Region with a Marked Instability in Yeast Artificial Chromosomes'

[CANcER RESEARCH54, 4183-4187, August 1, 1994)

A Homozygous Deletion in a Small Cell Lung Cancer Cell Line Involving a 3p2l Region with a Marked Instability in Yeast Artificial Chromosomes'

Klaas Kok, Anke van den Berg, Patrick M. J. F. Veldhws, Anneke Y. van der Veen, Marion Franke, Eric F. P. M. Schoenmakers, Miriam M. F. Huisbeek, Annemarie H. van der Hout, Lou de Le@j, Wim van de Ven, and Charles H. C. M. Buys2

DepartmentofMedicalGenetics[KK,A. v.LB.,P.M.J.F. V,A. Y. v. â‚¬1V.,M.F.,M.M.F.H.,A.H. v. d.H., C.H. C.M.B.JandClinicallmmunologyfL. d.L.J, University of Groningen, Groningen, the Netherlands, and Laboratory of Molecular Oncology, Center for Human Genetics, University of Leuven, Leuven, Belgium fE. F. P. M. S., W. v. S V.J

ABSTRACT MATERIALS AND METHODS

All types of lung carcinoma are characterized by a high frequency of Cell Lines. Lung cancer cell lines were established from biopsies of loss of sequences from the short arm of chromosome 3, the smallest region primary tumors by one of us (L. d. L.). Culture conditions were as described of overlap containingD3FJSS2 In band p21. Here we characterizea (12). 440-kilobase segment from this region, which we found homozygously Probes and Primers. Primers were made in this laboratory by the phos deleted In one of our small cell lung cancer-derived cell lines. The ho phite triester method using a commercial oligonucleotide synthesizer (Gene mozygous deletion maps between UBE1L and ZnF16,just centromenc to Assembler Plus; Pharmacia). Primer sequences for D3S643, D3S647, D3S663, D3F1SS2. Yeast artificial chromosomes with Inserts originating from the D3S771, D3S917, D3S1227, GNAI2, andD3S1235 were as published (13, 14). deleted region are very unstable and readily lose parts of their Insert DD1 primerswere5'-GGCTCFGCFTCCFGGTCCI'TG-3'and5'-11@GGAC AGCCTGTCTCCAGG-3' (229-base pair product); H1R primers were 5'-AA AGGTAGGTrAGTGGTfGC-3' and 5'-TCAGAACAmCATGACCCC-3' INTRODUCTION (113-base pair product); H1L primers were 5'-AGTCATACAGGAAC ACCCfG-3' and 5'-ATGAGGCATGGAGGAAC-3' (150-base pair product). Both by cytogenetic analysis (1, 2) and by loss of heterozygosity Southern Analysis. DNA was digested with restriction enzymes under studies (3â€”5),theregion 3p2lâ€”3p22ofthe short arm of chromosome conditions recommended by the suppliers. Agarose gel electrophoresis was 3 has been identified as the region most frequently involved in loss of carried out at low voltage using 0.7% gels. The DNA was transferred from the constitutional heterozygosity of the various types of lung cancer. This gel onto Hybond-N+ filters(Amersham)in 0.4 MNaOHfor 8 h. Filterswere strongly suggests that this chromosomal region is the location of a hybridized with probes labeled by random hexanucleotide priming in a solu lung-specific tumor suppressor gene. The finding that a human-mouse tion consisting of 0.5 M sodium phosphate (pH 7.2), 1 mM EDTA, and 7% hybrid cell line containing a 2-megabase DNA fragment from 3p2l sodium dodecyl sulfate (w/v) at 65Â°Cfor 16 h. PCR Analysis. PCR was carried out in a total volume of 30 @lusing100 had reduced tumorigenicity as compared with the parent mouse fi ng genomic DNA or 25 ng yeast DNA and 150 ng of each primer in a reaction brosarcoma cell line (6) further supports this suggestion. buffer consisting of 10 mM Tris-HCI (pH 9.0), 1.5 mM MgC12, 50 mM KC1, Heterozygous deletions in lung cancer as well as in other types of 0.01% (w/v) gelatin, 0.1% Triton X-100, 200 pi.i of each nucleotide, and 0.13 tumors are usually of a substantial size, and their common region of units Taq-polymerase (SphaeroQ; HT Biotechnology, Ltd.). Amplification