Lines Mutations in Human Mesothelioma Cell ras p53 and Kirsten-

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p53 and Kirsten-ras Mutations in Human Mesothelioma Cell Lines R. A. Metcalf, J. A. Welsh, W. P. Bennett, et al. Cancer Res 1992;52:2610-2615. Published online May 1, 1992.

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[CANCER RESEARCH 52, 2610-2615, May 1, 1992|

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p53 and Kirsten-ras Mutations in Human Mesothelioma Cell Lines R. A. Metcalf, J. A. Welsh, W. P. Bennett, M. B. Seddon, T. A. Lehman, K. Pelin, K. Linnainmaa, L. Tammilehto, K. Mattson, B. I. Gerwin, and C. C. Harris1 Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland 20892 ¡R.A. M., J. A. W., W. P. B., M. B. S., T. A. L., B. I. G., C. C. H.J; Institute of Occupational Health, Topeliuksenkatu 41 aA, 00250 Helsinki, Finland [K. P., K. L.]; and Department of Pulmonary Medicine, University of Helsinki, Haartmaninkatu 4, 00290 Helsinki, Finland [L. T., K. M.]

Abstract Twenty cell lines from 17 individuals with malignant mesothelioma have been examined for p53 alterations by direct sequencing of genomic DNA, by evaluation of mRNA expression levels, and by immunocytochemical analysis of p53 protein expression in comparison with normal human pleural mesothelial cells. The results of this study show p53 abnormalities in cell lines from 3 individuals. These include 2 point mutations and one null cell line. Interestingly, while both cell lines with point mutations exhibit high levels of p53 protein, normal mesothelial cells as well as 12 of the mesotheliomas evaluated express low but significant levels. In addition, sequencing of k-r«.v at codons 12, 13, and 61 reveals wild-type sequence in all 20 mesothelioma cell lines. The capacity to induce tumors in athymic nude mice did not correlate with the presence of a p53 mutation or elevated p53 protein levels. These data suggest that neither p53 alteration nor K-ras activation constitutes a critical step in the development of human mesothelioma.

Introduction Malignant mesothelioma is a rare cancer the development of which is strongly associated with asbestos exposure (1,2). This disease is characterized by a long latency from onset of exposure and a short survival after diagnosis (3, 4). The length of the latency period suggests that multiple genetic alterations may be required for tumorigenic conversion of mesothelial cells (5). A search for molecular changes which have relevance for the development of mesothelioma benefits from studies of other tumor types which suggest specific oncogenes and tumor sup pressor genes. One such gene is the Kirsten-ras protooncogene; examples of Ki-rai genes activated by mutations in codons 12, 13, and 61 have been found with substantial frequencies in adenocarcinomas of the exocrine pancreas, colon, and lung (610). If activated ras were involved in the genesis of meso thelioma, mutations in the p53 tumor suppressor gene might be expected to act synergistically for tumor development. It has been shown in murine systems that normal embryonic cells are converted to tumorigenicity by the combination of an activated ras oncogene and overexpression of a mutated p53 gene (1113). Many recent studies indicate that loss of function of the p53 suppressor gene through mutation or allelic loss is an event that is frequently associated with pathogenesis of human tumors including lung carcinomas (14-20). Evidence suggesting alter ation of the p53 suppressor gene in mesothelioma includes reports of deletions of chromosome 17p which contains thep53 locus (21, 22) in some malignant mesotheliomas (23-26). This study examines the hypothesis that activating mutations of Ki-ras and/or mutation or loss of p53 may be common events Received 1/29/92; accepted 3/19/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Laboratory of Human Carcinogenesis. National Cancer Institute, NIH, Building 37, Room 2C05, Be thesda, MD 20892.

in the carcinogenic process leading to malignant mesothelioma. We report here the analysis of 20 cell lines from 17 individuals for abnormalities in c-Ki-ras by direct sequencing of PCR2amplified genomic DNA at codons 12, 13, and 61 and for p53 alterations by immunocytochemical analysis of protein expres sion, Northern hybridization analysis for mRNA expression level, and genomic DNA sequencing after PCR amplification. In contrast to most other tumor types and a previous report on mesothelioma cell lines (27), the frequency of mutation in p53 is relatively low. Materials

