has also been assigned to 3p21, indicating that the rearrangement of human chromosome 3 in the hybrid occurred within this band. In situ hybridization of PCR-amplified single-copy DNA from the DIS2.6 hybrid to banded human lymphocyte metaphases re vealed that a small part of band p21 is the only chromosome 3 material present in the hybrid (Fig. la). Some other human sequences, including about 5% of the short arm of chromosome 1 (12), known to be present in this hybrid from gene expression and DNA blot hybridization assays, were also detected by this method. Human genomic clones were isolated from the DIS2.6 library and assigned to human chromosomes using somatic cell hybrids and in situ hybridization. A number were found which mapped to the short arm of chromosome 3, among them clone D2-X8. In situ hybridization using both tritium-labeled and fluorescent D2-X8 probes (Fig. Ib) shows that this clone originates from the 3p21 fragment in the hybrid. Because both the D3FI5S2 locus and genomic sequences homologous to the D2-X8 clone lie within the same small part of 3p21, an attempt was made to determine the physical distance between them by pulsed field gel electrophoresis. As shown in Fig. 2 the two loci are separated Fig. 1. a, human DNA in the hybrid DIS 2.6. Single-copy human sequences by a distance of about 140 kilobases. As expected from their from the hybrid DIS 2.6 were obtained by Alu-PCR (19), biotin labeled, and hybridized to normal lymphocyte chromosome preparations as described (18). proximity, quantitative DNA blot analysis (not shown) showed Arrow, band 3p21, which is strongly labeled by the DIS 2.6 probe. />.chromosome a concomitant reduction in copy number of D3F15S2 and D2- assignment of a cosmid isolated with the D2-A8 probe by fluorescent in situ X8 sequences in all 15 SCLC cell lines examined. hybridization. Fluorescent signals accumulate only over band 3p21. A single-copy fragment obtained from the D2-X8 clone was found which hybridized, under stringent conditions, to DNA same RNA blots to the APEH/D3S48E cDNA was carried out from a wide variety of species (Fig. 3a). The restricted evolu either subsequently or concurrently, inasmuch as this gene is tionary divergence of this sequence is consistent with the pres also included in the SCLC deletion and can be used for stand ence of coding sequences within the fragment, and it was ardization not only of RNA loading and integrity but also of therefore used to screen a lung cDNA library. A single clone possible quantitative differences in transcript abundance due to was isolated which was used to isolate further clones from reduced gene dosage. human pre-B-cell, T-cell, and liver cDNA libraries. Of the The expression of the D8 gene was further examined using several clones isolated, the longest was 3.3 kilobases. All clones the more sensitive PCR. Sufficient sequence information (to be detected a 3.5-kilobase transcript by gel blot (Northern) analysis published in full elsewhere) was obtained from cDNA clones to of normal adult lung RNA (Fig. 3Â¿>).Werefer to this gene as enable us to design a set of oligonucleotide primers for a D8. combined RT-PCR analysis in which uncloned cDNA, reverse Polyadenylated RNAs from various normal and tumor cell transcribed from normal and SCLC cell line RNA, was ampli lines were hybridized, after electrophoresis and blotting, to D8 fied. The primers originated from the 3' region of the D8 gene cDNA probes. A 3.5-kilobase transcript was detected in a wide and spanned at least one intron. In order to quantitate D8 variety of cell types, both normal and tumor (Fig. 4a; Table 1). mRNA levels, various amounts of recombinant D8 mRNA, However, under the same conditions, the transcript was unde- synthesized in vitro from a cDNA clone enlarged by insertion tectable in all 23 SCLC lines examined. Hybridization of the of a 102-base pair plasm id fragment into the D8 insert, were 1537

B4 1 H3E4 B4.1 (

Nuil Nrul Kb Pvul Noll R Pvul Noti Kb Kb Noti Noti Nrul R Noti Noti Nrul Kb

365 - 340 - .:

