lnlernalional Journal of Andrology, 1990, 13, pages 377-388

Cellular oncogenes in human teratocarcinoma cell lines H . T E S C H , R . FURBAB, J . CASPER*, J . LYONS?, C. R . BARTRAMI-, H. J . SCHMOLL" and D . L. BRONSONS Medizinische Klinik, Universitat Koln, Koln, * Hiimatologische Klinik, Medizinische Hochschule Hannover, ?Section of Molecular Biology Department of Pediatrics I , University of Ulm, FRG and $Department of Virology and Immunology, Southwest Foundation for Biomedical Research, Sun Antonio, Texas, U.S.A.

Summary We have analysed, by Northern blots, the expression of 14 cellular oncogenes in nine cell lines established from human teratocarcinomas. All lines expressed considerable amounts of p.53, c-Ki-ras2, c-Ha-ras 1, c-raf 1, N-myc, and c-fos. Low level expression of c-myc was detected in some lines. Southern blot experiments revealed no amplification or rearrangement of the c-Ki-ras2, N-myc or c-fos genes. Using a rapid dot-blot screening procedure, based on a combination of in-vitro amplification of ras-specific sequences and oligonucleotide hybridization, we could detect no activation of Ha-ras or Ki-ras or any unexpressed N-ras sequences secondary to a point mutation at codons 12, 13, or 61. Keywords: Northern blots, oncogenes, Southern blots, teratocarcinoma cell lines. Introduction Human testicular germ cell tumours are thought to arise from pre-meiotic germ cells. These tumours may contain a variety of cell types, from the very undifferentiated type known as embryonal carcinoma (EC), though recognizably differentiated tissues of any of the three germ layers (teratomas) and the extra-embryonic yolk sac and placental chorion (choriocarcinoma) (Damjanov, 1986). Cytogenetic analyses often reveal an isochromosome 12 (Fough & Trempe, 1975; Hogan et al., 1977), and a variety of other chromosomal aberrations, especially of chromosomes 1 and 7, have been described in cell lines derived from such tumours (Bronson et al., 1980, 1983). However, the genetic events that might contribute to tumourogenesis of germ cells remain to be investigated. Several cell lines have been established from human testicular germ cell tumours (Brinster, 1974; Mintz, 1974; Bronson et al., 1980; Cotte et al., 1981,1982; Atkin & Baker, 1983; Casper et al., 1987). Most of these lines represent EC with little or no differentiation potential, but others can be induced to differentiate, sometimes with production of alpha-fetoprotein (AFP), human chorionic gonadoCorrespondence: Dr H . Tesch, Medizinische Klinik, Universitat Koln, 5000 Koln 41, FRG.

377

378 H . Tesch et al. trophin (hCG), or both (Brinster, 1974; Damjanov, 1986), suggesting differentiation along extra-embryonic lines. The full developmental potential of these human EC cells is not known, but in the mouse E C system some lines are capable of participating in apparently normal embryonic development (Wang et al., 1980; Delozier-Blanchet, 1985). These human lines thus provide one of the few models of human embryogenesis (Damjanov, 1986). A family of genes has been identified in retroviruses that can directly transform certain cells in vivo or in vitro. The viruses have captured these oncogenes by transduction from eukaryotic genomes. The cellular homologues of the viral oncogenes - cellular or proto-oncogenes - are highly conserved during evolution, the same ones having been found in species as disparate as yeasts and humans (Bishop, 1987). This conservation and the expression of such genes in embryonic tissues suggests that cellular oncogenes play an important role in the proliferation and differentiation of normal cells. Cellular oncogenes encode proteins with many different functions, such as protein kinases, regulation of CAMP, and binding to DNA. Others have homology to growth factors or their receptors. The ability of cancer cells to produce and respond to their growth factors (autocrine stimulation) has become a central concept linking oncogene and growth factor research (Sporn & Roberts, 1985). Analyses of oncogenes in animal and human tumour cells frequently reveal aberrations and activation by various mechanisms. Thus somatic mutations of the ras gene family have been detected in a variety of human tumour cells (Reddy et al., 1982; Tabin et al., 1982; Santos et al., 1984; Sweet et al., 1984; Vousden et al., 1986). Translocations of the c-myc gene can put a cellular oncogene under the control of a heterologous enhancer or promotor (Dalla-Favera et al., 1982; Little et al., 1983; Schwab et al., 1983; Alitalo et al., 1984; Yokota et al., 1986). Integration of a retroviral promotor adjacent to a cellular oncogene has been shown in several virus-induced turnours. Amplification of the cellular oncogenes in N-myc, c-myc and c-erbB2 in human tumour cells can lead to over-expression of the gene product. Finally, transformation could result from the inactivation of a cellular suppressor gene. Activation of an oncogene can result in either over-expression of the oncogene or the formation of a structurally altered product (Bishop, 1987). To investigate the role of cellular oncogenes in human germ cell turnours, we analysed the expression of oncogenes in nine germ cell tumour lines by Northern blotting. We also sought specific mutations of the ras gene family. Our data indicate that the cells express a variety of oncogenes but do not bear point mutations at position 12, 13, or 61 of Ha-ras, Ki-ras, or N-ras. Materials and methods