was overlap is still large. Homozygous deletions are detected at a much for 30 cycles, each cycle consisting of denaturation at 92Â°Cfor30 s (except for lower frequency, and the homozygously deleted region is usually the first cycle 2 mm) and elongation at 72Â°Cfor60 s (except for the last cycle small. Therefore, mapping homozygous deletions may be of consid 5 mm) for all primer sets. Annealing was carried out at a temperature specific erable help for the fine localization and identification of tumor sup for the primers used for 60 s. pressor genes. The identification of the RB1 and W7'1 genes is PFGE. Agarose plugs containing 5 @gDNA were made as described illustrative in this respect (7â€”9).Homozygous deletions in 3p2lâ€”22, starting from either blood lymphocytes or cultured cell lines (15, 16). The plugs were digested with 40 units of the individual restriction enzyme as the common region of overlap of the heterozygous deletions, have recommended by the manufacturers for 4 h. The DNA plugs were applied onto been described in two recent reports (10, ii). a 1% agarose gel and electrophoresed in a Pulsaphor CHEF apparatus (Phar When screening a number of SCIC-derived cell lines with a new macia). Electrophoresis was at 150 V for 36 h using ramped pulse times. DNA probe from the distal 3p2i region, we found that its homologous was transferred onto Hybond N+ filters (Amersham) in 0.4 N NaOH. Filters sequences were completely missing from one of the cell lines. More were hybridized as above. over, YACS containing inserts from the region, which was homozy Screening and Analysis of YACs. A copy of the CEPH YAC library was gously deleted, showed a marked instability not found in YACS with screened as described (17) using a PCR-based screening strategy (18). Cal inserts from the adjacent nondeleted regions. turing of yeast cells, isolation of DNA from them (18), and generation of plugs were performed as described (19). The restriction digestion of the plugs was carried out as described previously (16) using conditions recommended Received 3/21/94; accepted 5/31/94. by the manufacturer (Pharmacia). PFGE and DNA transfer onto nylon The costs of publication of this article were defrayed in part by the payment of page membranes were carried out as described above. YAC-DNA was hybrid charges. This article must therefore be hereby marked advertisement in accordance with ized with both the 2.67-kilobase and the 1.69-kilobase PvuII X BamHl 18 U.S.C. Section 1734 solely to indicatethis fact. 1 This work was supported by Grant GUKC9O-15 from the Dutch Cancer Society, the fragment from pBR322 to define left and right end-fragments, respectively. â€œGeconcerteerdeOnderzoeksactie1992-1996,â€•theEC Biomed 1 concerted Action Pro YAC endclones were amplified by vectorette PCR according to Riley et a!. gram â€œMolecularCytogeneticsofSolid Tumorsâ€•(BMH1-CF92â€”0156),andan equipment (20) and sequenced. grant from the Maurits and Anna de Kock Foundation. 2 To whom requests for reprints should be addressed, at Department of Medical Fluorescence in Situ Hybrldlzatlon In situ hybridization was carried out Genetics, University of Groningen, A. Deusinglaan 4, 9713 AW, Groningen, the essentially as described previously for cosmids (21) and chromosome libraries Netherlands. (22). Chromosomes were identified in R-banded metaphases (23) using a filter 3 The abbreviations used are: SCLC, small cell lung cancer; YAC, yeast artificial chromosome;PCR,polymerasechainreaction;FISH,fluorescenceinsituhybridization; for propidium iodide fluorescence. In all cases, at least 50 chromosomes or 100 PFGE, pulsed-field gel electrophoresis. interphase nuclei were analyzed. 4183

