tPathology and of §Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111; ..... lators and Asbestos Workers Fund, by the Lucille P. Markey Chari-.
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 10854-10858, November 1995 Medical Sciences
High frequency of inactivating mutations in the neurofibromatosis type 2 gene (NF2) in primary malignant mesotheliomas ALBERT B. BIANCHI*t, SHIN-ICHIRO MITSUNAGAtt, JIN QUAN CHENG§, WALTER M. KLEIN§, SURESH C. JHANWARI, BERND SEIZINGER*, NIKoLAI KLEY*, ANDRES J. P. KLEIN-SZANTOt, AND JOSEPH R. TESTA§II *Department of Molecular Genetics and Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000; Departments of
tPathology and of §Medical Oncology, Fox Chase Cancer Center, Philadelphia, PA 19111; and IDepartment of Human Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021
Communicated by C. C. Tan, Fudan University, Shanghai, China, August 8, 1995
Both germ-line and somatic NF2 gene mutations have been extensively characterized in meningiomas and schwannomas (11-15). The observed mutations represent predominantly either nonsense or splice site mutations, or frameshift nucleotide deletions and insertions leading to truncated forms of merlin. In addition to these tumor types, somatic mutations have been identified in neoplasms seemingly unrelated to the NF2 disorder such as malignant melanoma (like schwannomas, however, this tumor type derives from tissue of neural-crest origin) as well as carcinoma of the breast (12) and colon (16). However, the frequency of NF2 mutations detected in breast and colon cancers is significantly lower than the incidence of allelic losses from chromosome 22q observed in these tumors (17). Malignant mesotheliomas (MMs) are mesodermally derived, primarily pleural tumors that respond poorly to current therapeutic approaches. Although MMs are relatively rare, their incidence has continued to increase, and considerable interest has focused on this neoplasm because of its association with asbestos exposure (18, 19). Loss of chromosome 22 is the single most consistent numerical cytogenetic change in MMs (20, 21). Rearrangement and loss of chromosomes lp, 3p, 6q, and 9p are also a prominent feature of this malignancy (20-24). The critically involved genes located within these deleted regions are currently unknown. A putative tumor suppressor gene p16/MTSI/CDKN2 is located in chromosome 9p, and we recently showed that alterations of p16 are a common occurrence in MM cell lines and, to a lesser extent, in primary tumors (25). While MMs have a different histogenic derivation than neuroectodermal neoplasms, the frequent abnormalities of chromosome 22 in MM prompted us to investigate whether alterations of the NF2 gene are involved in these tumors.
ABSTRACT Malignant mesotheliomas (MMs) are aggressive tumors that develop most frequently in the pleura of patients exposed to asbestos. In contrast to many other cancers, relatively few molecular alterations have been described in MMs. The most frequent numerical cytogenetic abnormality in MMs is loss of chromosome 22. The neurofibromatosis type 2 gene (NF2) is a tumor suppressor gene assigned to chromosome 22q which plays an important role in the development of familial and spontaneous tumors of neuroectodermal origin. Although MMs have a different histogenic derivation, the frequent abnormalities of chromosome 22 warranted an investigation of the NF2 gene in these tumors. Both cDNAs from 15 MM cell lines and genomic DNAs from 7 matched primary tumors were analyzed for mutations within the NF2 coding region. NF2 mutations predicting either interstitial in-frame deletions or truncation of the NF2encoded protein (merlin) were detected in eight cell lines (53%), six of which were confirmed in primary tumor DNAs. In two samples that showed NF2 gene transcript alterations, no genomic DNA mutations were detected, suggesting that aberrant splicing may constitute an additional mechanism for merlin inactivation. These findings implicate NF2 in the oncogenesis of primary MMs and provide evidence that this gene can be involved in the development of tumors other than nervous system neoplasms characteristic of the NF2 disorder. In addition, unlike NF2-related tumors, MM derives from the mesoderm; malignancies of this origin have not previously been associated with frequent alterations of the NF2 gene.
Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder characterized by the development of bilateral vestibular schwannomas of the eighth cranial nerve and by other brain tumors, including meningiomas and ependymomas (1). Affected individuals are also predisposed to develop spinal schwannomas and meningiomas (1-3). Genetic linkage and deletion mapping analyses of sporadic and familial NF2associated tumors suggested that inactivation of a tumor suppressor gene in chromosome 22q was involved in the etiology of this disorder (4-6). These studies, coupled with positional cloning approaches, have led to the recent isolation of the NF2 gene (7, 8). The NF2 gene is a tumor suppressor gene which encodes a 595-amino acid protein called merlin (for moesin-ezrinradixin-like protein) (8) or schwannomin (7). Merlin exhibits significant homology to a highly conserved family of proteins postulated to play a role in cell surface dynamics and structure by linking the cytoskeleton to components of the plasma membrane (9, 10).
MATERIALS AND METHODS Human Primary Tumors, Cell Lines, and Nucleic Acid Extraction. Fifteen human MM cell lines were established from primary cultures of surgically resected tumors as described previously (25). Matched primary tumor tissues were snap frozen and stored at -70°C. DNA was isolated from cell line and tissue samples by using standard methods (26). Normal diploid human mesothelial cell strain LP-9 (27) was obtained from the NIA Aging Cell Repository. Total RNA was extracted from frozen tumor specimens and from cell lines by lysis in guanidinium thiocyanate and extraction with phenol/ chloroform (28). Abbreviations: MM, malignant mesothelioma; NF2, neurofibromatosis type 2; RT, reverse transcriptase; SSCP, single-strand conformation polymorphism. tA.B.B. and S.-I.M. contributed equally to this work. ITo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 10854
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Reverse Transcriptase (RT)-PCR, Genomic PCR, and Single-Strand Conformation Polymorphism (SSCP) Analysis. cDNA synthesis was performed by denaturing total RNA at 70°C for 10 min in diethyl pyrocarbonate-treated water. After chilling on ice the denatured RNA was incubated in 20 mM Tris-HCl, pH 8.4/50 mM KCl/2.5 mM MgCl2/10 mM dithiothreitol containing bovine serum albumin at 100 ng/,ul, each deoxynucleoside triphosphate at 500 ,uM, primer 3m3 or 3m6 at 12 ng/,ul, and SuperScript (GIBCO/BRL) RT at 10 units/,ul for 10 min at room temperature followed by 60 min at 42°C. The reaction was terminated by heating to 95°C for 2 min and quenching on ice. PCR amplifications and SSCP analyses were performed as described previously (12). Oligonucleotide primers were identical to those reported in earlier studies for RT-PCR assays (12) and for genomic PCR amplification of individual exons (13). Sequence Analysis. Individual bands were carefully excised from dried SSCP gels and placed into 100 ,ul of deionized water, and the DNA was allowed to elute for 4-6 hr at room temperature with gentle shaking. Ten microliters of eluted DNA was reamplified with appropriate primers as described above. Amplified products were subcloned in the plasmid vector PCR II (Invitrogen), and inserts were sequenced, using double-stranded recombinant plasmids as template for the dideoxynucleotide chain-termination method (29). Reaction products were electrophoresed on 6% polyacrylamide/8 M urea/0.1 M Tris borate, pH 8.3/2 mM EDTA gels. Some of the clones were sequenced by dideoxynucleotide terminator chemistry in an Applied Biosystems 373-A automated DNA sequencer. Double-strand sequencing of three clones was performed for each mutant sample. Sequence data were analyzed by using the MACVEcTOR 4.1 software package (International Biotechnologies).
RESULTS To investigate the presence of mutations within the NF2 coding region, RT-PCR and SSCP analyses were performed with RNA extracted from 15 MM cell lines. Abnormal SSCP patterns were detected in cDNAs from 8 of the 15 (53%) cell lines. A representative subset of MM samples analyzed by RT-PCR/SSCP is shown in Fig. 1. Sequencing analysis of the '0
0
tps~ -~~~~~~~~~~~~~~~~.....
f. . . I.:. J
L::
m
"A
mw4So
.ml
FIG. 1. SSCP analysis of RT-PCR products made by using RNA from normal mesothelial control cells (Co) and the MM cell lines indicated. Arrows show some of the aberrant SSCP conformers sequenced. Oligonucleotide primer pairs used in this analysis were 5ml-3ml (MM 6-53); 5m3-3m3 (MM 1-55); 5m4-3m4 (MM 222); and 5m2-3m2 (MM 2-50), according to Bianchi et al. (12).