Nrul Pvul Mlul Pvul Mlul Not! Nrul Mlul Noll Mlul Nrul I

D2-8 D3FI5S2

Fig. 2. Long-range physical map showing the physical relationship of the D3FI5S2/D3S48E (APEH) and 1>Kloci. In a, gel transfers of pulsed-field gel electrophoresis experiments were hybridized to single-copy probes isolated from either side of the Mlul and Nrul sites in genomic clones containing the D8 and D3F15S2/D3S48E (APEH) regions. Autoradiographs produced by the probes B4.1 (from a D2-X8 cosmid) and H3E4 (which detects the D3S48E/APEH locus) are shown for illustration. Left, both probes recognize a 620-kilobase (Kb) Noti fragment; right, Nrul site within this fragment separates the two probes. The position of the Saccharomyces cerevisiae chromosome markers is indicated between the autoradiographs; their sizes are (top to bottom) 760, 580,430, 340,270, and 235 kilobases. Asterisk, compression zone, b, long-range physical map. The map, derived from experiments such as those in a, is shown as a continuous horizontal line; below it is a representation of the regions cloned, with the positions of D2-X8 and D3F15S2 marked. A high-resolution map around the D3F15S2 locus appeared in the paper of Welche/ al (17).

Fig. 3. Conserved and expressed sequences within LL U- the D2-X8 clone, a, hybridization of a single-copy Kb sequence from the D2-X8 clone to DNA from different 28S organisms. Genomic DNA (10 MB),isolated from tis 9.4 - sues of the indicated species, was digested with Taql, 6.6 - electrophoresed, blotted, and hybridized to the B4.1 probe (see Fig. 2). b, hybridization of the D8 lung 4.4 - cDNA clone to human adult lung RNA. The position of the 28S and 18S rRNA bands is shown. In the experiment shown here, the probe was the insert of a 1.6-kilobase (Kb) clone isolated from a T-cell cDNA 18S library. 2.3 2.2

added to a fixed amount of total RNA isolated from the cell DISCUSSION line under test. Subsequent RT-PCR analysis of the RNA mixtures gave rise to two products of different sizes. An analysis We have localized a new gene to the chromosomal region of the ratio of these two products in different gel lanes (Fig. 3p21 and studied its expression in SCLC cell lines. This gene 4/>)made it possible to estimate the amount of endogenous D8 lies within a region of the genome which both cytogenetic and mRNA. The results, summarized in Table 2, show that the D8 molecular studies have identified as a possible site for a putative mRNA concentration in SCLC cell lines is 0.5-3% of that in SCLC-specific tumor suppressor gene. This is the fourth gene normal lung tissue. assigned to this region which has been studied in this way. The 1538

_ CM r-- co 3 3 33

5Â° -> O OOO OC OO -3 O LJJ O OOZ Z O (JO 28So ^D8 T-JT " â€¢¿Â«D3S48E 18S â€¢¿.

LUNG GLC-2 GLC-1

pg recombinantRNA

recombinantD8-RNA cellularD8-mRNA(1 \ig)

Fig. 4. Expression of the Dti gene in SCLC cell lines, a, Northern blot analysis of lÃ¬timRNA levels in SCLC and other tumor cell lines. JW2 is a colorectal carcinoma cell line, EJ is a bladder carcinoma, and GCT27 is an embryonal carcinoma cell line (see Table 1). All other cell lines are derived from SCLC. Polyadenylated RNA gel transfers were hybridized concurrently with the D3S48E/APEH cDNA (lower signal in all lanes) and with the insert of a 1.6-kilobase D8 cDNA (upper signal in non-lung tumor lanes and absent in SCLC samples), b, quantitative RT-PCR analysis of D8 mRNA levels in SCLC cell lines. In viYro-synthesized recombinant D8 RNA (1 ng, 0.2 ng, 40 pg, 8 pg, and 1.6 pg, respectively) and total cellular RNA were mixed and subjected to the quantitative RT-PCR protocol outlined in "Materials and Methods." One-half of the PCR product was analyzed on a 3% agarose gel. For each cell line, the amount of DA mRNA present is estimated by determining the amount of recombinant RNA that will give an intensity similar to the cellular RNA sample. From the relative lengths of the recombinant and cellular D8 RNAs, we calculate that 1 pg of recombinant RNA is equivalent to 1.7 pg of cellular message (about 10' copies), bp, base pairs.