and Methods

Cell Lines and Culture Conditions. Human mesothelioma cell lines were cultured as described in LHC MM growth medium (28). Twenty cell lines derived from 17 patients were analyzed in this study: JMN and DND (24); VAMT 1 (29); HUT 28, HUT 226, and HUT 290 (30); MT 3 (31); M9K, M10K, M14K, M14P, M14M, M19, and M20 (32); and M15, M24K, M25K, M28K, M32K, and M33K. All mesothelioma cell lines were established from pleural fluid or tumor samples from patients whose diagnosis of malignant mesothelioma was pathologically confirmed. All cell lines with the M prefix in their designation were specimens from patients in whom the diagnosis of mesothelioma was reviewed by the Finnish National Mesothelioma Panel and the Euro pean Organization for Research and Treatment of Cancer Meso thelioma Panel. All tumor material was reviewed and grouped into epithelial, mixed, or fibromatous subtypes. Cell lines Ml 4M, Ml 4P, M14K, and M20 are derived from the same individual. Ml 4P and M20 were established from pleural effusions with Ml 4P being established before and M20 after mitoxantrone chemotherapy (total dose, 50 mg = 27 mg/m2) (32). In addition, M9K, Ml OK, and M15 were established from patients who had received previous mitoxantrone chemotherapy. M15 and Ml OKwere the only cell lines from patients who had received prior radiotherapy. All M-prefix cell lines with the exception of M19 and M25K were from patients who had documented asbestos exposure. All cell lines were unique by karyotypic analysis with the exception of Ml 4M which was a tetraploid subclone of Ml 4P. Primary mesothelial cell cultures were cultivated as described in LHC MM growth medium (28). The three cultures evaluated were derived from pleural fluid samples from three human subjects with noncancerous conditions. Tumorigenicity. In order to evaluate the tumorigenic potential of mesothelioma cell lines, 5 x IO6cells were inoculated s.c. into athymic nude mice. Mice were exposed to 350 rads 24 h before inoculation. Mice were maintained for 52 weeks and observed weekly for presence and size of tumor. Tumors were scored as positive when the crosssectional area was 5 mm or greater and regression did not occur. Immunocytochemical Analysis. Cells were seeded onto glass multiwell chamber slides (LAB-TEK No. 177402; Nunc, Naperville, IL) at an initial concentration of 10,000 cells/cm2. After incubating overnight at 37°Cthe cells were fixed in acetone at —¿20°C for 10 min and stored at —¿20°C. Endogenous peroxidase activity was quenched for 20 min at room temperature with a 0.3% H2O2 solution in phosphate-buffered saline. After copious washing in phosphate-buffered saline, antigenic 2The abbreviation used is: PCR, polymerase chain reaction.

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incorporation was achieved with preincubation at 42°Cfor 2 min with the "S-labeled deoxynucleotide (New England Nuclear) corresponding

cross-reactivity was blocked with a 1:50 dilution of normal horse serum for 30 min at room temperature. Saturating concentrations of murine monoclonal primary antibodies were incubated overnight at 4°Cand subsequently detected by a biotinylated secondary antibody and an avidin-biotin peroxidase system according to the manufacturer's pro tocol (Vecta-stain Elite Kit; Vector Laboratories, Inc., Burlingame, CA). The chromogen was diaminobenzidine (final concentration, 0.05 mg/ml) osmicated with nickel chloride (final concentration, 0.03%); there was no counterstain. A proliferation marker, Ki-67, demonstrated the viability of the cells under analysis (M722; Dakopatts, Glostrup, Denmark). A monoclonal antibody to SV40 large T-antigen served as an isotype-matched negative control (Pab 416, AB-2; Oncogene Sci ence, Inc., Manhasset, NY). p53 protein expression was demonstrated by concordant staining with two monoclonal antibodies in at least two separate experiments. An epitope near the amino terminus was recognized by Pab 1801 (AB2), while an epitope near the carboxyl terminus was recognized by Pab 122 (14091 A; Pharmingen, Inc., San Diego, CA). The absence of p53 protein expression was determined by three separate, concordant ex periments utilizing two monoclonal antibodies per experiment. Quantitation of p53 protein expression was done by manual cell counts of one representative Pab 1801 stain per cell line. Between 104 and 335 (average 211) cells were counted for 17 cell lines. Three separate cultures of primary mesothelial cells were similarly examined; a total of 844 sequential cells were evaluated. Three categories were recog nized: cells with intense nuclear, extranucleolar staining were called positive; cells with negative nuclei were called negative; cells with faintly stained nuclei with or without cytoplasmic staining were called +/-; fragmented and mitotic cells were excluded. The percentage of positive nuclei was calculated excluding the +/- nuclei (data not shown). The resulting percentages were grouped into quartiles (i.e., 1+ to 4+) to facilitate comparison. DNA Amplification and Dideoxy Sequencing. Genomic DNA (250500 ng) was amplified by PCR (33) using either p53-specific primers in the intron regions surrounding exons 2 through 11 or k ras primers (20). PCR conditions: 1.875 HIM deoxynucleotide triphosphates; 40 pmol of each primer; 5 units AmpliTaq (Cetus, Emeryville, CA); 50 mM Tris (pH 9.0) 3 mM MgCl. PCR program: denature at 100°Cfor 10 min; 85°Cfor 3 min (add AmpliTaq); cycles 1 to 35: 94°Cfor 30 s, 60'C for 1 min, 78°Cfor 30 s. Each DNA sample was subjected to at least 2 separate PCR reactions for DNA sequencing. Gel-purified DNA was sequenced directly by a modification of the dideoxy chain termi nation method of Sanger et al. (34). Template DNA was denatured at 98°Cfor 3 min, annealed with 3 pmol sequencing primer, and sequenced with the Sequenase Kit reagents (I . S. Biochemical). Radioactive label

to the first base of the nascent chain (35). Samples were run on 8% gels (Gel-Mix 8; BRL) for 2-5 h. Dried gels were placed against Kodak XAR 5 film at room temperature for 1-2 days (20). RNA Analysis. Total cell RNA was isolated according to the method of Chomczynski et al. (36). Twenty-jig samples were fractionated on 1% agarose/formaldehyde gels and transferred by electroblotting to Gene Screen (Dupont) membranes which were hybridized as described previously (31) utilizing sequentially a 1.8-kilobase human p53 wildtype probe purified from the pC53-SN plasmid (37) and a glyceraldehyde-3-phosphate dehydrogenase complementary DNA insert (38). Re sults were quantitated using The Image Quant Software on a Molecular Dynamics laser densitometer with volume quantitation. p53/glyceraldehyde-3-phosphate dehydrogenase mRNA ratios were calculated for each cell line and the value for M9K was arbitrarily equated to 1.0 for purposes of comparison.