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variant SSCP conformers revealed nucleotide deletions and insertions and one nonsense mutation in the NF2 gene transcript, predicting in all cases truncated forms of the merlin protein. For 7 of the 8 MM cell lines with NF2 cDNA alterations, matched primary tumor specimens were available and analyzed at the genomic DNA level; NF2 mutations were confirmed in 6 of the 7 matched primary tumors (Table 1). NF2 cDNA aberrations found in cell lines 222 and 1-53 were not detected at the genomic level. NF2 cDNA frameshift deletions of 1 and 7 bp were found in cell lines 6-53 and 2-63, with a stop codon occurring 93 and 48 nt, respectively, downstream of the deletion breakpoint. Genomic PCR amplification and sequencing analysis of exon 3 and exon 6 of the NF2 gene by using DNA extracted from matched primary tumors confirmed intragenic deletions of 1 and 7 bp in MM 6-53 (Fig. 2) and 2-63, respectively. An 18-bp in-frame deletion encompassing nt 691-708 was detected in cDNA from cell line 217 and confirmed at the genomic level by amplification of exon 8 using DNA from the corresponding primary tumor. Sequencing of the variant conformer detected in cDNA from cell line 1-55 (Fig. 1) revealed a nonsense mutation in codon 198. This point mutation was also confirmed in the matched primary tumor by genomic PCR amplification of exon 6. Complete skipping of exon 10 in the NF2 gene transcript was found in cDNA from cell line 222 as compared with that observed in normal mesothelial cells (Figs. 1 and 3a). Analysis of genomic DNA from cell line 222 did not reveal any mutations that may account for the skipping of exon 10. No mutations were detected in either exon 10 or the flanking intron 9 splice donor (not shown) and acceptor sites (cag/G; Fig. 3b), or in the intron 10 splice donor site (not shown), or the branch point element (TATTAAC; consensus YNYURAY) positioned 26 nt upstream of exon 10 (Fig. 3b), as compared with genomic DNA from mesothelial control cells. As shown in Table 1, the remaining NF2 cDNA alterations detected by RT-PCR/SSCP analyses of cell lines 1-53, 2-50, and 1-51 represent frameshift nucleotide insertions of 43, 119, and 55 bp, respectively, positioned at different splice junction sites. Genomic PCR analysis of the intron sequences flanking exons 3 and 4 in DNA from cell line 2-50, and exons 1 and 2 in primary tumor 1-51, revealed that these nucleotide insertions actually corresponded to partial intronic sequences. Sequence comparison with normal mesothelial cells also revealed point mutations in the intron 3 splice donor site (CAGgtacat CAGctacat) and the exon 1 splice donor site (GAGitaacg GAAgtaacg) in cell line 2-50 and tumor 1-51, respectively (Table 1). These mutated G nucleotides have been shown to be conserved 100% and 80% (positions 1 and -1, respectively, relative to the cleavage site) as part of the mammalian 5' splice consensus site, and mutations in this site have been reported to often activate nearby cryptic splice sites in vivo, thereby causing partial intron retention in the spliced
transcript (30). The 43-bp nucleotide insertion at the junction of exons 13 and 14 detected in cDNA from cell line 1-53 was not observed at the genomic DNA level (Table 1), and the inserted sequence was not homologous to any portion of the intron 13 flanking segments examined. Similar to previous findings in various tumor types, the NF2 mutations reported in this study showed a uniform distribution along the coding sequence. Whereas two of the eight NF2 cDNA alterations described here would lead to interstitial in-frame deletions of the peptide sequence, six of the remaining mutations predict a truncation of the encoded protein at various positions leading to the removal of the C-terminal region of merlin. Interestingly, of the eight MMs with NF2 cDNA alterations, six were of the biphasic type. Among the seven tumors with
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Table 1. Analysis of NF2 mutations in human MMs Mutation Cell-line cDNA* Sample 1-bp deletion at nt 271; exon 3 6-53 18-bp deletion at nt 691-708; exon 8 217 2-63 7-bp deletion at nt 568-574; exon 6 Nonsense mutation at nt 592, CGA -- IGA; exon 6 1-55 119-bp insertion at exon 3-exon 4 junction 2-50 1-51
G
--
A at nt 114; 55-bp insertion at exon 1-exon 2 junction
Tumor DNAt Confirmed Confirmed Confirmed Confirmed nt 363 Ggt -> Gft; (5' SS of intron 3) jgt -+ Agt; (5' SS of intron 1) ND
(1995)
Predicted effectt Frameshift; protein truncated at aa 122 Deletion of aa 231-236 Frameshift; protein truncation at aa 208 Arg -- stop; protein truncation at aa 198 161-aa truncated protein altered at aa 121 138-aa truncated protein altered at aa 38
