targets through which this growth phenomenon may be regulated. We would like to express our appreciation to Dr. Robert Weinberg for the pEJ6.6 plasmid and ...
Proc. Nati. Acad. Sci. USA Vol. 85, pp. 3875-3879, June 1988 Cell Biology
Characterization of mutagen-activated cellular oncogenes that confer anchorage independence to human fibroblasts and tumorigenicity to NIH 3T3 cells: Sequence analysis of an enzymatically amplified mutant HRAS allele CRAIG W. STEVENS, T. HERBERT MANOHARAN,
AND
WILLIAM E. FAHL*
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706
Communicated by Van R. Potter, February 24, 1988
induced anchorage independence is likely to represent one of these discrete steps, where this acquired phenotype is related mechanistically to the final tumorigenic state. Our interest here was to determine whether mutageninduced soft agar growth could be assigned to an activating mutation at a specific human gene. We found that mutagen treatment induced soft agar growth in human fibroblasts and that cellular DNA from the majority of the anchorageindependent clones could also tumorigenically transform NIH 3T3 cells. In two clones, an activated human HRAS oncogene was identified in the NIH 3T3 tumor cells; in addition, in the majority of the anchorage-independent human clones, an as yet to be identified gene or genes was responsible for the observed human cell anchorage independence and associated NIH 3T3 cell transformation.
ABSTRACT Treatment of diploid human fibroblasts with an alkylating mutagen has been shown to induce stable, anchorage-independent cell populations at frequencies (11 X 10-4) consistent with an activating mutation. After treatment of human foreskin fibroblasts with the mutagen benzo[alpyrene (±)anti-7,8-dihydrodiol 9,10-epoxide and selection in soft agar, 17 anchorage-independent clones were isolated and expanded, and their cellular DNA was used to cotransfect NIH 3T3 cells along with pSV2neo. DNA from 11 of the 17 clones induced multiple NIH 3T3 cell tumors in recipient nude mice. Southern blot analyses showed the presence of human Alu repetitive sequences in all of the NIH 3T3 tumor cell DNAs. Intact, human HRAS sequences were observed in 2 of the 11 tumor groups, whereas no hybridization was detected when human KRAS or NRAS probes were used. Slow-migrating ras p21 proteins, consistent with codon 12 mutations, were observed in the same two NIH 3T3 tumor cell groups that contained the human HRAS bands. Genomic DNA from one of these two human anchorage-independent cell populations (clone 21A) was used to enzymatically amplify a portion of exon 1 of the HRAS gene. Direct sequence analysis of the amplified DNA indicated equal presence of a wild-type (GGC) and mutant (GTC) allele of the HRAS gene. The results demonstrate that exposure of normal human cells to a common environmental mutagen yields HRAS GC -+ TA codon 12 transversions that have been commonly observed in human tumors. This oncogene as well as an as yet to be identified oncogene are also shown to stably confer anchorageindependence to human cells.
MATERIALS AND METHODS Cells, Mutagen Treatment, Soft Agar Cloning. A diploid human foreskin fibroblast culture (25sk) was initiated in our laboratory as described (19), and these cells (passage 2-3, 2 x 106 cells per group, 11 replicate groups) were treated with an LD70 (0.24 ,uM) of (±+)7f3,8a-dihydroxy-9a,10a-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene (anti-diol-epoxide) or solvent control as described (19). Anchorage-independent clonal cell populations were selected in agar as described in detail elsewhere (20). Anchorage-independent colonies (162 colonies; diameter, >75 ,um) were picked, and 17 of these clonal populations were expanded and used for genomic DNA isolation. NIH 3T3 Transfections and Nude Mouse Injections. DNA transfection was done by the calcium phosphate coprecipitation method as described (21). NIH 3T3 cells (7.5 x 10' cells per 100-mm dish) were exposed to the calcium phosphate-DNA coprecipitate (40 p.g of genomic DNA plus 3 ,ug of pSV2neo per dish) for 4 hr. Two days later, each dish was trypsinized and reseeded into a 175-cm2 flask. For the next 10 days, cultures were selected in G418 (400 jug/ml), and the flasks were then trypsinized and cells were replated in the same flask to disperse the G418-resistant colonies into a diffuse lawn ofcells. Two days later, the cells were harvested and washed with serum-free medium prior to injection. One injection of 5 x 106 cells into the right flank and one injection of 1 x 107 cells into the left flank, each in a volume of 200 Al, were done on each nude mouse. Injection sites were monitored at 3- or 4-day intervals for 100 days. Southern Blot Analyses. DNAs were digested with restriction enzymes, separated by agarose gel electrophoresis, transferred to nitrocellulose filters (22), and hybridized with
The majority of human tumor cells, and particularly those of mesenchymal origin, have the ability to grow in semisolid medium (i.e., anchorage-independent growth; refs. 1-5), a phenotype that is rarely observed in nonneoplastic human cells. Treatment of rodent or human fibroblasts with mutagens has been shown to induce anchorage-independent growth at frequencies consistent with mutation at a single gene locus (6-8), or perhaps pool of loci, any locus of which is permissive in its mutant form. Mutagen-induced acquisition of anchorage-independent growth in many aneuploid immortal rodent cell lines has been shown to correlate well with acquisition of tumorigenic growth (7, 9-11), whereas in diploid primary human fibroblasts, mutagen treatment has been shown to induce anchorage-independent cell populations (8, 12-14) that do not yield progressively growing tumors (15-17), and, consistent with this, the anchorageindependent cell clones senesce at passage levels similar to non-anchorage-independent cell clones (17). When neoplastic transformation is viewed in the context of a multiple-step transition from normal to neoplastic cell (18), mutagen-
Abbreviations: anti-diol-epoxide, (±+)7P,8a-dihydroxy-9a,10aepoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; LD70, a dose lethal to 70o of treated cells. *To 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.