Results Twenty cell lines derived from 17 individuals with malignant mesothelioma (see "Materials and Methods") were sequenced for p53 exons and adjacent consensus splicing regions as indi cated in Table 1 and for Kirsten-ras codons 12, 13, and 61 and flanking regions. The examined cell lines exhibited wild-type K-ras genotypes for codons 12, 13, and 61 and surrounding regions. Only two single base pair mutations were detected in the p53 gene (Table 1). These were in the cell line JMN, where a G to A transition of codon 245 of exon 7 changed the sequence from GGC to AGC, causing a glycine to serine amino acid change, and, in the cell line M15, at codon 278 of exon 8, where a C to T transition converted CCT to TCT, causing a proline to serine change (Fig. 1). For JMN, only a single band in the A lane was observed for the first base pair of codon 244, indicating that the cells were effectively homozygous for this mutation, perhaps as a result of a concomitant chromosome 17p deletion. For Ml5 (Fig. 1), both a wild-type C band and a mutant T band were present in the first base pair of codon 278, indicating that the cells were probably heterozygous for the wild-type and mutant alÃ-ele. The results of immunocytochemical analysis of cultured cells for p53 protein expression are presented in Table 1 and repre sentative photomicrographs are shown in Fig. 2. The JMN cell

Table 1 Analysis of mesothelioma cell lines for p53 status and tumorigenicity Cell lineDNDHUT

mutation cytochemistry"2+1 (p53/GAPDH)*1.1I.I4.11.94.90.81.02.31.2ND2.41.35.24.47.31.34.79.70.40Tumo sequenced2-112-112-114-114-115-95-92-112-115-92-114-112-112-112-112-112-112-112-112-11Codon. acidWild amino (wk)142012312028455445364

typeWild 28HUT typeWild +NDfND4+1 226HUT typeWild type245 290JMNMT3M9KM10KM14K(tumor)M14PMl Gly-SerWild GGC-AGC +3+1 typeWild typeWild typeWild +1+3+3+3+1 typeWild typeWild (subclone)MISM19M 4M type278 Pro-SerWild CCT-TCT typeWild +1+1+1+1+2+1+NegmRNA 20M24KM25KM28KM32KM33KVAMT typeWild typeWild typeWild typeWild typeWild typeWild 1Exons typeInumimi °Immunostain criteria: 4+, more than 75% nuclei intensely stained; 3+, 50 to 75% nuclei stained: 2+, 25 to 50% nuclei stained; 1+, less than 25% nuclei stained; Neg, not stained. p53/GAPDH ratios were determined as detailed in "Materials and Methods." ' ND, not done.

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Wild Type (M9K)

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Mutant (M15)

ACGT

ACGT

•¿^M

^

'

i9/A ' ~

Codon 278 Pro

-^

Hétérozygote Pro/Ser

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Fig. \. Sequence analysis of two mesothelioma cell lines showing wild-type sequence or heterozygous mutation at codon 278. Sequencing gels of a wild-type mesothelioma cell line (M9K) or a heterozygous mutant mesothelioma cell line (Ml5). Sequences were obtained after PCR amplification as described in "Materials and Methods" and proceed from the -V direction. The figure displays sequence from codons 269 to 281 and shows the presence of two bands (G and A) at equal intensity in the first base pair of codon 278 in the M15 cell line.

Fig. 2. Immunocytochemical analysis of pS3 protein expression in cells derived from human pleural mesothelium. 1. intense nuclear staining in most of the JMN cells shown. There is variation in the intensity of nuclear staining, and two nuclei near the center of the field are unstained. B, characteristic extranucleolar, nuclear staining pattern as well as giant cell formation by some MIS cells. Although all the nuclei in this field are positive, there is variation in the staining intensity and unstained nuclei were observed in other fields. C. unstained nuclei in the null cell line VAMT 1. /). normal human pleural mesothelial cells including one darkly stained nucleus, several faintly stained nuclei, and scattered unstained nuclei. In all panels, the primary antibody was Pab 1801, and the original magnification was x 630.

line demonstrated high levels of p53 protein in 85% of the cell population: this staining (Fig. 2A) is explained by the presence of a missense mutation. M15 (Fig. 2B) also contained a missense mutation, and 62% of the nuclei were positively stained. Neither p53 mRNA nor protein were detected in the VAMT 1

cell line (Fig. 2C; Table 1). The remaining tumor cell lines lacked mutations within the coding regions examined and ex hibited a range of levels of p53 protein expression: three had 3+ levels of protein expression; two had 2+ levels; and ten had 1+ levels. Three samples of normal human pleural mesothelial