aminoacylase gene (ACY1) shows reduced expression in about recently been assigned to this region and is deleted in 5 of the 20% of SCLC cell lines (15, 27), but the significance of this 10 primary non-SCLC tumors and one of the two SCLC cell finding for a gene whose product participates in general cellular lines examined (28). This gene was apparently well expressed metabolism is unclear. The APEH (D3S48E) gene, also de in all the lung tumor cell lines examined (histopathological type scribed here, has been found in a single study (IS) to be not specified). underexpressed in one of four SCLC cell lines studied; the With regard to the D8 gene described here, the question exceptional cell line was also deficient in ACY1 expression. naturally arises as to whether the observed pattern of expres Again, the significance of this is not clear. In the present study sion, namely strong expression in normal lung and dramatically we found consistently high levels of expression of this gene in reduced expression in SCLC, indicates its primary involvement SCLC cell lines. In one study (23), some reduction in the in the origin and/or progression of this tumor. It may be, for expression of the APEH gene has been found in renal cell example, that the reduced expression of this gene in SCLC cell carcinomas. A protein-tyrosine phosphatase (PTPG) has also lines reflects a differentiated state that they share with a sub- 1539

Table 1 Expression of the D8 gene in SCLC and other cell lines deletion, the remaining alÃeleisassumed to have suffered a loss The presence of the D8 gene 3.5-kilobase transcript was assessed by Northern blot hybridization using either polyadenylated or total RNA as the target. Probes of function mutation by one of a variety of mechanisms. Al were either the entire insert of a 2.8-kilobase cDNA or shorter fragments con though many of these might be expected to be point mutations, taining more 3' sequences. Where the same cell lines were tested as both total and hence only detectable by sequencing, grosser events, includ and polyadenylated RNAs and where different probes were used on the same samples identical results were obtained. All RNA samples gave strong 2.4-kilobase ing genomic rearrangements or even deletions, might well occur signals when reprobed with the APEH/D3S48E cDNA probe. in a minority of tumors. These have not thus far been found 3.5-kilobase within the 1)8 gene or in its immediate vicinity. The extent to DB transcript" which this should be taken as evidence against the primary Cell line Name involvement of this gene in SCLC tumor suppression cannot Normal be assessed, because such homozygous genomic events may, as Diploid skin fibroblast HFL121 in the case of the WT1 suppressor in Wilm's tumor, be very B-lymphoblastoid MGL8B2 rare (34, 35) or even nonexistent. A homozygous deletion has Tumor Cervical carcinoma HeLa been found in a single SCLC cell line in the more proximal Bladder carcinoma EJ region 3pl4 (36), but the significance of this is not clear, since ColorÃ©ela!carcinoma JW2 the weight of evidence favors a more distal localization for a CaC02 Colo320 putative SCLC suppressor. Although the location of the D8 gene in the 3p21 chromosomal region, close to the D3F15S2 Breast carcinoma MCF7 Burkitt's lymphoma Raji locus, and its dramatically reduced expression in SCLC are Embryonal carcinoma GCT27 consistent with its role as an SCLC tumor suppressor gene, in SCLC 23 independent lines'' the absence of a functional assay these should for the moment " +, positive hybridization signal at 3.5 kilobases using D8 cDNA probes; -, be regarded as circumstantial evidence for such a role. no detectable 3.5-kilobase signal on Northern blots. * Two different strains of HeLa (in London and Groningen) gave quantitatively different results, one having consistently higher niRNA levels than the other. ' The colorÃ©ela!carcinoma cell line Colo320 contained very low levels of D8 ACKNOWLEDGMENTS mHNA as assessed by Northern blot hybridization. Methods were as in Fig. 3. ''The 23 independent SCLC cell lines included: COR-L23, COR-L24, COR- We wish to acknowledge the contribution made in the early stages L27, COR-L41, COR-L42, COR-L47, COR-L51, COR-L88, COR-L103, FRE of this work by the late Alan D. Jonas. We thank P. Twentyman, L. de (30); NCI-N417, NCI-H69 (32); GLC1, GLC2, GLC3, GLC4, GLC8, GLC14, Leij, B. Wainwright, T. H. Rabbitts, and A. Bernards for cell lines and GLC19, GLC28, GLC34, GLC42, GLC44 (7). cDNA libraries and T. G. Draaijers for carrying out the Alu-PCR of DIS 2.6.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1992 American Association for Cancer Research. A Gene from Human Chromosome Region 3p21 with Reduced Expression in Small Cell Lung Cancer

Ben Carritt, Klaas Kok, Anke van den Berg, et al.

Cancer Res 1992;52:1536-1541.

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