Carcinogenesis vol.21 no.2 pp.205–211, 2000
Localization of tumor suppressor gene candidates by cytogenetic and short tandem repeat analyses in tumorigenic human bronchial epithelial cells
D.A.Weaver, T.K.Hei1, B.Hukku2, J.P.DeMuth, E.L.Crawford, J.A.McRaven, S.Girgis and J.C.Willey3 Department of Medicine, Medical College of Ohio, 3120 Glendale Avenue, Rupert Health Center, Room 0012, Toledo, OH 43614, 1Center for Radiological Research, VC11-218, Columbia University, 630 West 168th Street, New York, NY 10032 and 2Cell Culture Laboratory, Children’s Hospital of Michigan, 3901 Beaubien Boulevard, Detroit, MI 48201, USA 3To
whom correspondence should be addressed Email:
[email protected]
Radon exposure is associated with increased risk for bronchogenic carcinoma. Mutagenesis analyses have revealed that radon induces mostly multi-locus chromosome deletions. Based on these findings, it was hypothesized that deletion analysis of multiple radon-induced malignant transformants would reveal common mutations in chromosomal regions containing tumor suppressor genes responsible for malignant transformation. This hypothesis was supported by a previous study in which tumorigenic derivatives of the human papillomavirus 18-immortalized human bronchial epithelial cell line BEP2D were established following irradiation with 30 cGy of high linear energy transfer radon-simulated α-particles. Herein, we describe the analyses of 10 additional tumorigenic derivative cell lines resulting from the irradiation of five additional independent BEP2D populations. The new transformants have common cytogenetic changes, including the loss of chromosome (ch)Y, one of three copies of ch8, one of two copies of ch11p15–pter and one of three copies of ch14. These changes are the same as those reported previously. Analysis of PCR-amplified short tandem repeats of informative loci confirmed the loss of heterozygosity (LOH) at 12 loci spanning the length of ch8 in cell lines from four of the total of eight irradiation treatments to date and the loss of chY in all cell lines (8 of 8). LOH analysis with a total of 17 informative loci confirmed loss on ch14 in transformants from seven of eight irradiation treatments and indicated a 0.5–1.7 cM region of common involvement centered around locus D14S306. No LOH was detected at any of the informative loci on ch11. The overall results support our stated hypothesis. Further studies are currently in progress to determine whether the ch8 and ch14 regions contain genes with tumor suppressor function in bronchial epithelial cells. Introduction As lung cancer is the most common cause of cancer death for men and women in the USA, it is important to identify the mechanisms involved in human bronchogenic carcinogenesis at both the cellular and molecular levels. The relationship between residential radon exposure and human bronchogenic Abbreviations: ch, chromosome; HPV, human papillomavirus; LET, linear energy transfer; LOH, loss of heterozygosity; STR, short tandem repeat. © Oxford University Press
carcinoma has been strengthened by both epidemiological and experimental criteria in recent years. Epidemiological evidence includes a large study in which a significant risk of lung cancer was observed per unit of exposure to radon (1). The risk identified was comparable with that observed in other studies (2,3) and is consistent with earlier estimates based on data in miners. However, in other smaller studies no such excess risk was observed (4–6). All of these were case–control studies which were limited in that they were retrospective and it is very difficult to prove or disprove a small risk confined to rarely occurring exposure levels. In an effort to circumvent this problem, a meta-analysis of data from eight individual studies