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cells exhibited a composite of 2+ level staining (i.e., 27.5% total positive nuclei) with individual positive percentages of 25, 37, and 20% (Fig. ID). The proliferation (Ki-67) and negative (SV40 large T-antigen) controls showed that the cells were viable and that an isotype-matched negative control was nega tive as expected (see "Materials and Methods"). Discussion Utilizing direct genomic DNA sequencing after PCR ampli fication, this report presents an analysis of the frequency of single base alterations in codons 12, 13, and 61 of the K-ras gene and in major portions of the coding sequence (Table 1) of the p53 gene in 20 human mesothelioma cell lines from 17 individuals. Cytogenetic analyses of mesothelioma have shown numerous abnormalities involving almost every chromosome including 3p deletions and trisomy 7 (24, 25, 32), but no specific lesions which might serve as markers of this disease. The present study was designed to examine the possibility that activating mutations in K-ras, perhaps in combination with alterations of p53, might be associated with the genesis of mesothelioma. The involvement of ras was suggested by exper iments showing that expression of mutant EJ-ras can provide growth factor independence to normal human mesothelial cells (39) and convert an SV40 large T-antigen-immortalized human mesothelial cell line (Met-5A) to tumorigenicity (40). All 20 cell lines were wild-type for K12-13 and K-61, suggesting that ras mutations are uncommon in mesothelioma. Immunocytochemical analysis as well as DNA sequencing were used to detect p53 mutations in mesothelioma cell lines. Two missense point mutations were identified in two cell lines expressing high levels of protein. Several lines of evidence indicate that overexpression of p53 protein within a tumor may signal the presence of missense mutation, but the correlation between p53 protein overexpression and mutation is imperfect (16, 20, 41, 42). In this series, the JMN cell line had the highest protein expression (i.e., 85% positive nuclei) and a mutation. The second mutation occurred in M15, which was one of four cell lines with 3+ expression of p53 protein (i.e., 50-75% positive nuclei). The remaining cell lines with 3+ staining (i.e., M9K, Ml 4M, Ml 4P) were derived from two patients and contained wild-type sequences. Mechanisms which may explain p53 protein overexpression in cells with wild-type genomic sequence include (a) inactivation of an enzymatic pathway responsible for p53 protein degradation (43), (b) stabilization of wild-type protein through complex formation with a DNA tumor virus protein (44) or a cellular oncogene; and (c) overexpression of the myc oncogene product (45). The last mecha nism is relevant since myc amplification and/or overexpression is common in lung cancer (46, 47). Lower levels of p53 protein (i.e., 1+ to 2+ staining) were detected in the remaining tumor cell lines as well as in primary mesothelial cell cultures. Wild-type p53 protein expression in normal human mesothelial cells is compatible with several observations in other nonneoplastic cell types. For example, the wild-type protein appears to play a role in normal prolifer ation of nontransformed cells such as human lymphocytes, normal mouse thymocytes, and NIH 3T3 fibroblasts (48-50). Wild-type p53 protein may be expressed at significant levels in cells in which it contributes to maturation and differentiation (51). The meaning of p53 protein expression in a small fraction of a tumor cell population is not clear. These data suggest that low levels of p53 protein may not signal p53 abnormality but

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may reflect physiological expression of a wild type protein. Alternatively, the previously cited mechanisms for p53 protein accumulation may apply. Clearly, many questions remain re garding the interpretation of p53 protein expression in normal and tumor-derived human mesothelial cells. DNA sequence, RNA expression, and immunocytochemical analyses presented in Table 1 indicate that alteration of the/75.? tumor suppressor gene occurs infrequently in the development of human mesothelioma. p53 protein accumulated in a majority of tumor nuclei in cell lines from only four individuals. In two cases, missense point mutations explain the protein overexpres sion; in the remaining pair, the examined sequences were wildtype, and some other alteration may exist. The remaining abnormality produced a p53 null cell (VAMT 1) as judged by analysis of p53 mRNA by Northern blot techniques and of p53 protein by immunocytochemistry. Genomic DNA sequencing after PCR amplification of exons 2-11 detected only a wildtype sequence in this cell line suggesting that the lack of p53 expression results from rearrangement, intronic mutation, or an alteration in downstream processes which regulate p53 gene expression. Since cell lines, as opposed to tumors, might be expected to develop mutations during extended passaging, the low frequency of mutations observed in the cell lines studied here strengthens the suggestion that loss of wild-type p53 expression is not a frequent occurrence in this tumor type. These results contrast with a recent report of three p53 altera tions in a total of four mesothelioma cell lines (27). In agree ment with the data presented here, two alterations were single base changes while a third resulted in a p53 null cell. The reported base changes in mesothelioma are all G:C to A:T transitions in codons 175 and 245 (27) and 245 and 278 (Table 1). Only the JMN transition mutation occurs at a CpG site which would be expected to have a high mutability because of modification of C in this pair to 5-methylcytosine (19, 52). In addition to these changes, two p53 null cells without docu mented mutations have been reported in these two studies. Therefore, combining the previous (27) and present data, four point mutations and two p53 null cells have been documented in 24 cell lines from 21 individuals. This frequency of approx imately 29% is to be compared to frequencies of 45-79% reported for tumors of the lung (23 of 51) (15), stomach (19 of 24) (42, 53), bladder (9 of 16) (54), liver (13 of 26) (55, 56), and skin (14 of 28) (35). A low frequency of p53 mutations has been reported for medulloblastomas (0 of 12 tumors, 0 of 8 xenografts, and 1 of 3 cell lines) (57). Examination of the spectrum of p53 point mutations associ ated with a known carcinogenic agent has been utilized to produce a "molecular footprint" providing information con cerning the nature of the molecular interactions which may be of importance in the carcinogenic process (19, 35, 58). Malig nant mesothelioma is strongly associated with asbestos fiber exposure (1, 2). It was hoped that the sequencing analysis performed for this study might reveal a mutational spectrum found in mesothelioma containing patterns suggestive of mo lecular routes involved in the carcinogenic interaction of asbes tos with the mesothelium. The relative infrequency of single base changes in mesothelioma supports the hypothesis that oxy radical-generated 8-hydroxydeoxyguanine adducts are not im portant in the genesis of this tumor. Earlier reports have shown that, while exposure to asbestos in vitro supplies a growth advantage to and induces chromosomal abnormalities in nor mal human mesothelial cells (59), it does not generate oxy radicals (60). However, it is known that oxy radicals are pro-