RESULTS denoted D8 (16). Both the 1.7-kb Not! X BamHI fragment of DD1 and the GNAJ2 PCR product recognized a ilO-kilobase Not! frag From a 3p14-specific microdissection library (kindly provided by ment. These data, as well as those obtained with double digests of G. Senger, H-J. LÃ¼decke,U. Claussen, and B. Horsthemke, Institute Not! X MluI and Not! X Nru! (results not shown), strongly suggest FÃ¼rHumangenetik, Essen, Germany), we isolated a few clones that that the 1.7-kilobase fragment from DD1 on the one hand and GNAJ2 turned out to map far outside 3pl4. One of these hybridized to DNA on the other hand lie on different ends of the same 110-kilobase Not! from the human-Chinese hamster hybrid cell line Dis2.6. Since this fragment (Fig. 2c). cell line contains part of 3p2l as its only chromosome 3 material (16), Plugs containing DNA from GLC2O were routinely included in the the microdissection clone must map to this chromosomal subregion. PFGE analyses. As expected, we detected aberrant bands upon hy By screening a cosmid library with the clone, we obtained a cosmid bridization of UBE1L to Nru!- and Nod-digested GLC2ODNA (Fig. from which we isolated a 2.9-kilobase single copy BamHI fragment, 2b), the Not! fragment being 680 kilobases in length. Aberrant bands DD1. When DD1 was used in a Southern analysis of 15 SCLC were also detected with ZnF16. A 2.2-kilobase single-copy EcoRI derived cell lines, one of these, GLC2O, completely failed to give a clone from this locus recognizes a 360-kilobase Not! fragment in signal (Fig. 1). Rehybridization with probes from other loci in 3p2l, control DNA, but in GLC2O DNA again a 680-kilobase Not! frag namely UBE1L (16), ZnFJ6 (24), and D3S32 (25) resulted in positive ment. The same clone recognizes a 110-kilobase NruI fragment both hybridization signals with all cell lines. Also, upon PCR amplffication in control DNA and in GLC2O DNA. Thus, one end of the homozy with primer sets for MYL3, D3S1235, D3S643, D3S647, D3S663, gous deletion appears to map within the 360-kilobase Not! fragment D3S917, D3S771, and D3S1227 that all map in 3p21-p22 (13, 14), a detected by ZnF16, not in the 110-kilobase Nru! fragment. Detailed product of the expected length was seen in all the SCLC-derived cell pulsed field analysis, including double digests with the enzymes used lines examined. A primer set for GNAI2, however, failed to detect any to construct the map (see Fig. lc), indicated that the 360-kilobase Not! PCR product from GLC2O. Thus, the homozygous deletion in GLC2O fragment is adjacent to the ilO-kilobase Not! fragment detected by encompasses the loci defined by DDJ and GNAI2. DD1 and GNAJ2. Thus, in control DNA, the two Not! sites flanking The restriction enzyme Not! cuts the 2.9-kilobase BamHI fragment the homozygous deletion span three NotI fragments of 650, 110, and DDJ in two fragments of 1.2 and 1.7 kilobases, respectively. The 360 kilobases, respectively, i.e., 1120 kilobases in total. In GLC2O, result of a PFGE analysis with both BamHI X Not! subfragments of they define a single Not! fragment of 680 kilobase. This implies that DDJ is shown in Fig. 2a. The hybridization pattern of the 1.2-kb the size of the deletion in GLC2O must be 440 kilobases. Its approx BamHI X Not! fragment used as a probe turned out to be identical to imate position (the exact breakpoints have