was recently conducted which strongly supports the extrapolation of risk from uranium miner data to residential exposure (7). Although many questions remain, the results of the first meta-analysis indicate that extrapolation of miner data to indoor residential radon exposure is an acceptable basis for risk determination and management (8). Experimentally, we have previously reported the initial results of a model system consisting of cell lines from independent populations of the human papillomavirus (HPV)18-immortalized human bronchial epithelial cell line BEP2D (9) that were established following treatment with radon-simulated high linear energy transfer (LET) 4He ionizing radiation (10,11). Further, it has been determined that passage of a single α-particle through the nucleus of a human–hamster hybrid (Al) cell was only slightly cytotoxic but was highly mutagenic (12). Experimental data obtained in this laboratory indicate that exposure to a 30 cGy dose of α-irradiation with a LET of 150 keV/µm results in a stepwise malignant transformation of the non-tumorigenic HPV18-immortalized BEP2D cell line at a frequency of ~4.1⫻10–7. Based on the average nuclear crosssectional area of 115 µm2 for BEP2D cells, it was estimated that on average, ~1.4 α-particles would have transversed each nucleus. This dose is consistent with the particle fluence experienced by underground miners, where most nuclei are expected to be transversed by multiple particles. In contrast, most environmental radon exposure occurs at a dose where it is unlikely to exceed more than one particle per nuclear transversal (13). Through a combination of cytogenetic and short tandem repeat (STR) loss of heterozygosity (LOH) analyses of these tumorigenic cell lines, common areas of deletion on chromosome ch8 and ch14 have been identified. The region commonly deleted on ch14 has been localized to 0.5–1.7 cM. The deleted regions on ch8 and ch14 contain multiple tumor suppressor gene candidates. Materials and methods Cell culture and derivation of tumorigenic cell lines The HPV18-immortalized human bronchial epithelial cell line BEP2D (9–11,14) used in this study is an anchorage-dependent line that is nontumorigenic in nude mice. Briefly, BEP2D-derived tumorigenic cell lines were established from independent populations of BEP2D cells following treatment
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Table I. Chromosome copy number/most karyotypesa Ch
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
Parent lines
Tx.b 1
Tx. 2
BEP2D p17
BEP2D p35
H2BT p21
R30T1L p15
2 2 2 2 3 2 2 3 2 2 3 1 1 3 2 2 2 2 2 3 2 2 1 1
2 2 2 2 3 2 2 3 2 1 2 1 1 3 1 2 2 2 2 2 2 2 1 1
2 2 2 1 3 2 2 2 2 1 1 1 1 2 1 2 2 1 2 2 2 2 1 0
2 2 2 1 3 2 2 2 2 1 1 1 1 2 2 2 2 2 1 2 2 2 1 0
Tx.3
Tx. 4
Tx. 5
Tx. 6
Tx. 7
Tx. 8
R30T2 R30T3L p6 p6
H1ATN H1ATBA1 p22 p22
R30-2C p8
R30-3C p8
R30-4B p8
R30-5B p8
R30-6A p8
2 2 2 1 3 2 2 2 2 1 1 1 1 2 2 2 2 2 1 2 2 2 1 0
2 2 2 1 3 2 2 1 2 1 1 1 1 0 2 2 2 1 1 1 2 2 1 0
2 2 2 1 3 2 2 2 2 1 1 1 1 2 1 2 2 2 2 2 2 2 1 0
2 2 2 1 2 2 2 1 2 1 1 1 1 2 1 2 2 2 2 2 2 2 1 0
2 2 2 1 3 2 2 2 2 1 0 1 1 2 1 2 2 1 2 2 2 2 1 0
2 2 2 1 2 2 2 2 2 1 1 1 1 2 2 2 2 2 2 2 2 2 1 0
2 2 2 1 3 2 1 2/3c 2 1 1 1 1 2 2 2 2 1/2d 2 2 2 2 1 0
3 2 2 1 3 2 2 2 2 1 1 1 0 2 2 2 2 2 1 2 2 2 1 0
4 4 3 2 6 4 4 4 4 2 2 2 2 4 2 4 4 4 4 4 4 4 2 0
aThe
number of karyotypes evaluated for each line are as follows: BEP2Dp17, 8; BEP2Dp35, 8; H2BTp21, 9; R30T1Lp15, 10; R30T2p6, 10; R30T3Lp6, 9; H1ATNp22, 9; H1ATBA1p22, 10; R30-2Cp8, 10; R30-3Cp8, 10; R30-4Bp8, 10; R30-5Bp8, 11; R30-6Ap8, 10.
bTx., irradiation treatment. cOf the 10 karyotypes evaluated, dOf
five contained two copies and five contained three copies. the 10 karyotypes evaluated, five contained one copy and five contained two copies.