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duced by the interaction of asbestos and macrophages or tra chéalepithelial cells (61-63). Perhaps the mesothelial cell has sufficient DNA repair capability or oxy radical-trapping mech anisms to render exposure from this source of minor conse quence. Alternatively, it may be that, when the critical genetic targets for mesothelioma are identified, they may reveal a different mutational spectrum. The finding of frequent and multiple chromosomal abnormalities in human mesothelioma cells, coupled with the observations that asbestos associates with chromosomes (64) and induces chromosomal damage in tissue culture (59, 65), is consistent with the present report, suggesting that structural changes and chromosomal re arrangements may be of great importance in the development of mesothelioma after fiber exposure. In support of this hy pothesis, Cora and Kane (66) have recently reported that fre quent deletions and two point mutations in the p53 gene were observed in tumorigenic cell lines isolated from tumors devel oped in C57BL/6 mice after weekly i.p. injections of 200 ng of UICC crocidolite asbestos. Thus, the interaction of fibers and mesothelial cells appears to yield structural chromosomal changes with high frequency and, perhaps, with a greater prob ability than base pair mutations. Interestingly, a recent study of mutational spectra resulting from aerobic incubation of DNA with Fe2* documented primarily single-base substitutions (67). In contrast to these results, a study which examined mutagenesis of a plasmid in H2O2-treated simian cells reported dele tions in 45% of spontaneous or induced mutants and single or multiple base changes in 68 or 57% of induced or spontaneous mutants (68). The differences observed point out the impor tance of evaluating such spectra in carefully controlled studies. Thus, the factors involved in the lack of base substitutions in mesothelioma cell lines may be related to uncontrolled factors involved in asbestos carcinogenesis. It will be of interest in the future to examine mutational spectra resulting from interaction of asbestos and mesothelial cells or with mixed cultures of mesothelial cells and macrophages. The findings of this report suggest that loss of wild-type p53 expression is less commonly associated with human meso thelioma than with many other tumor types. An additional study showing no abnormalities in retinoblastoma gene protein or mRNA expression in human mesothelioma cell lines' makes it probable that these two tumor suppressor genes do not play critical roles in growth regulation in mesothelial cells. Thus, loss of function of the retinoblastoma gene has been associated with some but not all tumor types (69) while p53 alterations have been more commonly found (70). It is probable that specific cell types will differ with regard to which genes are most critical in the regulatory processes which define the "nor mal" cell. In mesothelial cells, it would appear that loss of function alterations in the p53 or the retinoblastoma not rate limiting for tumor development.

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Acknowledgments We wish to acknowledge helpful discussions with Dr. Bonita Taffe and the assistance of Dorothea Dudek in preparation of this manuscript.

References 1. Wagner, J. C., Sleggs, C. A., and Marchand, P. Diffuse pleural mesothelioma and asbestos exposure in the North Western Cape Province. Br. J. Ind. Med., / 7: 260-271, 1960. 2. Craighead, J. E., and Mossman, B. T. The pathogenesis of asbestos-associ3A. Van der Meeren et al., manuscript in preparation.

9. 10.

11. 12. 13. 14.

15.