Cell lines The human germ cell tumour lines used (Table 1) have been described previously in detail (Bronson et al., 1980, 1983; Casper et al., 1987). They were grown either in RPMI 1640 with 15% fetal bovine serum, 10% tryptose phosphate broth, 2 mM L-glutamine, penicillin, and streptomycin or subcutaneously by injection of 1 x 10’ cells into NMRI nu/nu mice. Xenografts were explanted and frozen at -70°C until

Cellular oncogenes 319 Table 1. Properties of the human germ cell tumour lines

Cell line

Site and histology of origin

Histology of tumours in nude mice

Tumour marker produced in nude mice

571 MR 1156 Q 1428 A 2102 EP 2102 ER H 12.1 H 12.S H 12.7 H 23.1

Testis - TC Testis - T+EC+CC Testis - EC+YS+STGC Testis - TC+YS+P Retrop. - TC Testis - EC+S+T+CC Testis - EC+S+T+CC Testis - EC+S+T+CC Testis - EC

EC+T EC+T+CC EC+STGC EC EC+ STGC+T EC+ STGC+T EC EC+ STGC+T+ Y S ECiYS

AFP+PHCG AFP AFP+PHCG AFP+PHCG AFP+PHCG AFP+PHCG AFP+@HCG AFP+PHCG

AFP. alpha-fetoprotein; BhCG, human chorionic gonadotrophin; CC, choriocarcinoma; EC, embryonal carcinoma; P, polyembryona; S , seminoma; STGC, syncytiotrophohlastic giant cells; T, teratonia; TC, teratocarcinorna; YS. yolk sac.

use. Human placenta was obtained from the Department of Gynaecology, Cologne, FRG).

Isolation of RNA and DNA Isolation of RNA and DNA was performed as described elsewhere (Maniatis e t a f . , 1982). Briefly, to extract RNA, washed cells were lysed (0.01 M Tris, pH 7.5; 0.01 M KCI, 0.001 M MgCI2, 1% Triton ~ 1 0 0 )and the supernatant from the centrifuged lysate (2000 g for 10 min) was mixed with an equal volume of urea buffer (0.01 M Tris, p H 7.5; 0.3 M NaCI, 0.01 M SDS, 7 M urea). RNA was extracted twice each with phenolkhloroformhso-amylalcohol and chloroformhsoamylalcohol, precipitated in ethanol, and stored at -70°C. To recover the DNA, the cells were incubated in 10 mM Tris, 150 mM NaCI, 10 mM EDTA, proteinase K (200 yg ml-I), and 0.2% SDS at 37°C for 6 h and then extracted with phenol/ chloroformhso-amylalcohol and precipitated in ethanol. The DNA was dissolved in 10 mM Tris with 1 mM EDTA, pH 7.4, and stored at 4°C. The concentrations of RNA and DNA were measured by absorbance at 260 nm. Northern blot analysis RNA (10 or 20 pg) denatured for 30 min at 65°C was applied to 1% agarose gels containing 1x MOPS (20 mM 3-(N-morpholine)propane-sulphonicacid), 5 mM Na-acetate, 1 mM EDTA p H 7.0 and 0.23 M formaldehyde. The RNA was transferred to nitrocellulose or Nytran membranes (Schleicher & Schuell, Dassel, FRG) by blotting overnight in 2 0 x SSC (1x SSC = 0.15 M NaCl, 0.015 M Na-citrate pH 7.0). Blots were washed in 2X SSC, dried and baked for 2 h at 80°C. To control for different amounts of RNA, the filters were rehybridized with an actin-specific probe. Southern blot analysis High molecular weight DNA (10 yg) was digested with the appropriate restriction enzyme (Boehringer Mannheim, Eggenstein, FRG), and the fragments were separated on 0.8% agarose gels and transferred to nitrocellulose or Nytran membranes

380 H . Tesch et al. as described elsewhere (Maniatis et al., 1982). The intensity of the restriction fragments was analysed by densitometry, and the amplification of a cellular oncogene was calculated as the ratio of the relative densitometry units of the oncogene and the single copy gene, bcr. To verify that each "P-labelled DNA probe could bind specifically to human sequences, human genomic DNA was cut with appropriate restriction enzymes, transferred to nitrocellulose and hybridized to the probe. This step served as a positive control in cases where proto-oncogenes were not expressed. H y bridizafion Fragments on filters were pre-hybridized for 3-4 h in 4X SSC, 0.1% Denhardt's solution (Denhardt, 1966), 0.05 M Na-phosphate, denatured and sonicated salmon sperm DNA (500 pg ml-I), 0.1% SDS and 50% formamide. Overnight hybridization at 42°C was performed with labelled denatured probes at a final concentration of 2 x 10' c.p.m. ml-' in 50% formamide, 4x SSC, 0.02 M Na-phosphate, 0.2% SDS, 0.02% Denhardt's solution, 10% dextran sulphate and denatured sonicated salmon sperm DNA (150 pg ml-I). The filters were then washed at high stringency ( 0 . 2 ~SSC and 0.1% SDS at either 52 or 65°C). Cloned probes