with radon-simulated high LET 4He ionizing radiation. Irradiated cells were expanded in culture, xenotransplanted into Nu/Nu mice to produce tumors (latency period ~10–13 weeks) and tumorigenic cell lines were established from tumors that developed (~6–8 months post-injection) as previously described (10,11,14). BEP2D and derived tumor cell lines were cultured in serum-free LHC-8 medium (15) as previously described (16). For these studies, passages 21–22 were used. Chromosomal analysis Cytogenetic and isozyme analyses were done in the Cell Culture Laboratory Facility at the Children’s Hospital of Michigan (Detroit, MI). Briefly, exponentially growing cultures were treated with 0.04 µg/ml colcemid for 1–2 h, trypsinized and treated with 0.0375 M KCl for 9 min and fixed in 3:1 methanol:glacial acetic acid, centrifuged and finally dropped onto cold wet slides, as previously described (17). Slides were air dried and stained with 4% Giemsa solution. Chromosomes were examined and counted to establish the ploidy distribution and constitutional aberrations. For trypsin/Giemsa banding of chromosomes, the slides were aged at 60°C on a slide warmer for 18 h, immersed in 0.025% trypsin for 1–2 s, stained with 4% Gurr-Giemsa solution for 11 min (18), washed in buffer and then dried and mounted in permount. Well-banded metaphases were photographed using the AKSII image analysis system. A minimum number of eight karyotypes were prepared from these prints for each cell line. The karyotypes were described according to the International System of Cytogenetic Nomenclature 1991. PCR amplification-based STR analysis Methods for the PCR amplification of STRs and analysis of polymorphisms have been described previously (19,20). Primers were obtained from either Research Genetics (Huntsville, AL) or Keystone/Biosource International (Menlo Park, CA). Briefly, PCR was performed in 10 µl volumes containing 500 ng of genomic DNA, 1 µM each primer, 0.5 U Taq polymerase (Promega, Madison, WI), 1 µl of 10⫻ PCR buffer (500 mM Tris, pH 8.3, 2.5 mg/ml BSA, 5% Ficoll 400, 10 mM tartrazine and 30 mM MgCl2), dNTPs at a concentration of 200 µM and water. Thirty-five cycles of denaturation (5 s at 94°C), annealing (10 s at 74°C) and elongation (15 s at 72°C) were performed on a Rapidcycler air thermocycler (Idaho Technology Inc., Idaho Falls, ID). Following amplification, PCR fragments were electrophoresed on 4 or 5%
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Metaphor agarose gels (FMC, Rockland, ME) containing 0.05 µg/ml ethidium bromide in recirculating, cooled 25 mM Tris–acetate, 0.5 mM EDTA, pH 7.5, running buffer containing 0.05 µg/ml ethidium bromide at 300 V as described by the manufacturer. Gels were visualized with a Foto/Eclipse image analysis system (Fotodyne, Hartland, WI) and digital images were stored on a Power Mac 7100/66 computer. Collage software (Fotodyne) was employed for densitometric analysis. All primer sets were initially tested in the parental BEP2D cell line for informativity at each locus. A heterozygous state at an informative locus was defined by the ability to discern two distinct gel bands by the procedure described above.