not yet been determined) is that obtained after hybridization of the same filter with a genomic indicated with a hatched bar in Fig. 2c. probe from the UBE1L gene (Fig. 2a). Both probes detect the same As a first step in the analysis of the region homozygously deleted 650-kilobase Not! fragment and the same 350-kilobase M1uI X Not! from GLC2O, the CEPH YAC library was screened with DD1-specific and 350-kilobase Nru! X Not! fragments. This positions DD1 in an primers. Thirty individual yeast colonies, all originating from the already established long range map (16) at a distance of slightly less same DDJ-positive library address 181H1, contained YACs varying than 350 kilobases from UBE1L (see also Fig. 74, which we formerly in length from 60â€”340 kilobases (Fig. 3). FISH analysis with. the longest YAC proved that it was nonchimeric. By applying a vector SCLC ette-PCR protocol to four of these YACs, we could demonstrate that they had identical endclones. For 19 individual 181H1 YACS, we HP * constructed an M1uI,NruI, and Not! restriction map. A comparison of these maps with the pulsed-field map showed that the left endclone I (H1L) of all 181H1 YACS mapped at 80 kilobases from DD1 in the @@ UBE1L ilO-kilobase genomic Not! fragment, i.e., within the GLC2Odeletion. Indeed, amplification of GLC2O DNA with primers derived from H1L failed to produce a PCR product. The right endclone (H1R) mapped at 45 kilobases from the Nru!/Mlu! site between UBE1L and APEH. Thus, in control DNA, the endclones of 181H1 span a genomic region @ DD1 â€”.â€¢q of about 475 kilobases, implying that even the longest (340-kilobase) YAC we obtained still carries a deletion of some 135 kilobases. In 8 of 15 YACS, the deletion included the DD1 homologous sequence. We subsequently screened the YAC library with primers specific for H1L, H1R, and UBE1L, respectively. This resulted in four more YACS. 407A1 1 (nonchimeric; 300 kilobases) is positive for H1R and @ ZnF1 6 â€”â€”.@-@ _ .. UBE1L but not for DD1. This YAC also overlaps with the loci APEH and D3F15S2, and thus extends away from DDJ. 191D3 (chimeric) is positive for H1R but not for UBE1L. As this YAC had already been reported to be positive for D3F15S2 (14), it also maps away from DD1. YAC 529B2 is positive for UBE1L but not for H1R. Although @@@ D3S32 . â€”â€” â€”â€” * this YAC is only 60 kilobases long, it contains a sequence homolo gous to H1L, the most distant endclone of the 181H1 YAC. Therefore, this YAC, too, most likely has been derived from a larger YAC which Fig. 1. Southern analysis of BamHl-digested DNA from SCLC-derived cell lines with lost part of its human insert. 181B3 was isolated with HJL. This 3p2l probes. The cell lines are from left to right: GLC45, GLC44, GLC42, GLC36, GL@35, GLc34, GLC28, GLC2O, GLC16, GLC14, GLC8, and GLC4. HP is human 160-kilobase YAC was positive for GNAI2 as well. Although by FISH placenta DNA. The probes used were a 3.3-kilobase UBE1L complementary DNA, the analysis it appeared to be nonchimeric, its left endclone, isolated by 2.9-kilobase BamHi fragment DDI, a 2.3-kilobase EcoRI fragment from ZnFJ6 (24) and pEFD145.1, defining D3S32. The lane containing DNA from GLC2Ois indicated by an vectorette PCR, failed to hybridize to single human chromosome 3 asterisk. hybrid DNA. Since primers derived from the right endclone of 181B3 4184