The probes used are listed in Table 2. Inserts were excised from the plasmid vectors, purified in the Biotrap chamber (Schleicher & Schuell) and labelled by random hexanucleotide priming (Feinberg & Vogelstein, 1983) to a specific activity of 1-2 X 10' c.p.m. pg-' DNA. Table 2. Probes used in the analysis of teratoma cell lines

Oncogene

Insert

Reference

c-myc c-myb N-myc c-me! c-rafl v-fos c-erbB 1 c-erbB2 c-K i - rm2 c-Ha-ras 1 N - ras c-src2

1.5kb Sac1 2.0kb EcoRI l.0kb BamHI 2.3kb PstI 3.5kb EcoRI I.Okb PstIO 2.4kb ClaI 0.5kb BamHI 3.0kb EcoRI 6.6kb BarnHI l.Okb HindIII 1.7kb BarnHI 1.8kb EcoRI 1.7kb BamHI 3.6kb HindIII

Eick er a/. (1985) Franchini et d.(1983) Schwab e! a/. (1983) Cooper er al. (1984) Jansen er al. (1984) Curran ef a/. (1 983) Xu el a/. (1984) Semba el al. (1985) Chang el al. (1982) Shih & Weinberg (1982) Shimizu et a/. (1983) Parker et a/. (1985) Wolf ef a/. (1985) Dalla-Favera et a/. (1982) Moos & Gallwitz I (1983)

p.53

c-sis Actin

Oligonucleotides

Oligomers (20-mers) for priming and hybridization were synthesized by the solidphase triester method (Janssen et a f . , 1987a). For hybridization analyses, oligimers were end-labelled to a high ( > 2 X 10" c.p.m. pmol-') specific activity using an

Cellular oncogenes 381 a(”P]dATP (New England Nuclear, Boston. U.S.A.) and T4-polynucleotide kinase (Pharmacia, Freiburg, FRG) and separated from unincorporated dATP on Sephadex G50. Polymerase chain reaction Genomic DNA (150 ng) was incubated in the presence of Taq polymerase (Biolabs, Boston, U.S.A.) with 100 ng of each amplimer complementary to sequences upstream and downstream of the codon to be screened. The reaction mixture consisted of 1 mM of each dNTP, 10 mM Tris HCI pH 7.5, 5 mM NaCl, and 10 mM MgClz in a total volume of 30 pl. The reaction was carried out according to the recommendations of the manufacturer. Fifteen cycles of amplification using an outer set of amplifiers was followed by 15 cycles with an inner set (Janssen et al., 1987b). Oligomer hybridization Amplified DNA ( 5 ng) was spotted onto nylon filters (Gene Screen Plus, New England Nuclear, Bad Homburg, FRG) and fixed by UV illumination. The DNA was pre-hybridized overnight at 50°C in 5 x SSPE (1X SSPE = 10 mM Naphosphate p H 7.0, 0.18 M NaCl and 1 mM EDTA), 7% SDS, sonicated denatured salmon sperm DNA (100 pg/ml), and 5 x Denhard’s solution and hybridized for 3 h at 50°C in the presence of 1 ng of ”P-labelled oligomer probe. The oligomer panel included probes specific for the wild-type allele and all possible amino acid substitutions at codon 12, 13, and 61 of N-ras, Ki-ras, and H a m s (Janssen et al., 1987a,b). (A complete list is available from the corresponding author on request.) The filters were then washed twice in 2 x SSPE and 0.1% SDS for 5 min at room temperature and once in 5 x SSPE and 0.1% SDS for 30 rnin at 50°C. A final high stringency wash was performed for 10 min at 50°C for N-ras and Ki-ras 61 probes; at 63°C for N-ras 12/13, and H a m s 61 probes, and at 72°C for Ha-ras 12/13 probes. Filters were exposed to Kodak XAR film for 12 h at -70°C using intensifying screens.

Results No mRNA specific for c-sis, c-src2, c-met, c-srcl, c-erbB1, c-erbB2, or c-myb could be detected in any of the germ cell tumour lines. On the other hand, normal-size (2.8 kb) mRNA specific for the nuclear oncogene p53 was detected in similar amounts in all the lines. Similarly, all lines expressed c-rafl transcripts of the expected 3.6 kb size. Within the ras gene family, expression of the 1.4 kb c-Ha-rasl and the 4.6 kb c-Ki-ras2 genes, but not of the N-ras gene, was detected in all lines (Fig. 1; Table 3). Expression of the 2.2-kb c-fos mRNA was found in all lines, most abundantly in H12.1, and, to a lesser extent, in human placenta. To determine whether expression of c-fos was an artefact of culture conditions, cells of lines H12.1, H23.1, and 2102ER were injected subcutaneously into nude mice, and RNA from the resulting tumours was extracted and analysed. The tumours derived from H12.1 and H23.1 expressed c-fos in amounts similar to those found in these lines in culture, whereas in the 2102ER tumours, only a small amount could be detected after long exposure (data not shown).