CELL LINES

ated diseases. N. Engl. J. Med., 306: 1446-1455, 1982. Selikoff, I. J., Hammond, E. C, and Seidman, H. Latency of asbestos disease among insulation workers in the United States and Canada. Cancer (Phila.). 46:2736-2740, 1980. Gross, P., and Braun. D. C. Asbestos, talc, inorganic fibers, man-made vitreous fibers, and organic fibers. In: Toxic and Biomedicai Effects of Fibers, pp. 94-96. Park Ridge. NJ: Noyes Publishing, 1984. Fearon, E. R.. and Vogelstein, B. A genetic model for colorectal tumorigenesis. Cell, 61: 759-767, 1990. Bos, J. L. ras oncogenes in human cancer: a review. Cancer Res.. 49: 46824689, 1989. Barbacid, M. ras genes. Annu. Rev. Biochem., 56: 779-827. 1987. Kobayashi. T.. Tsuda, H., Noguchi, M., Hirohashi, S.. Shimosato, Y.. Goya, T.. and Hayata, Y. Association of point mutation in c-Ki-ras oncogene in lung adenocarcinoma with particular reference to cytologie subtypes. Cancer (Phila.), 66: 289-294, 1990. Almoguera, C., Forrester, K.. Winter, E., Lama, C., and Perucho, M. Acti vated ras genes in pulmonary carcinoma. Lung Cancer, 4: 168-170, 1988. Rodenhuis, S.. Slebos. R. J., Boot, A. J., Evers. S. G., Mooi, W. J., Wagenaar, S. S., van Bodegom, P. C., and Bos, J. L. Incidence and possible clinical significance of K-ras oncogene activation in adenocarcinoma of the human lung. Cancer Res., 48: 5738-5741, 1988. Eliyahu, D., Raz, A., Gruss, P., Givol, D.. and Oren, M. Participation of p53 cellular tumour antigen in transformation of normal embryonic cells. Nature (Lond.), 312: 646-649, 1984. Jenkins. J. R.. Rudge, K . and Currie, G. A. Cellular immortalization by a cDNA clone encoding the transformation-associated phosphoprotein p53. Nature (Lond.), 312: 651-654, 1984. Parada, L. F.. Land, H., Weinberg, R. A., Wolf, D., and Rotter, V. Cooper ation between gene encoding p53 tumour antigen and ras in cellular trans formation. Nature (Lond.), 312: 649-651. 1984. Nigro, J. M., Baker, S. J., Preisinger, A. C., Jessup, J. M., Hosteller, R., Cleary, K., Bigner, S. H., Davidson, N.. Baylin, S., Devilee, P., Glover, T., Collins, F. S., Weston, A., Modali. R.. Harris. C. C., and Vogelstein. B. Mutations in the p53 gene occur in diverse human tumor types. Nature (Lond.), 342: 705-708. 1989. Chiba, I., Takahashi.T., Nau, M. M., D'Amico. D.,Curiel. D. T.. Mitsudomi.

T., Buchhagen, D. L., Carbone. D., Piantadosi. S., Koga, H.. Reissman, P. T., Slamon, D. J., Holmes, E. C., and Minna, J. D. Mutations in the p53 gene are frequent in primary, resected non-small cell lung cancer. Oncogene, 5: 1603-1610, 1990. 16. Iggo, R., Gatter, K.. Bartek, J., Lane, D.. and Harris. A. L. Increased expression of mutant forms ofp53 oncogene in primary lung cancer. Lancet, 335: 675-679, 1990. Takahashi, T., D'Amico. D.. Chiba. I., Buchhagen, D. L., and Minna, J. D. 17. Identification of intronic point mutations as an alternative mechanism for p53 inactivation in lung cancer. J. Clin. Invest.. 86: 363-369, 1990. 18. Takahashi, T., Nau, M. M., Chiba, L, Birrer, M. J., Rosenberg, R. K., Vinocour, M., Levitt, M., Pass, H., Gazdar, A. F.. and Minna, J. D. p53: a frequent target for genetic abnormalities in lung cancer. Science (Washington DC), 246: 491-494. 1989. 19. Hollstein. M.. Sidransky, D., Vogelstein, B., and Harris, C. C. p53 mutations in human cancers. Science (Washington DC), 253: 49-53, 1991. 20. Lehman, T. A., Bennett, W. P., Metcalf. R. A., Reddel, R., Welsh, J. A., Ecker, J., Modali, R. V., Ullrich, S., Romano. J. W., Appella, E., Testa, J. R., Gerwin, B. I., and Harris, C. C. p53 mutations, ras mutations and p53heat shock 70 protein complexes in human lung cell lines. Cancer Res., 5/: 4090-4096. 1991. 21. McBride, O. W.. Merry, D., and Givol, D. The gene for human p53 cellular tumor antigen is located on chromosome 17 short arm (17pl3). Proc. Nati. Acad. Sci. USA, 83: 130-134, 1986. 22. Isobe, M., Emanuel. B. S., Givol, D., Oren, M., and Croce, C. M. Localization of gene for human p53 tumour antigen to band 17pl3. Nature (Lond.), 320: 84-85, 1986. 23. Gibas, Z., Li, F. P., Animan. K. H., Bernal. S., Stahel. R., and Sandberg, A. A. Chromosome changes in malignant mesothelioma. Cancer Genêt. Cytogenet., 20: 191-201, 1986. 24. Popescu, N. C., Chahinian, A. P., and DiPaolo, J. A. Nonrandom chromo some alterations in human malignant mesothelioma. Cancer Res., 48: 142147, 1988. 25. Tiainen, M., Tammilehto. L.. Mattson. K.. and Knuutila. S. Nonrandom chromosomal abnormalities in malignant pleural mesothelioma. Cancer Ge net. Cytogenet.. 33: 251-274, 1988. 26. Flejter, W. L., Li, F. P., Amman. K. H., and Testa, J. R. Recurring loss involving chromosomes 1, 3, and 22 in malignant mesothelioma: possible sites of tumor suppressor genes. Genes Chromosomes Cancer, /: 148-154, 1989. 27. Cote, R. J., Jhanwar, S. C., Novick, S.. and Pellicer, A. Genetic alterations of the p53 gene are a feature of malignant mesotheliomas. Cancer Res., 51: 5410-5416,1991. 28. LaVeck, M. A., Somers, A. N. A., Moore. L. L., Gerwin, B. I., and Lechner. J. F. Dissimilar peptide growth factors can induce normal human mesothelial cell multiplication. In Vitro, 24: 1077-1084, 1988. 29. Tibbetts, L. M. Loss and restoration of tumorigenicity in a human meso thelioma cell line. Proc. Am. Assoc. Cancer Res., 25:45, 1984. 30. Carney, D. N., Gazdar. A. F., Bepler, G., Guccion, J. G., Marangos, P. J., Moody, T. W., Zweig, M. H., and Minna, J. D. Establishment and identifi-

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Downloaded from cancerres.aacrjournals.org on July 9, 2011 Copyright © 1992 American Association for Cancer Research

p53 STATUS IN HUMAN MESOTHELIOMA

3!.