a ,f b NniI Neil NruI Noti @ !:@Y:!@:!/1/1 t-i-t z-rr z:i-;: z-;-t

.. â€¢. -650 450-. . @- _Is.

@ S. â€¢@ 350@S. *@ noâ€” â€¢ 465â€” 0@.

@ 110- @p :11 I @ U L R I. u z

@ 650 I â€œÂ°I 360 N N WA NRMNAM A RN I II I 1 I I I I J@JL I I 100165 APEH UBE1L DOl GNAI2 ZnFI6 C

Fig. 2. a-c, PFGE analysis and resultinglong-range physical map ofthe region containing theAPEH, UBEIL, DDJ, GNAJ2, and ZnFJ6 loci. a, successive hybridizations ofa single PFGEfilterwith a 4.0-kilobaseBamHl fragmentfromUBE1L(U), the 1.2-kilobaseBamHl X Nod fragmentfromDDI (L), andthe 1.7-kilobaseNotl X BamHIfragmentfromDDJ (R), respectively. The restriction enzymes used for DNA digestion are indicated above the lanes. Each digestion was carried out on lymphocyte DNA from two unrelated individuals. UBEJLandDDJ-L detect the same fragments;DDI-R detects differentones. b, successive hybridizationsof a single PFGEfilter with a 4.0-kilobase BainHIfragmentof UBEIL (U) andwith a 2.3-kilobaseEcoPJ fragmentof ZnFIO(Z), respectively.The restrictionenzymesused for DNA digestionare indicatedabove the lanes.The lanes containeitherGLC2O DNA (S) or lymphocyteDNA (L). c, long rangemapderivedfromPFGEanalysisas describedin the text andshown above.The positionof the loci is indicatedby small blocks (not drawn to scale). Restriction sites for Mlul, NotI, and NruI are indicated by M, N, and R, respectively. Numbers indicate the length of the NotI fragments in kilobases. The NruI site indicated by an asterisk is consistently partially digested, as can be seen below a and b. The approximate position ofthe region homozygously deleted in GLC2Ois indicated by a hatched bar below the map.

were able to amplify both 181H1 and GLC2O DNA, this end of the 26) suggests the presence of a lung-specific tumor suppressor gene in YAC should map between UBE1L and DD1. Sequences homologous the vicinity. Our search for genes in this region had already yielded a to DDJ were absent from this YAC, indicating that it must have novel gene, UBE1L, formerly denoted D8 (16), which shows a sig undergone some rearrangement(s). nificant homology with the gene coding for the activating enzyme in In order to determine the orientation of the pulsed-field map with the ubiquitin pathway (27). Although this gene is well expressed in respect to the chromosome 3 centromere, we applied bicolor fluores normal lung tissue, expression is virtually absent from lung cancer cell cent in situ hybridization using combinations of two of three YACS, lines (28). In the structural part of the gene, no mutations could be namely 407A11 and 181B3, isolated with the endclones of 181H1, detected. Tumor suppressor genes can, however, be distinguished in plus one at a larger distance, for which we used the nonchimeric YAC two classes (29). The so-called class ! tumor suppressor genes are 264C5, isolated with MYL3. By first hybridizing YACS 407A11 and those in which loss of function results from mutation or deletion of 264C5 to prometaphase chromosomes from blood lymphocytes, YAC DNA. UBE1L, therefore, cannot be a class I tumor suppressor gene. 407A1 1 containingAPEH and UBE1L turned out to map centromeric For class II tumor suppressor genes, loss of function is caused by a to 264C5, containing MYL3 (Fig. 4). From a subsequent interphase regulatory block of expression. Such genes are supposed to be regu FISH analysis of 407A1 1 and 181B3 in combination with 264C5 lated from a different locus. Also for UBEJL, there is still the (results not shown), the order of the YACS from telomere to centro possibility that its expression is under direct positive control of a class mere could be established as 264C5â€”407A11â€”181B3.This implies I gene, which would defme UBEJL as a class II tumor suppressor that the UBE1L locus flanks the homozygous deletion at the telomeric gene. One or more class I gene(s) are, however, most likely present in end, whereas the ZnFJ6 locus flanks the deletion at its centromeric the 3p2l region in view of the results of microcell-mediated chromo end. Using cosmid clones from these loci in a bicolor FISH analysis some transfer experiments. The introduction of either the whole of on interphase nuclei from normal lymphocytes in combination with chromosome 3 (30) or only part of it (6, 31) into tumor cell lines the cosmid containing DDJ, we could confirm that DD1 mapped resulted in a reduction of tumorigenicity. This is readily explained by between UBE1L and ZnFJ6 (Fig. 4b). When the same analysis was the assumption that a class I tumor suppressor gene must have been carried out on GLC2O cells, no signal was caused by the DD1- containing cosmid in any of the interphase nuclei analyzed (Fig. 4c). introduced. Hybridization to GLC2O metaphase spreads of a fluorescently labeled The 440-kilobase homozygous deletion described here to occur in chromosome 3 library revealed chromosome 3 material on three one of our SCLC-derived cell lines, GLC2O, maps in the distal part of chromosomes, one of which consisted almost completely of chromo 3p2l between UBE1L and ZnFJ6 centromeric to both D3S15F2 and some 3 material. YACS 264C5, 407A11, and 181H1, as well as the UBE1L. The results of our FISH analysis of GLC2O made clear that UBE1L cosmid all hybridized to the central part of the presumed short the deletion in the short arm of chromosome 3 is an interstitial one. arm ofthat chromosome (Fig. 4d). No signals could be detected on the The finding of a homozygous deletion in a region so frequently other two chromosomes that actually contained each only a small affected by heterozygous losses corroborates the idea of the involve fragment of chromosome 3. ment of a tumor suppressor gene. Moreover, it clearly restricts the area in which to search for such a gene. Also, for known tumor suppressor genes like RB1 and TP53, homozygous deletion has been DISCUSSION reported as an inactivating mechanism in a number of cases (32, 33). The high frequency with which loss of heterozygosity for H3H2 For lung tumors, there have been some previous reports on ho defining D3F15S2 has been found in all types of lung carcinoma (3, mozygous deletions. In the SCLC-derived cell line SK-C-17, homozy 4185