382 H. Tesch et al. L

28s28s 18s18s -

c - fos

N- myc

c- K i - r a s 2

c- m y c

18s -

Fig. 1. Expression of N - m y , c-myc. c-Ki-rus2 and c-Jbs in human germ cell tuinour lines. Total cellular RNA (10 big) from the given cell lines were separated o n 0.8% formaldehyde-containing agarose gels, transferred t o nitrocellulose membranes and hybridized with the indicated labelled probes. The size of the 28 and 18s ribosomal RNAs are given. The size of N-myc, c-Ki-rus 2 and c-myc specific bands are 2102 E R T, H12. I T and H23.1 T RNAs were derived from tumours in nude mice of indicated by 0). the given lines.

Table 3. Expression of cellular oncogenes in human germ cell tumour lines Cell line

H12.1 H12.S H12.7 H23.1 577MR 1 1560 2102ER 2 102EP I428A Placenta

p53

c-fos

+

+ + (+) + + + + + +

ND

ND

+ + + + + + + -

~

~~~

c-rufl

+ + + + + + + +

ND

ND

c-myc

N-myc

c-Ki-rus2

+ (+) +

ND

+ + + + + + + +

ND

ND

ND

+

+

ND

(+I

-

(+I

-

-t

+ -

+

+ + +

c-Ha-rasl

ND

~

- , n o expression. even after long exposure time (3 weeks); (+), low expression. faint bands after long exposure (3 weeks); +. expression detected after 4-7 days. Negative: c-sis, c-src2. c-srcl, c-mel, c-erbB1. c-erbB2, N-rus, c-myb.

Cellular oncogenes 383 Expression of the c-myc gene was found in lines H12.1, H12.7, 2102ER, 2102EP, and 1428A but not in H23.1, 577MR, or 1156Q. c-myc RNA could be detected in xenografts of 2102 E R cells but not H12.1 cells in nude mice. On the other hand, a second gene of the family, N-myc, was detected regularly in various amounts, with 1156Q cells expressing the most and H12.5 cells the least (Fig. 1). Since the over-expression of certain oncogenes could be the result of gene amplifications, which had been described in the case of N-myc in human neuroblastoma, we investigated whether the high expression of the c-Ki-rus2, N-myc, and c-fos genes in the cell lines was attributable to amplifications or structural aberrations. EcoRI-digested DNA from the tumour lines digested with EcoRl were hybridized sequentially with the cellular oncogenes and the single copy gene bcr, and the intensity of the labelled fragments was compared by densitometry. There was no indication of amplification or rearrangement of the c-Ki-rus2, N-myc, and c-fos genes in any of these lines (Fig. 2; other data not shown).

2.0

-

18.0 -lp

-

L

bcr

Fig. 2. Organization of N-myc and c-Ki-rus2 genes in human germ cell turnour lines. High molecular weight DNAs of the given cell lines were cut with EcoRI. The digested fragments were separated on 0.8% agarose gels, transferred to nitrocellulose filters and hybridized sequentially to N-myc, c-Ki-rus2 and bcr specific probes. The size of the respective restriction fragments is indicated. The intensity of the labelled fragment was scanned by densitometry. Amplification was calculated as the ratio of the relative densitometry units of the cellular oncogene to the single copy gene bcr.

384 H . Tesch et al. Using the polymerase chain reaction technique, we screened lines H12.1, H23.1. 577MR, 1156Q, and 2102EP for the presence of point mutations at codons 12, 13, and 61 of Ha-rus, Ki-rus. or N-ras. None of these lines contained such structural alterations (for an example of the results, see Fig. 3).

codonl2

N-ras

-GGT-

Illy -GAT-

a=P

1

mut

Fig. 3. Hybridization of N-rus specific oligorners to in-vitro amplified DNAs obtained from six teratorna cell lines. DNAs (5 ng) were spotted and hybridized to oligorners representing wild type (wt) and a mutation-specific sequence of N-rus ar codon 12.

Discussion In addition to growth factors and their receptors, proto-oncogenes may regulate the proliferation and differentiation of normal and malignant cells. There are several lines of evidence which suggest that the activation of certain cellular oncogenes is involved in the transformation of human tumours (Bishop, 1987). We have therefore analysed the expression of a panel of 14 cellular oncogenes in human germ cell tumour lines by Northern blot experiments to detect both the amount and size of the respective transcripts. Our data indicate that p53-, c-rufl-, c-Ha-rasl-, c-Ki-rus2, c-fos and N-myc gene transcripts of the normal size are expressed in similar amounts in all germ cell tumour lines analysed. Two transforming mechanisms have been postulated for the viral analogues of the c-rus genes: over-production of