Results Karyotypic analysis of the malignant transformants of BEP2D One or more tumorigenic lines resulting from irradiation of eight different populations of BEP2D cells (treatment groups 1–8) were subjected to a karyotypic analysis of at least eight metaphases. Results of isozyme analysis (human LDH, G6PD, PGM1, PGM3, ESD, Me-2, AK-1 and GLO-1) are consistent with a shared origin of the parental and tumorigenic populations of cells. The results of the karyotypic analyses are presented in Tables I–III. All of the cell lines were predominantly near diploid except for the previously reported H1ATBA1, which was near tetraploid (11). After comparing the normal and marker chromosomes in the malignant transformants with the parental BEP2D cell line, karyotypic analysis was used to identify the commonly deleted areas (Table III). LOH analysis of the malignant transformants of BEP2D Of the over 150 sets of microsatellite STR loci primers obtained from Research Genetics (Huntsville, AL) which were tested for informativeness, 82 loci were shown to be
Cell culture model for lung carcinogenesis
Table II. Marker chromosomes present in at least 50% of metaphases Cell line Parental lines BEP2D p17 (8 metaphases) BEP2D p35 (8 metaphases)
Tx. 1b H2BT p21 (9 metaphases)
Tx. 2 R30T1L p15 (10 metaphases)
R30T2 p6 (10 metaphases)
R30T3L p6 (9 metaphases)
Tx. 3 H1ATN p22 (9 metaphases)
H1ATBA1 p22 (10 metaphases)
Tx. 4 R30-2C p8 (10 metaphases)
Tx. 5 R30-3C p8 (10 metaphases)
Tx. 6 R30-4B p8 (10 metaphases)
Tx. 7 R30-5B p8 (11 metaphases)
Tx. 8 R30-6A p8 (10 metaphases)
Probable origins
Presencea
M1 ⫽ t(12q;13q) M1 ⫽ t(12q;13q) M2 ⫽ 15p⫹ M3 ⫽ 10p⫹ M3A ⫽ t(4qter⬎4p16::10q21⬎10qter)
8k 8k 6k 4k 4k
M1D ⫽ t(12q;?) M3A ⫽ t(4qter⬎4p16::10q21⬎10qter) M4 ⫽ small metacentric (?) M5H ⫽ t(11qter⬎11p15::?::13q13⬎13qter) M10D ⫽ t(3qter⬎3q26/q25::15q22/23⬎15pter) M18 ⫽ t(18q;?)
9k 9k 9k 9k 9k 9k
M1 ⫽ t(12q;13q) M3A ⫽ t(4qter⬎4p16::10q21⬎10qter) M4 ⫽ small metacentric (?) M17A ⫽ more q⫹ material than M17 M1 ⫽ t(12q;13q) M3A ⫽ t(4qter⬎4p16::10q21⬎10qter) M4 ⫽ small metacentric (?) M5G ⫽ t(11qter⬎11p15::15q12⬎15qter) M17A ⫽ more q⫹ material than M17 M1 ⫽ t(12q;13q) M3A ⫽ t(4qter⬎4p16::10q21⬎10qter) M4 ⫽ small metacentric (?) M5G ⫽ t(11qter⬎11p15::15q12⬎15qter) M8 ⫽ 13p⫹ 17A ⫽ more q⫹ material than M17
10k 10k 9k 10k 10k 10k 10k 10k 10k 9k 9k 9k, 2 copies/1k 9k 7k 8k
M1 ⫽ t(12q;13q) M3A ⫽ t(4qter⬎4p16::10q21⬎10qter) M4 ⫽ small metacentric (?) M5G ⫽ t(11qter⬎11p15::15q12⬎15qter) M9 ⫽ t(8q;14q) M9B ⫽ 14p⫹ M17 ⫽ 19p⫹ M18 ⫽ t(18q;?) M1 ⫽ t(12q;13q) M3A ⫽ t(4qter⬎4p16::10q21⬎10qter) M4 ⫽ small metacentric (?) M5G ⫽ t(11qter⬎11p15::15q12⬎15qter) M10C1 ⫽ i(3q)
9k 9k 8k 9k 9k 9k 9k 5k 10k, 10k, 10k, 10k, 9k
M1 ⫽ t(12q;13q) M3A ⫽ t(4qter⬎4p16::10q21⬎10qter) M4 ⫽ small metacentric (?) M5G ⫽ t(11qter⬎11p15::15q12⬎15qter) M20 ⫽ der(D)
10k 10k 9k 10k 10k
M1 ⫽ t(12q;13q) M3A ⫽ t(4qter⬎4p16::10q21⬎10qter) M4 ⫽ small metacentric (?) M5G ⫽ t(11qter⬎11p15::15q12⬎15qter) M16A ⫽ t(8,?)(8q10:?)/dup?(8) (8qter⬎8p22::8q21⬎8qter) M20 ⫽ der(D) M21 ⫽ i(5p?)