Deletion of aa 296-333 Deletion of exon 10 222 507-aa truncated protein altered at aa 482 ND§ 43-bp insertion at exon 13-exon 14 junction 1-53 *Mutations detected by RT-PCR/SSCP analysis of cDNA from MM cell lines. Nucleotide (nt) numbers correspond to those reported by Rouleau et al. (7). tMutations detected by PCR of genomic DNA from the primary tumors from which cell lines were derived; confirmed, detection of the same mutation as the one reported in the cell line cDNA; lowercase letters represent introns; SS, splice site; ND, not detected. tAmino acid (aa) numbers according to Rouleau et al. (7). §DNA from the primary tumor was not available. Genomic DNA from the cell line was analyzed instead.
normal NF2 cDNA status, six were of either epithelioid or sarcomatous type, and only one was biphasic.
DISCUSSION To determine whether the high frequency of loss of chromosome 22 previously reported in MM is associated with alteration of the NF2 tumor suppressor gene, we screened a set of MM cell lines and matched primary tumors for mutations within the NF2 coding region. Alterations in the NF2 cDNA were detected in 8 of the 15 cell lines analyzed. Whereas 6 of the 8 NF2 gene transcript alterations were confirmed at the genomic level in primary tumors, the NF2 cDNA aberrations found in cell lines 222 and 1-53 were not detected in the corresponding genomic DNAs. Our findings clearly implicate NF2 in MM tumorigenesis and provide evidence that this gene can be involved in the development of malignancies other than the nervous system neoplasms characteristic of the NF2 disorder. Upon completion of our studies, Sekido et al. (31) reported NF2 mutations in 6 of 14 (43%) MM cell lines. Three of their cell lines derived from matched tumor samples had a deletion not detected by PCR analysis in the tumor specimen, presumably because of the presence of normal cells admixed with tumor cells in the fresh sample. Thus, the investigators were unable to rule out the possibility that these alterations occurred during cell culture. However, they did identify an NF2 mutation in an unmatched primary tumor specimen. We have confirmed their cell line findings, and in addition we demonstrate the frequent involvement of NF2 mutations in the pathogenesis of primary MMs. Our data indicating a mutation incidence of at least 40% in primary MMs rule out the possibility that these alterations occurred as a result of in vitro culture of the tumors, as has been suggested in the case of p16 (32).
TUMOR 6-53 S' ICIAAGGAG
The fact that MMs are not associated with the NF2 disorder is also reminiscent of other hereditary cancer syndromes such as familial retinoblastoma and Li-Fraumeni syndrome, in which the corresponding tumor suppressor gene has been found to be somatically mutated in seemingly unrelated malignancies (33). Similar to the retinoblastoma and adenomatous polyposis coli tumor suppressor genes which are commonly inactivated by nonsense mutations, the NF2 gene alterations found in MM in this study consisted mainly of frameshift nucleotide deletions, insertions, and nonsense mutations, with absence of missense mutations, typically leading to a truncation of the C terminus of the merlin protein. This mutation spectrum closely resembles that found in other tumor types, including schwannomas and meningiomas, and indicates that in most cases major structural alterations are necessary to impair merlin function. One exon-skipping alteration found in this study corresponds to the in-frame deletion of exon 10 in MM cell line 222, as indicated by the variant SSCP conformer shown in Fig. 1. Interestingly, analysis of genomic DNA from MM cell line 222 revealed no mutations in the flanking intron and exon sequences analyzed, including the splice donor, splice acceptor, and lariat branchpoint elements (Fig. 3) that may account for the generation of a truncated transcript lacking exon 10 (NF2-A10 mRNA). Previously, the NF2-A10 transcript was shown by RT-PCR to be expressed at very low levels in RNA isolated from normal leptomeningeal tissue and other sources (34). However, this transcript form was not detected in any of the other tumor samples or normal mesothelial control studied here, or in previously analyzed tissues, including cDNA from the eighth cranial nerve (12). Therefore, the presence of low expression levels of alternatively spliced transcripts previously detected in normal cells (34) might reflect background levels CONTROL
TCACCACT 3'
AIr 5' TTCAAAIc
3'
FIG. 2. Intragenic 1-bp deletion in DNA from MM cell line 6-53. Mutant (Left) and normal mesothelial control (Right) sequences are shown above density tracings of the sequencing gels. The arrow indicates the position of the nucleotide deleted, C-271.