the normal gene product and structural mutations that could lead either to inappropriate regulation of the normal function (such as constitutive interaction with the effector target even in the absence of an environmental signal), or inability to carry out a normal p21 function (Lowy & Willumsen, 1986). Point mutations are relatively common in malignant human tumours, with ‘hot spots’ at amino acid residues 12, 13, 59, 61, and 63, all of which are within the catalytic domain of the protein. However, only a minority of turnours of a given histological type have activated r m genes (Lowy & Willurnsen, 1986); tumours of similar histological type have as much as a 10-fold difference in the levels of particular c-ras products, and the same type of tumour from different patients may have different rus genes activated (Fiorucci & Hall, 1988). Interestingly, the c-Ki-rus2 gene is encoded on chromosome 12 in humans and all germ cell tumour lines analysed here have an isochromosome 12. We have analysed, by the polymerase chain extension method, whether mutations of codon 12, 13 and 61 of the Ha-ras, Ki-ras, and N-rus genes occurred in the germ cell tumour lines. Our data do not show any aberration of these genes in the germ cell tumour lines analysed. Tainsky et al. (1984) observed a G-A mutation at codon 12 of N-rus in a late but not in an early passage of human PA1

Cellular oncogenes 385 teratocarcinoma cells, which indicates that the mutation was acquired during cell culture. In the human teratoma lines TeraI and TeraII the c-Ki-ras gene is amplified without point mutations at codon 12 (Tobaly-Tapiero et al., 1986). High expression of c-fos mRNA was detected in all germ cell tumour lines and in human placenta. The c-fos oncogene, an analogue of a v-onc found in two mouse sarcoma viruses, encodes a 55 kD nuclear protein that undergoes considerable post-translational modification, including phosphorylation and association with a smaller protein, p39. While c-fos expression is usually very low but transiently inducible by growth factors, other cells (e.g. amnion cells, yolk sac, fetal liver, postnatal bone-marrow, differentiated macrophages) exhibit an apparently constitutive expression in vivo (Sassone-Corsi & Verma, 1987). It has been suggested recently that the expression of c-fos in embryonic cells might be due to exogeneous factors derived from placenta- o r embryo-conditioned medium (Miiller et al., 1986). The gene is also expressed in several human tumours, including gastrointestinal cancer, breast, lung and kidney tumours (Slamon et a f . , 1984). Low levels of c-myc mRNA were detected in some but not all germ cell tumour lines, whereas high expression of c-myc can be detected in a variety of other human tumour cell lines (Little et al., 1983). In mouse Yeratocarcinoma-derived stem cells there is a temporal inverse correlation between the expression of c-myc and that of genes encoding markers of terminal differeptiation. The amount of c-myc transcripts in proliferating nullipotent F9 stem cells in 25-fold higher than in non-tumourigenic 3T3 cells (Griep & De Luca, 1986) and higher than that in proliferating pluripotent PCC3 stem cells (Sejersen et al., 1985). A second gene of the myc gene family, N-myc, is expressed readily at various levels in all germ cell tumour lines tested. The line 1156Q carries a homogeneous staining region (HSR) on chromosome 1, where the N-myc gene is encoded. However, we were unable to demonstrate amplification of N-myc by Southern blot. The N-myc gene is amplified up to 300-fold in almost half of advanced, but not early, human neuroblastomas, with a corresponding increase in the extent of expression (Brodeur et al., 1984; Schwab et al., 1984). Moreover, when cultured neuroblastoma cells are induced to differentiate, the expression of N-myc decreases dramatically. Jakobovits et al. (1985) also found abundant expression of N-myc in two lines of mouse teratocarcinoma stem cells, PSA-1 and F9, and a human teratocarcinomaderived line, NTera-2 clone D1. However, the gene was not amplified in any of these lines. When the two lines of mouse cells were induced to differentiate into endoderm, the amount of N-myc mRNA declined. It appeared from these data that N-myc expression in the stem cell lines was a manifestation of their embryonic nature rather than of their tumourigenicity as mouse embryos continued to express N-myc mRNA in considerable amounts until after day 11.5 of gestation, after which expression declined. What is the significance of the over-expression of cellular oncogenes in human tumour cells? To date we do not know whether over-expression of cellular genes is a cause or effect of the malignant transformation. As it is generally believed that a single event is not sufficient to transform a normal cell to a tumour cell fully (Land et al., 1983), it is possible that the altered expression or deregulation of genes which