10k 10k 10k 10k 9k 9k 10k
M1 ⫽ t(12q;13q) M3A ⫽ t(4qter⬎4p16::10q21⬎10qter) M4 ⫽ small metacentric (?) M5G ⫽ t(11qter⬎11p15::15q12⬎15qter) M5I ⫽ der(11) t(11q;M20?) M18A ⫽ der(18) t(18;M20)(18q12::?)
10k 10k 10k 10k 10k 10k
M1 ⫽ t(12q;13q) M3A ⫽ t(4qter⬎4p16::10q21⬎10qter) M4 ⫽ small metacentric (?) M5G ⫽ t(11qter⬎11p15::15q12⬎15qter)
9k 10k 10k 10k
M1 ⫽ t(12q;13q) M3A ⫽ t(4qter⬎4p16::10q21⬎10qter) M4 ⫽ small metacentric (?) M5G ⫽ t(11qter⬎11p15::15q12⬎15qter)
9k 10k 10k 10k
2 2 2 2
copies/10k copies/9k copies/9k copies/8k
aPresent in no. of karyotypes. bTx., irradiation treatment.
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Table III. Common cytogenetic chromosomal deletions in all tumor linesa 1 1 1 1
of of of of
3 3 1 2
copies of ch8 copies of ch14 copy of chY copies of ch11p15–pter
aCell
lines H2BTp21, R30T1Lp15, R30T2p6, R30T3Lp6, H1ATNp22, H1ATBA1p22, R30-2Cp8, R30-3Cp8, R30-4Bp8, R30-5Bp8 and R30-6Ap8.
number of irradiation treatment groups and the total number of cell lines that show LOH at these loci presented in Table VI. LOH was observed for multiple loci on ch8 in the malignant transformants, as shown in Table IV. LOH at the majority of loci was observed in seven of 16 lines from four of eight treatments, while in one line (R30-5B) there was LOH at only two loci (D8S344 and D8S347) in the q22–24 region. As shown in Table V, LOH was observed at locus D14S306 in eight of 16 tumorigenic transformants from five of eight irradiation treatments. Five of 16 lines presented with no LOH at D14S306, but had LOH at loci on either side of the D14S306 locus. In addition to the changes in the area of D14S306, loci D14S283 and D14S274 were deleted in R30-4C, and loci D14S285 and D14S290 were lost in R30-5B. One line (H2BT) had no LOH at any of the informative loci tested. LOH at two of two informative loci was revealed for chY in all tumor lines. Of the five loci evaluated on ch11 in the region 11p12–pter, no LOH was observed in any of the tumorigenic cell lines. LOH was seen at only one out of 10 informative loci for ch11 at locus D11S910 in only one cell line, R30-5A. Discussion
Fig. 1. LOH analysis of locus D14S306. This Laplacian image was created by Collage software (Fotodyne) and is representative of LOH experiments. PCR reaction products, utilizing DNA from the identified cell lines at locus D14S306 (190–210 bp) were electrophoresed on a 4% Metaphor agarose gel containing 0.05 µg/ml ethidium bromide. The cell lines exhibiting LOH at this locus are underlined.