32.

33. 34. 35.

36. 37. 38.

39. 40.

41. 42.

43.

44.

45. 46.

47. 48. 49.

cation of small cell lung cancer cell lines having classic and variant features. Cancer Res., 45: 2913-2923, 1985. Gerwin, B. I., Lechner. J. F., Reddel, R. R., Roberts, A. B.. Robbins. K. C., Gabrielson, E. W., and Harris, C. C. Comparison of production of transform ing growth factor-fi and platelet-derived growth factor by normal human mesothelial cells and mesothelioma cell lines. Cancer Res.. 47: 6180-6184, 1987. Pelin-Enlund, K., Husgafvel-Pursiainen, K., Tammilehto, L., Klockars, M., Jantunen. K.. Gerwin, B. I., Harris, C. C., Tuomi, T., Vanhala, E., Mattson. K.. and Linnainmaa, K. Asbestos-related malignant mesothelioma: growth, cytology, tumourigenicity and consistent chromosome findings in cell lines from five patients. Carcinogenesis (Lond.), //: 673-681, 1990. Mullis, K. B., and Faloona. F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol., 155: 335-350, 1987. Sanger, F., Nicklen, S., and Coulson, A. R. DNA sequencing with chainterminating inhibitors. Proc. Nati. Acad. Sci. USA, 74: 5463-5467, 1977. Brash. D. E., Rudolph, J. A., Simon, J. A., Lin, A., McKenna, G. J., Baden, H. P., Halperin. A. J., and Ponten. J. A role for sunlight in skin cancer: UVinduced p53 mutations in squamous cell carcinoma. Proc. Nati. Acad. Sci. USA,*«: 10124-10128. 1991. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156-159. 1987. Baker. S. J.. Markowitz, S., Fearon, E. R., Willson. J. K., and Vogelstein. B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science (Washington DC), 249: 912-915. 1990. Piechaczyk, M., Blanchard. J. M., Marty, L., Dani, C., Panabieres, F., el Sabrouty, S., Fort. P., and Jeanteur. P. Post-transcriptional regulation of glyceraldehyde-3-phosphate-dehydrogenase gene expression in rat tissues. Nucleic Acids Res.. 12: 6951-6963, 1984. Tubo. R. A., and Rheinwald. J. G. Normal human mesothelial cells and fibroblasts transfected with the EJras oncogene become EGF-independent. but are not malignantly transformed. Oncogene Res., /: 407-421, 1987. Reddel, R. R., Malan-Shibley, L., Gerwin, B. I., Metcalf, R.. and Harris, C. C. Tumorigenicity of a human mesothelial cell line transfected with the II ras oncogene. J. Nati. Cancer Inst., 81: 945-948, 1989. Rodrigues, N. R., Rowan, A.. Smith. M. E. F., Kerr, I. B.. Bodmer. W. F., Gannon, J. V., and Lane, D. P. p53 mutations in colorectal cancer. Proc. Nati. Acad. Sci. USA, 87: 7555-7559. 1990. Bennett, W. P., Hollstein, M. C, He, A., Zhu, S. M.. Resau, J.. Trump. B. F., Metcalf, R. A., Welsh. J. A., Gannon. J. V.. Lane. D. P., and Harris, C. C. Archival analysis of p53 genetic and protein alterations in Chinese esophageal cancer. Oncogene, 6: 1779-1784, 1991. Ciechanover, A., DiGiuseppe, J. A., Bercovich, B., Orian, A., Richter, J. D., Schwartz, A. L., and Brodeur, G. M. Degradation of nuclear oncoproteins by the ubiquitin system in vitro. Proc. Nati. Acad. Sci. USA, 88: 139-143, 1991. van den Heuvel, S. J., van Laar. T., Käst,W. M., Melief, C. J., Zantema, A., and van der Eb, A. J. Association between the cellular p53 and the adenovirus 5 ElB-55kd proteins reduces the oncogenicity of Ad-transformed cells. EMBO J, 9: 2621-2629, 1990. Roñen.D., Rotter, V., and Reisman. D. Expression from the murine p53 promoter is mediated by factor binding to a downstream helix-loop-helix recognition motif. Proc. Nati. Acad. Sci. USA. «A: 4128-4I32, 1991. Kok, K., Osinga. J.. Schotanus, D. C.. Berendsen, H. H., de Leij, L. F., and Buys. C. H. Amplification and expression of different /nyc-family genes in a tumor specimen and 3 cell lines derived from one small-cell lung cancer patient during longitudinal follow-up. Int. J. Cancer, 44: 75-78, 1989. Wong, A., Ruppert, J., Eggleston. J., Hamilton. S.. Baylin, S., and Vogelstein, B. Gene amplification of c-myc and N-myc in small cell carcinoma of the lung. Science (Washington) 233:461-464, 1986. Mercer, W. E., and Baserga. R. Expression of the p53 protein during the cell cycle of human peripheral blood lymphocytes. Exp. Cell Res., 160: 31-46, 1985. Oren, M., Maltzman, W., and Levine, A. Post-translational regulation of the

50. 51.