386 H . Tesch et al. are important for proliferation and/or differentiation is one of several events that can contribute to the conversion of a normal cell to the malignant phenotype. It is not clear yet whether low or enhanced levels of an oncogene can contribute directly to tumourigenesis or simply reflect a certain stage of differentiation of the cells. Indeed, it has been shown recently that the differentiation of teratocarcinoma cells into neurones induced by retinoic acid leads to altered expression of pp60 C-SYC protein (Lynch et af., 1986). In-situ hybridization experiments on primary human germ cell tumours should be performed to analyse this question at the single cell level. Further analyses are required to determine the function of the expressed proteins and the significance of over-expression of a particular gene. Acknowledgments The authors thank Drs D . Eick, F. Wong-Staal, M. Schwab, M. Dean, K. Bister, T . Curran, I. Pastan, T . Yamamoto, K. Willecke, R. Gallo, R. Weinberg, V. Rotter, D . Gallwitz for gifts of DNA probes, and R . Hippler-Altenburg, B. Feldmeier and I. Pahl for expert technical help. This work was supported by the Ministeriurn fur Wissenschaft und Forschung des Landes Nordrhein Westfalen and by the Deutsche Forschungsgemeinschaft. References Alitalo, K., Winqvist. R.. Lin. C. C . , D e La Chapelle. A , , Schwab, M. & Bishop, J. M. (1984) Aberrant expression of an amplified c-my6 oncogene in two cell lines from a colon carcinoma. Proceedings of the Naiional Academy of Sciences. U.S.A. 81, 4534-4538. Atkin. N. B. & Baker, M. C. (1983) i (12p): Specific chromosomal marker in the seminoma and malignant teratoma of the testis. Cancer Genetics and Cyiogeneiics, 10, 199-204. Bishop, J. M. (1987) The molecular genetics of cancer. Science, 235. 305-311. Brinster, R. L. (1974) Embryo development. Journal of Animal Sciences, 38, 1003- 1012. Brodeur, G . M.. Seeger, R. C.. Schwab. M.. Varmus, H. E . & Bishop, J. M. (1984) Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science. 224, 1121-1124. Bronson. D. L.. Andrews, P. W.. Solter. D.. Cervenka. J.. Lange. P. H. & Fraley, E . E. (1980) Cell line derived from a metastasis of a human testicular germ cell tumor. Cancer Research, 40. 2500- 2506. Bronson. D . L., Bronson. J . G . & Fraley, E . E . (1983) Germ cell tumors of mice and men: the teratocarcinoma models and their clinical implications. In: Testi.s Tumors, pp. 77-91. Williams & Wilkins, Baltimore. Casper, J . . Schmoll, H. J.. Schnaidt, U . & Fonatsch, C . (1987) Cell lines of germinal cancer. Infernational Journal of Andrology, 10. 105- 114. Chang, E . H . , Gonda, M. A , . Ellis, R . W., Scolnick. E. M. & Lowy, D. R. (1982) Human genome contains four genes homologous to transforming genes of Harvey and Kirsten murine sarcoma viruses. Proceedings of the National Academy of Sciences, U . S . A . 79,4848-4852. Cooper, C., Blair, D . . Oskarsson. M.. Tainsky, M.. Eader, L. & Vande Woude, G . (1984) Characterization of human transforming genes from chemically transformed, teratocarcinoma, and pancreatic carcinoma cell lines. Cancer Research, 44, 1- 10. Cotte. C. A.. Easty. G . C. & Neville, A . M. (1981) Establishment and properties of human germ cell tumors in tissue culture. Cancer Research, 41, 1422- 1427. Cotte. C . , Raghavan, D . , McIlhinney, R . A . J . & Monaghan, P. (1982) Characterization of a new human cell line derived from a xenografted embryonal carcinoma. In V i m . 18, 739-749. Curran, T., MacConnell, W. P., van Straaten, F. & Verma, I . M. (1983) Structure of the FBJ murine oseosarcoma virus genome: molecular cloning of its associated helper virus and the cellular homolog of the v-fos gene from mouse and human cells. M o k u l a r and Cellular Biology, 3, 914-921. Dalla-Favera, R., Gelmann, E. P.. Gallo. R. C . & Wong-Staal, F. A . (1981) Human onc gene homologous to the transforming gene (v-sis) of simian sarcoma virus. Nature, 292, 31 -35.