heterozygous in the DNA of the parent BEP2D cell line and were evaluated in the 16 malignantly transformed cell lines. The results from a representative experiment assessing LOH at locus D14S306 are presented in Figure 1 as a Laplacian transformed image (Collage 4.0 software; Fotodyne Inc., Hartland, WI) of an ethidium bromide stained agarose gel. Of the 82 informative loci evaluated, only the loci for ch8 and ch14 are listed in Tables IV and V with a summary of the 208
The HPV18-immortalized BEP2D bronchial epithelial cell line represents a useful model to study the molecular changes at various stages in radiation-induced human bronchial carcinogenesis. While it would be ideal to work with cells that are not expressing HPV18 E6/E7 proteins, they are required for the immortalization process. It is recognized that these proteins may select for particular tumorigenic pathways, however, it is likely that such pathways are among those responsible for naturally occurring bronchogenic carcinoma. Results from this study support the hypothesis that tumorigenic cell lines derived from BEP2D following high LET irradiation will enable us to identify genetic changes associated with tumorigenesis in human bronchial epithelial cells. Thus far, LOH has been observed at multiple loci on ch8 and ch14 in the majority of radon-induced malignant transformants of BEP2D. Karyotypic analysis of the BEP2D cell line indicates that three copies of both ch8 and ch14 are present, which would consist of a single copy of the chromosome from parent a and two copies of the chromosome from parent b. Karyotypic analyses of all of the tumorigenic transformants indicate that one of three copies of both ch8 and ch14 were lost. In several of the tumorigenic cell lines, the chromosome lost was the only copy from parent a, resulting in LOH at a large number of loci. In other cell lines, the lost chromosome was one of the two copies from parent b, resulting in either no observed LOH or limited LOH at several loci. The basic understanding in LOH analysis is that at a specific locus, LOH was selected for in association with inactivation of the remaining allele of a tumor suppressor gene. This tumor suppressor gene inactivation is the result of a submicroscopic deletion or point mutation. When LOH was not observed, it was concluded that both alleles were inactivated through such smaller alterations. One possible explanation for the frequent occurrence of large ch8 deletions in the tumorigenic cell lines is that multiple tumor suppressor genes reside on ch8 and that they were selected for during tumorigenesis. Platelet-derived growth factor receptor β-like tumor suppressor is one such possible candidate gene located in the 8p21.3–p22 region (21,22).
Cell culture model for lung carcinogenesis
Table IV. LOH in ch8 as determined by STR analysisa
The number in the column under a specific cell line indicates the copy number of chromosomes in that cell line and the presence of marker chromosomes are indicated with M and a multiple of the number of markers present for that specific chromosome. The ⫹ or – symbols represent the presence or absence, respectively, of an amplified PCR microsatellite product on Metaphor agarose gels, thereby indicating the presence or absence of an allele. ND indicates that LOH was not determined at the loci. Tx., irradiation treatment. aData for this table were compiled from Comprehensive Human MapPairs List and Cooperative Human Linkage Center (CHLC) Fluorescein Labeled Screening Set/Weber v.6a (Research Genetics, Huntsville, AL, http://206.26.163.2 ), QUICKMAP infoclone database (Fondation Jean Dausset-CEPH, Paris, France, http://www. cephb. fr/infoclone. html), CHLC Genetic Mapping Markers database (CHLC, University of Iowa, Iowa City, IA, http://www. chlc. org:80/ChlcMarkers. html), WAIS Polym Query database, GDB Human Genome Data Base INFOBIOGEN Node (John Hopkins University School of Medicine, Baltimore, MD, http://gdbwww. gdb. org/gdb-bin/gdb/wais/bin/waisq/GDB/gdb-polym) and Bray-Ward et al. (23). The following cell lines were evaluated: BEP2Dp22, BEP2Dp54, H2BTp21, R30T1Lp11, R30T2p6, R30T3Lp6, H1ATNp22, H1ATBA1p22, R30-2Cp7, R30-3Ap7, R30-3Bp7, R30-3Cp7, R30-4Ap7, R30-4Bp7, R30-4Cp7, R30-5Ap7, R30-5Bp7 and R30-6Ap7.