A HOMOZYGOUS 3p2l DELETION IN AN SCLC CELL LINE

gous deletions occurred on chromosomes 5, 8 and X/Y, respectively (34). The latter two were found repeatedly in a small number of cell lines. The X/Y deletion was also detected in a lymph node metastasis. Since it did not occur in the primary tumor, it cannot be a primary event. Instead, this kind of homozygous deletion may reflect an increased genomic instability during progression of lung cancer in vivo or in vitro. Homozygous deletions on chromosome 9 were detected in 4 of 26 non-SCLC-derived cell lines but not in 6 SCLC derived cell lines (35). This is in line with the relatively high fre quency of loss of heterozygosity of 9p in non-SCLC (36). The smallest region of overlap of these homozygous deletions on chro mosome 9 is between the MTAP gene and the IFN gene cluster in band p22. From this region, a gene has been isolated which shows mutations in a variety of tumors, including non-SCLC (37, 38). A large homozygous deletion at 3pl2â€”l3 has been described to occur in the SCLC cell line U2020 (14, 39). As this deletion is not in 3p2l, the most commonly deleted region in lung cancer, its significance is unclear. More recently, homozygous 3p2l deletions have been reported in lung cancer-derived cell lines (10, 11). A cosmid that was mapped to 3p2l.3 by in situ hybridization detected homozygous deletions in 5 of 36 lung cancer cell lines (10). Thirty-nine more cosmids from 3p2l.3â€”

indopendont colonies of YAC 181H1 A V A kb @.**.@@*â€¢â€¢@â€¢ ..i @@@ @,,.@e .: *@@5 4

500

300 200

Fig. 4. Bicolor FISH analysi& a, hybridization of YACS 264C5 (detected with fiuo rescein) and 407Al 1 (detected with Texas red) to a prometaphase chromosome 3. The centromere (arrow) and both telomers (thin bars) are indicated. 264C5 mapped telomeric ID to 407A1l on all 50 chromosomes analysed. b and c, hybridization of COSD8A1.4 containing the UBE1L gene and ZnFI6 (both green) in combination with the cosmid It II I containing DDJ (red) to interphase nuclei from lymphocytes (b) or GLC2O (c). DD1 maps 0/194/19 11/191 Qf19 3/15 between UBEJL and ZnFJ6. The situation as depicted in b was seen in 75% of the interphases examined. DD1 is absent from GLC2O.d hybridization of a chromosome 3 M WA NR MN library (red) in combination with the (JBE1L cosmid (green) to a GLC2O metaphase. @ I IIII Chromosome 3 material is visible on three chromosomes (arrowheads). GLC2Ocontains APEH Hi R UBEIL DDI HI L 50kb one copy of UBE1L. Its position, central on the presumed short arm of the red-stained chromosome, implies that the deletion eliminating DDJ from this chromosome must be