Cellular oncogenes

387

Dalla-Favcra, R.. Wong-Staal. F. B Gallo, R. C . (1982) Onc gene amplification in promyclocytic leukaemia cell line hL-60 and primary leukaemic cells of the same patient. Nature, 299, 61-63.

Damjanov, I. (1980) Testicular germ cell tumors as a model of carcinogenesis and embryogenesis. In: Principles and Managernrnt (if Testicular Cancer (ed. N. Javadpour). pp. 73-87. Thieme, New York. Delozier-Blanchet. C. D.. Engel. E. & Walt. H . (1985) Isochromosome 12p in malignant testicular tumors. Cuncer GencJrics und Cytogenetics, 15. 375-376. Denhardt. D . T. (1966) A membrane-filter technique for the detection of complementary DNA. Biochemicul arid Biuphysicol Research Commimicarion.~.23. 64 1-646. Eick. D.. Piechaczyk, M., Henglein. B., Blanchard. J. M.. Traub, B.. Kofler. E., Wiest. S., Lcnoir, G. & Bornkamm. G. W . (1985) Aberrant c-myc RNAs of Burkitt’s lymphoma cells have longer half-lives. E M B O Journal, 4. 3717-3725. Feinherg, A . P. B Vogclstein. B. (1983) A technique for radiolabeling D N A restriction endonuclcase fragments to high specific activity. Analyrical Biochemistry, 132. 6- 13. Fiorucci, G. & Hall. A . (1988) All three human ras genes are expressed in ii wide range of tissues. Biuchimica, Biophysicu Aclu. 950, 8 1-83. Fough. J. & l’rempe, G. (1975) New human tumor cell lines. In: tfuman Tumor Cell Lines In Virro (ed. J . Fough), pp. 1 15- 159. Plenum Press. New York. Franchini. G.. Wong-Staal, F.. Baluda. M. A , . Lengel. C. B Tronick, S. R . (1983) Structural organization and expression of human D N A sequences related to the transforming gene o f avian myeloblastosis virus. Proceedings of the National Academy of Sciences, U . S . A . 80, 7385-7389. Griep. A . E . B D e Luca, H. F. (1986) Decreased c-myc expression is an early event in retinoic acid-induced differentiation of F9 tcratocarcinoma cells. Proceedings (if rhe National Academy of Sciences. U . S . A . 83. 5539-5543. Hogan. B . , Fellous. M., Avner. P. B Jacob. F. (1977) Isolation o f a human terato cell line which expresses F9 antigen. Nuture. 270. 515-518. Jakobovits, A., Schwah, M., Bishop. J . M. & Martin. G. R . (1985) Expression of N-myc in teratocarcinoma stem cells and mousc embryos. Nature. 318. l8X-191. Jansen. H. W., Lurz. R . , Bister, K., Bonner, T. I.. Mark, G. E. & Rapp, U . R. (1984) Homologous cell-derived oncogenes in avian carcinoma virus MH2 and murine sarcoma virus 361 I.Nulure, 307. 281 -284. Janssen, J. W. G . , Lyons, J . , Steenvoorden, A . C. M., Seliger. H . & Bartram, C. R. (1987a) Concurrent mutations in two different ras genes in acute myelocytic leukemias. Nucleic Acids Rrseurch, 14, 5669-5680. Janssen. J . W . G.. Steenvoorden. A . C. M., Lyons, J . , Anger. B.. Biihlke. J. U.. Bos, J. L.. Seliger. H . & Bartram, C . R . (1987b) Ras gene mutations in acute and chronic niyelocytic leukemias. chronic myeloproliferative disorders. and myelodysplastic syndromes. Proceedings of the Na~ionalAcademy of Sciences, U . S . A . 84, 9228-9232. Land, H.. Parada, L. F. B Weinberg. R . A. (1983) Tumorigenic conversion o f primary embryo fibroblasts requires at least two cooperating oncogenes. Nature, 304. 596-602. Lee, W . H.. Murphree, A . L. & Bcnedict, W. F., Expression and amplification of the N-mvc gene in primary retinoblastoma. Nature. 309. 458-460. Little, C . D . , Nau, M. M.. Carney. D. N., Gazdar. A . F. & Minna, J. D . (1983) Amplification and expression of the c-myc oncogene in human lung cancer cell lines. Nature, 306. 194- 195. Lowy, D. R. & Willumsen, B. M . (1986) The ras gene family. Cancer Surveys, 5 , 275-289. Lynch. S . A . . Brugge, J . S. B Levine, J. M. (1986) Induction of altered c-src product during neural differentiation of embryonal carcinoma cells. Science. 234, 873-876. Maniatis, T . , Fritsch. E. F. B Sambrook, J. (1982) Molecular Cloning. Cold Spring Harbour Laboratory, New York, U.S.A. Mintz. B. (1974) Gcne control of mammalian differentiation. Annual Review sf Genrtics, 8. 41 1-470. Moos, M. & Gallwitz, D . (1983) Structure of two human P-actin-related processed genes one of which is located next to a simple repetitive sequence. E M B O Journal, 2, 757-761. Miiller. R . , Miiller, D., Verrier. B . , Bravo. R . B Herhst. H. (19x6) Evidence that expression of c-fos protein in amnion cells is regulated by external signals. E M B O Journal, 5 . 311-316. Parker, R. C., Mardon, G.. Lebo, R. V . , Varmus. H . E . & Bishop, J. M. (1985) Isolation of duplicated human c-src genes located on chromosomes 1 and 20. Molecular and Cellular Biology, 5 . 831-838.