In addition, the deletion present in the R30-5B line in the 8q22–q24 region may provide the first step towards developing a minimal interval for a putative tumor suppressor gene. The 8q22–q24 region contains several genes whose function could characterize them as candidate tumor suppressor genes. Possible genes include the cyclin-D related core-binding factor α subunit 2, translocated to 1, thyrotropin-releasing hormone receptor, fibronectin-tenascin-related undulin, glutaminepyruvate transaminase, PTK2 protein tyrosine kinase and adenylyl cyclase-8 [Online Mendelian Inheritance in Man (OMIM) database, National Center for Biotechnology Information, National Library of Medicine, Bethesda, MD, http://www3.ncbi. nlm. nih. gov/Omim/searchomim. html]. The results of the LOH studies have indicated that the potential missing portion of 11p15–pter in the tumorigenic cell lines, as identified by karyotypic analyses, must be present in the form of an unidentified marker or portion of a marker. As this change is common to all tumorigenic cell lines, it is possible that disruption of a gene at the breakpoint on ch11 is related to malignant transformation. After analyzing the data on ch14, it is apparent that there was a translocation at locus D14S306 between one of the duplicated chromosomes from one parent and the single chromosome from the other parent. This translocation most likely arose prior to irradiation and is represented graphically in Figure 2. After this event, one of the three ch14 was lost from each malignant transformant, but it was not the same
chromosome in each case. The D14S306 translocated chromosome representing the only copy from parent b was lost from lines H1ATBA1, R20-2C, R30-3A, R30-3B and R30-3C. As a result, the only chromosome 14 material from parent b in these lines is that from and adjacent to locus D14S306 (situation 1). One of the two chromosomes representing parent a, which had the D14S306 translocation, was lost from eight lines from five different treatment groups (situation 2, cell lines R30T1L, R30T2, R30T3L, H1ATN, R30-4A, R30-4B, R30-5A and R30-6A). Both ‘complete’ parental chromosomes were apparently present in lines H2BT, R30-4C and R30-5B by LOH analysis (situations 3–5). In these lines, the D14S306 translocated chromosomes (one from each parent line) were present, thus copies of each allele at each locus evaluated were present. It is likely that a tumor suppressor gene (or genes) was disrupted through a combination of the translocation event prior to irradiation and chromosome loss after irradiation. In summary, the regions of common LOH on ch14 contain several genes which have functions appropriate for tumor suppressors. Possible candidates in the 14q12–q13 region are CCAAT/ enhancer binding protein epsilon, transglutaminase 1, paired box homeotic gene 9, apurinic/apyrimidinic (abasic) endonuclease and somatostatin receptor-1 (OMIM database). A second region of interest, 14q21–q23, contains cyclin-dependent kinase inhibitor 3, myc-associated factor X and transforming growth factor β3 (OMIM database). It is expected that more detailed evaluation of these chromosomal regions, 209
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Table V. LOH in ch14 as determined by STR analysisa
aSee
legend for Table IV.
Table VI. Summary of LOH on ch8 and ch14 Map primer pair Ch8 D8S1130 D8S1145 D8S136 D8S137 D8S1110 D8S1113 D8S1119 D8S556 D8S1132 D8S1128 D8S373 D8S344 D8S347 Ch14 D14S72 D14S283 D14S297 D14S599 D14S253 D14S69 D14S75 D14S306 D14S129 D14S556 D14S288 D14S276 D14S285 D14S274 D14S290 D14S611 D14S267
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No. of cell lines with LOH
No. of irradiation treatment groups with LOH
7 7 7 6 7 3 7 7 7 7 No LOH in tested lines 8 8
4 4 4 4 4 2 4 4 4 4
No LOH 6 5 5 5 5 5 8 5 5 5 5 5 6 6 4 No LOH
5 5 4 3 3 3 3 3 5 3 3 3 3 3 4 4 2
Fig. 2. Chromosome 14 translocation. Letters a and b represent the two parental chromosomes. For situations 1–5 depicted above, lines that exemplify the LOH observed are as follows: situation 1, H1ATBA1, LOH at all but D14S306; situation 2, R30T1L, LOH at only D14S306; situation 3, H2BT, no LOH; situation 4, R30-4C, LOH at D14S283 and D14S274; situation 5, R30-5C, LOH at D14S285 and D14S290.
currently in progress, will identify genes that serve as tumor suppressor genes in human bronchial epithelium. Acknowledgements We thank Adam Matuszak, Andrew Smith and Parul Raizada for their technician skills involved in this work. This work was supported in part by NIEHS grant ES-05719 and NCI grant CA49062 from the US National Institutes of Health. Cell characterization studies by isozyme and chromosomal
Cell culture model for lung carcinogenesis analyses were carried out under NCI contract NOI-CB-33063 to the Children’s Hospital of Michigan, Detroit.
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