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a
Proc. Natl. Acad. Sci. USA 92 (1995)
Control
Tumor 2m .xo IIown 9 sn aGLA
exon 10
ca r
exon 9
3' ATAOCTAAA CAGCTTATTAACA '
ron 9
b Tumfo
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222
Contl
FIG. 3. (a) Sequence of the RT-PCR-amplified product from MM 222 and normal mesothelial control cDNAs. Sequences of the junctions of exon 9 to exon 10 or 11 are shown. The sequence of the 3' end of exon 9 is joined directly to the 5' end of exon 11 in the tumor cDNA (exon 10 is skipped) as compared with the 5' end of exon 10 in the control. (b) Sequence of the intron 9/exon 10 boundary in genomic DNAs from MM cell line 222 and normal mesothelial control. No mutations were detected in exon 10, intron 9 (partial sequences shown), or intron 10 (not shown) including the splice recognition sites (3'SS, 3' splice site of intron 9: CAG/A), branchpoint sequence (BPS; TATTAAC), and polypyrimidine tract (Py), marked by solid lines.
of exon skipping, the physiological relevance of which remains to be established. Although we cannot rule out the possibility that the NF2-A1O truncated transcript was generated by a mutation in a region other than the sequences analyzed, which included the critical splicing consensus elements, our data suggest that this form might have resulted from aberrant splicing of the NF2 gene transcript. Whether this' is due to specific defects in the splicing machinery regulating the processing of the NF2 transcript remains to be established. A similar mechanism of tumor suppressor inactivation by abnormal splicing in the absence of gene mutation has previously been advanced for the Wilms tumor 1 (WT1) tumor suppressor gene (35). In addition, aberrant splicing without gene mutation has also been proposed as a possible mechanism for disruption of the transcription factor IRF1 in human myelodysplasia/
leukemia (36). Interestingly, in a study of 75 lung carcinomas, no NF2 gene mutations were detected despite the presence of chromosome
22 losses in many cases (31). This suggests that other tumor suppressor gene(s) may be involved in some of the malignancies associated with chromosome 22 allelic losses in which NF2 mutations were either not detected or found at very low frequencies,- including pheochromocytoma, astrocytoma, ependymoma, as well as ovarian, colon, and breast carcinomas (12, 16, 37, 38). The pattern of frequent chromosomal losses in MM suggests that multiple tumor suppressor genes may be involved in the pathogenesis of this neoplasm. However, there have been few reports demonstrating recurrent involvement of such genes in MM. We observed alterations of p16 in 5 of 23 (22%) primary tumor specimens from MM patients (25). Mutations of other tumor suppressor genes such as TP53 and WTJ have also been reported in a few cases of MM (39, 40). The frequent involvement of NF2 alterations in both MM cell lines and primary tumors suggests that this gene could play an important role in the pathogenesis of a large subset of MMs. These data further
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demonstrate that the NF2 gene has a more widespread role in tumorigenesis than in the brain tumors typically associated with the NF2 syndrome. Moreover, the availability of NF2mutant and null cell lines should provide a useful model system for the functional analysis of merlin. We thank Xavier Villareal and Larry Gelbert for their collaboration in the sequencing analysis of the samples reported here. This research was supported by National Cancer Institute Grants CA-45745 and CA-06927, by the International Association of Heat and Frost Insulators and Asbestos Workers Fund, by the Lucille P. Markey Charitable Trust, and by an appropriation from the Commonwealth of Pennsylvania. J.Q.C. is a Special Fellow of the Leukemia Society of America.
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