388 H . Tesch et al. Reddy. E . P., Reynolds, R . K.. Santos. E . & Barbacid, M . (1982) A point mutation is responsible for the acquisition of transforming properties by the T24 human bladdar carcinoma oncogene. Nuture. 300, 149- 152. Santos. E . . Martin-Zanca. D . . Reddy, E. P . , Pierotti. M. A.. Della-Porta, G . & Barbacid, M. (1984) Malignant activation of a K-rus oncogene in lung carcinoma hut not in normal tissue of the same patient. Science. 223. 661-664. Sassone-Corsi. P. & Verma, I . M. (1987) Modulation of c-fcis gene transcription by negative and positive cellular factors. Nature, 326, 507-5 10. Schwah. M., Alitalo, K., Klempnauer. K. H., Varmus. H. E.. Bishop, J . M.. Gilbert, F., Brodeur, G., Goldstein. M. & Trent, J . (1983) Amplified DNA with limited homology to myc cellular oncogene shared by human neuroblastoma cell lines and a ncuroblastoma tumour. Nature, 305. 245-248. Schwab. M., Ellison, J . , Busch. M.. Rosenau, W . , Varmus. H. E. & Bishop. J . M. (1984) Enhanced expression of the human gene N-myc consequent to amplification of D N A may contribute to malignant progression of neuroblastoma. Proceedings of the Narionul Academy of Sciences, U.S.A. 81, 4940-4944. Sejersen, T.. Sumegi. J . & Ringerts. N. R. (1985) Expression of cellular oncogenes in teratoma-derived cell lines. Experinientul CelI Research. 160. 19-30. Semba, K.. Kamata, N., Toyoshima. K. & Yamamoto. T. (1985) A v-erbB-related proto-oncogene, c-erh-2, is distinct from the c-erbB-llepidermal growth factor-receptor gene and is amplified in a human salivary gland adenocarcinoma. Proceedings of the National Academy of Sciences, U.S.A. 81, 6497-6501. Shih. C. & Weinberg. R. (1982) Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell. 29, 161-169. Shiniizu. K., Goldfarb. M.. Perucho. M. & Wigler. M. (1983) lsolation and preliminary characterization of the transforming gene of a human neuroblastoma cell line. Proceedings of fhe National Acatlerny qf Sciences, U.S.A., 80, 383-387. Slamon. D . J . . DeKernion. J . B . , Verma. I . M. &Cline, M. J . (1984) Expre5sion of cellular oncogenes in human malignancies. Science, 224. 256-262. Sporn. M. B. & Roberts. A . B. (1985) Autocrine growth factors and cancer. Ntmire. 313. 745-747. Sweet, R. W.. Yokoyama, S.. Kamata. T.. Reramisco. J. R.. Rosenherg. M. & Gross. M. (1984) The product of rus is a GTPase and the T24 oncogenic mutant is deficient in this activity. Nature, 311, 273-275. Tabin, C . J . . Bradley, S. M.. Bargmann. C. I . . Weinberg. R. A.. Papageorge. A. G . . Scolnick. E. M.. Dhar. R., Lowy, D . R. & Chang. E. H. (1982) Mechanism o f activation of a human oncogene. Nurure, 300. 143- 149. Tainsky. M. A.. Cooper, C . S . , Giovanella, B. C. & Vande Woude, G . F. (1984) An activated rasN gene: detected in late but not early passage human PA1 teratocarcinoma cells. Science. 225. 643-645. Tobaly-Tapiero. J.. Saal, F.. Peries, J. & Emanoil-Ravier, R. (1986) Amplification and rearrangement of Ki-rus oncogene in human teratocarcinoma-derived cell lines. Riochiniie, 68. 1019- 1023. Vousden. K. H.. Bos. J . L.. Marshall, C. J. & Phillips. D. H. (1986) Mutations activating human c-Ha-rusl protooncogene (HRASI) induced by chemical carcinogens and depurination. Proceedirigr of the National Academy of Sciences, U.S.A. 83, 1222- 1226. Wang. N.. Trend. B., Bronson, D. L. & Fraley, E. E. (1980) Nonrandom abnormalities in chromosome 1 in human tcsticular cancers. Cancer Research. 40. 796-802. Wolf. D . . Laver-Rudich, Z . & Rotter. V. (1985) In vitro expression o f human pS3 cDNA clones and characterization of the cloned human p53 gene. Molecltlur und Cellular Biology, 5 . 1887-1893. Xu. Y . . Ishii. S . . Clark. A. J . L.. Sullivan, M., Wilson. R . K.. Ma, D . P., Roe. B. A.. Merlino. G. T. & Pastan, I. (1984) Human cpidermal growth factor receptor cDNA i h homologous to a varietv of RNAs overproduced in A431 carcinoma cells. Nulure. 309. 806-810. Yokota. J . . Tsunetsugu-Yokota. Y.. Battifora. H . , Le Fevre. C. & Cline. M. J . (1986) Alterations of myc. rnyh and ras-Ha proto-oncogenes in cancers are frequent and show clinical correlation. Science, 231. 261-265.

Received 30 October 1989; accepted 6 February 1990