â€” 181B3 interstitial. 407A11 ______529B2 p22 did not detect any homozygous deletions. Since no reference Fig. 3. a and b, instability of the DD1-positive YAC l8lHl. a, PFGE ofDNA from 13 individual colonies from the address 181H1. The reference lanes contain either DNA from markers had been included, the position of the homozygous deletions Saccharomyces cerevisiae YNN295 (Y), or lambda-concatamers (A). The size of the relative to ours cannot be determined. A homozygous deletion in the YACSpresent in the individual colonies varies from 60 to 340 kilobases. YAC positions SCLC cell line NCI-H740 encompasses the loci GNAJ2 and mfd93, were confirmed by Southern analysis using total human DNA as a probe. b, position of YACSrelative to part of the long range map. Indicated are the probes used in the analysis defining D3S1235 (11). FISH analysis with a YAC positive for the of the YACS, as well as the endclones of 181H1, H1R, and H1L. The position of YAC latter locus showed that it lies centromeric to ZnF16.4 Since D3S1235 l8lHl is indicated. The arrows indicate the position of DNA sequences analyzed for is not deleted in GLC2O, this implies that, at the centromeric side, the presence or absence in YACS from individual yeast colonies. The fractional numbers indicate the number of individual colonies that had lost this sequence relative to the homozygous deletion of cell line NCI-H740 extends much further number of colonies analyzed. The middle part, shown as an open bar, was lost in all than our homozygous deletion. The region deleted from NCI-H740 YACS. Below the map, the position of three other YACS is shown. Again, the open bar indicatesthatpartof thegenomicsequencewhichis notpresentintheYACS.Sequences present in the YACS are indicated by filled bars. 4 Unpublished results. 4186

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. A HOMOZYGOUS 3p2l DELETION IN AN SCLC CELL LINE overlaps with a human chromosome 3 fragment of approximate 2 van der Veen, A. Y., Rabbitts, P. H., Gulati, K., and Buys, C. H. C. M. A gene from megabases transfected into a mouse fibrosarcoma cell line by micro human chromosome region 3p21 which reduced expression in small cell lung cancer. Cancer Res., 52: 1536â€”1541,1992. cell fusion (11). The resulting hybrid cell line, HA(3)BB9F, had a 17. Schoenmakers, E. F. P. M., Kools, P., Crisis, P., Mols, R., Kazmierczak, B., much lower tumorigenicity than its parental mouse fibrosarcoma cell Bartnitzke,S.,Bullerdiek,J.,DalCm,P.,deiong,P.1.,vandenBerghe,H.,andvan line (6). Our homozygously deleted region shares GNAI2 with both de Ven, W. J. M. Physical mapping of chromosome 12q breakpoints in lipoma, pleomorphic salivary gland adenoma, uterine leiomyoma, and myxoid liposarcoma. the region homozygously deleted in NCI-H740 and the chromosome Genomics, 20: 210â€”212,1994. 3 fragment present in the hybrid cell line HA(3)BB9F. These various 18. Green, E. D., and Olson, M. V. Systematic screening of yeast artificial-chromosome independent indications for the presence of a tumor suppressor gene in libraries by use of the polymerase chain reaction. Proc. Nati. Acad. Sci. USA, 87: 1213â€”1217,1990. the same small region of band p21 of chromosome 3 warrant a 19. Schwartz, D. C., and Cantor, C. R. Separation of yeast chromosome-sized DNASby detailed genomic analysis of this region. The construction of a YAC pulsed-field gradient gel electrophoresis. Cell, 37: 67â€”75,1984. contig overlapping the homozygous deletion revealed a marked insta 20. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. A Homozygous Deletion in a Small Cell Lung Cancer Cell Line Involving a 3p21 Region with a Marked Instability in Yeast Artificial Chromosomes

Klaas Kok, Anke van den Berg, Patrick M. J. F. Veldhuis, et al.

Cancer Res 1994;54:4183-4187.

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