52. 53. 54.

55. 56. 57. 58. 59.

60.

61. 62.

63. 64.

65.

66. 67. 68. 69. 70.

CELL LINES

54K cellular tumor antigen in normal and transformed cells. Mol. Cell. Biol., /. 101-110, 1981. Rotter, V., Witte, O. N., Coffman, R., and Baltimore. D. Abelson murine leukemia virus-induced tumors elicit antibodies against a host cell protein, P50. J Virol., 36: 547-555, 1980. Kastan, M. B., Radin, A. I., Kuerbitz, S. J., Onyekwere, O., Wolkow, C. A., Civin, C. I., Stone, K. D., Woo, T., Ravindranath, Y.. and Craig, R. W. Levels of p53 protein increase with maturation in human hematopoietic cells. Cancer Res., 5/.- 4279-4286, 1991. Ehrlich. M., and Wang, R. Y. 5-Methylcytosine in eukaryotic DNA. Science (Washington DC). 212: 1350-1357, 1981. Hollstein, M., Metcalf. R. A., Welsh, J., Montesano. R., and Harris, C. C. Frequent mutation of the p53 gene in human esophageal cancer. Proc. Nati. Acad. Sci. USA, 87: 9958-9961, 1990. Sidransky, D., Von Eschenbach, A., Tsai, Y. C., Jones, P.. Summerhayes, I., Marshall, F.. Paul, M., Green. P., Hamilton. S. R., Frost, P., et al. Identifi cation of p53 gene mutations in bladder cancers and urine samples. Science (Washington DC), 252: 706-709, 1991. Hsu, I. C. Metcalf. R. A.. Sun, T., Welsh, J., Wang, N. J., and Harris, C. C. p53 gene mutational hotspot in human hepatocellular carcinomas from Qidong, China. Nature (Lond.). 350:427-428, 1991. Bressac, B., Kew, M.. Wands, J., and Ozturk. M. Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature (Lond.), 350:429-431. 1991. Saylors, R. L., Sidransky, D.. Friedman, H. S., Bigner. S. H., Bigner. D. D., Vogelstein, B., and Brodeur, G. M. Infrequent p53 gene mutations in medulloblastomas. Cancer Res., 51: 4721-4723, 1991. Soussi, T., Carón de Fromentel. C., and May, P. Structural aspects of the p53 protein in relation to gene evolution. Oncogene, 5: 945-952, 1990. Lechner, J. F.. Tokiwa, T., LaVeck, M. A., Benedict, W. F., Banks-Schlegel, S. P., Yeager. H., Jr., Banerjee. A., and Harris, C. C. Asbestos-associated chromosomal changes in human mesothelial cells. Proc. Nati. Acad. Sci. USA. 82: 3884-3888. 1985. Gabrielson, E. W., Rosen, G. M., Grafstrom, R. C.. Strauss, K. E., and Harris, C. C. Studies on the role of oxygen radicals in asbestos-induced cytopathology of cultured human lung mesothelial cells. Carcinogenesis (Lond.), 7: 1161-1164, 1986. Goodglick, L. A., and Kane, A. B. Role of reactive oxygen metabolites in crocidolite asbestos toxicity to mouse macrophages. Cancer Res., 46: 55585566, 1986. Hansen, K., and Mossman, B. T. Generation of Superoxide (Oj~) from alveolar macrophages exposed to asbestiform and nonfibrous particles. Can cer Res., 47: 1681-1686, 1987. Mossman. B. T., Marsh. J. P.. and Shatos, M. A. Alteration of Superoxide dismutase activity in trachéalepithelial cells by asbestos and inhibition of cytotoxicity by antioxidants. Lab. Invest., 54: 204-212, 1986. Wang, N. S.. Jaurand, M. C., Magne. L.. Kheuang. L., Pinchón, M. C., and Bignon. J. The interactions between asbestos fibers and metaphase chromo somes of rat pleural mesothelial cells in culture. A scanning and transmission electron microscopic study. Am. J. Pathol., 126: 343-349, 1987. Linnainmaa. K., Gerwin. B. I., Pelin, K., Jantuene. K., LaVeck, M. A., Lechner, J. F., and Harris, C. C. Asbestos-induced mesothelioma and chro mosomal abnormalities in human mesothelial cells in in vitro. In: The Changing Nature of Work and the Work Place, pp. 119-122. Cincinnati, OH: NIOSH, 1986. Cora, E. M., and Kane. A. B. Alterations in a tumor suppressor gene, p53, in mouse mesotheliomas induced by crocidolite asbestos. Eur. Resp. Rev., in press, 1992. McBride, T. J., Preston, B. D., and Loeb, L. A. Mutagenic spectrum resulting from DNA damage by oxygen radicals. Biochemistry, 30: 207-213. 1991. Moraes. E. C., Keyse, S. M., and Tyrrell, R. M. Mutagenesis by hydrogen peroxide treatment of mammalian cells: a molecular analysis. Carcinogenesis (Lond.).//: 283-293. 1990. Weinberg, R. A. Oncogenes, antioncogenes. and the molecular bases of multistep Carcinogenesis. Cancer Res.. 49: 3713-3721, 1989. Vogelstein, B. Cancer. A deadly inheritance. Nature (Lond.). 348: 681-682, 1990.

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