3875
3876
Cell Biology: Stevens et al.
nick-translated 32P-labeled probes. The human HRAS probe 0.6-kilobase (kb) Sma I fragment isolated from the pEJ6.6 plasmid; the human KRAS probe was a 1.1-kb Pst I/BamHI fragment isolated from the KRAS2 plasmid (Oncor, Gaithersburg, MD); the human NRAS probe was purchased from Oncor. The BLUR-8 plasmid, which contains a human Alu repetitive sequence insert (23), was obtained from W. R. Jelinek (Rockefeller University). DNA Amplification. Amplification of DNA was done by the described procedure (24). Briefly, a 50-plI reaction containing 1 ,g of genomic DNA; 10% dimethylsulfoxide; 2 ,ug of each primer; 33 ,uM each dATP, dCTP, dGTP, and dTTP; 67 mM Tris-HCI (pH 8.8); 16.6 mM ammonium sulfate; 6.7 mM MgCl2; 10 mM 2-mercaptoethanol; 6.7 ,uM EDTA was incubated at 95°C for 7 min. After allowing 5 min at 60°C to anneal the primers, 1 unit of DNA polymerase from Thermus aquaticus (New England Biolabs) was added and the primers were extended at 70°C for 5 min. Subsequently, denaturation was done at 93°C for 1.5 min, annealing was at 60°C for 2 min, and polymerization was at 70°C for 2 min. After 10 cycles, an additional unit of DNA polymerase was added. After 25 cycles, primers (2 ug each) and dNTPs (33 uM each) were added. Typically, 40 cycles were done, and the desired product [144-base-pair (bp) fragment] was purified on a 3% agarose (Nusieve) or 5% polyacrylamide gel. The fragment was a
Proc. Natl. Acad. Sci. USA 85 (1988) end-labeled and sequenced by Maxam and Gilbert reactions (25).
was 32P
RESULTS Anti-Diol-Epoxide-Induced Anchorage Independence in Dip-
loid Human Fibroblasts. Eleven replicate groups of 25sk diploid human fibroblasts were treated with a LD70 (dose lethal to 70% of treated cells) (0.24 ttM) of anti-diol-epoxide, a dose that induces 6-thioguanine-resistant mutants at a measurable frequency ( 5 x 10-4) in these same cells (19, 20). The surviving cells in the treatment groups were independently carried and seeded into soft agar to select anti-diolepoxide-induced anchorage-independent cell populations. After 4 weeks growth, all colonies >75 Aum in diameter (range, 75-140 ,um) were scored. When corrected for plating efficiencies and the original anti-diol-expoxide-induced cytotoxicity, the agar colony frequency was 113 colonies per 105 normalized viable cells (11 x 10-4) in the mutagentreated groups, which is 22-fold higher than background (20). A large number of agar colonies (162 colonies) >75 ,m in diameter were individually picked from the 11 treatment groups with a sterile micropipette and micromanipulator and then were individually expanded. Of these, 17 colonies that grew well were expanded to the level of 200-300 x 106 cells.
Table 1. Induction of NIH 3T3 cell tumors after transfection of NIH 3T3 cells with genomic DNA from anti-diol-epoxide-induced anchorage-independent human fibroblasts and partial characterization of the transforming genes Oncogene aberrations detected in DNARNA from original Days postinjection human anchorage-independent Mutant codon in Detection of human when average coloniest human RAS tumor area RAS genes in NIH 3T3 Tumors/injection gene* reached 100 mm2 tumor cells Amplification Overexpression sites Donor DNA Anchorage-independent colonies 19K 0/6 48 19L 2/4 + (HRAS) 12 35 5/5 20H 74 20I 1/6 12 97 + (HRAS) 21A 2/6 21F 0/3 -22A 0/6 74 23G 1/6 55 24K 1/6 25D 0/6 25L 0/6 67 26C 1/6 74 26F 2/6 47 28K 5/6 28M 0/2 97 29A 1/2 60 29L 3/6 Negative/positive controls 25sk 0/8 NT 12 NT + (HRAS) 9 6/6 pEJ6.6 NT NT 19 pSM-1 6/6 12 NT + (HRAS) NT 25 T24 6/6 NT NT + (NRAS) 61 28 HT1080 5/6 NIH 3T3 cells were transfected with genomic DNA isolated from anchorage-independent human fibroblast clones (19K-29L, column 1) along with the selectable marker pSV2neo (see Materials and Methods). After selection in G418, all drug-resistant colonies in each transfection group (typically 2500-4000 G418-resistant colonies per group) were pooled and injected (typically 5 x 106 cells per site, three flank sites; 10 x 106 cells per site, three flank sites) into nude mice. The negative tumor control involved transfection of NIH 3T3 cells with DNA from the untreated human fibroblasts (25sk) along with pSV2neo, G418 selection, and injection; no tumors were observed through 100 days of observation. Positive tumor controls consisted of pSV2neo cotransfection of NIH 3T3 cells with either plasmid DNA [pEJ6.6, HRAS codon 12 mutation (26); pSM-1, overexpressed human SIS gene (27)] or cellular DNA (T24, HRAS codon 12 mutation; HT1080, NRAS codon 61 mutation) that contained known oncogenes. *Based on p21 electrophoretic analysis (data not shown). tThese results are summarized from Stevens et al. (20); DNA and RNA from human anchorage-independent colonies (column 1) were subjected to Southern and RNA blot analysis with probes for the following oncogenes: HRAS, KRAS, NRAS, MOS, FOS, FES, ABL, SIS, MYB, ERBA, ERBB, SRC, RAF, NMYC. -, No gene amplification or over-expression events were detected. NT, not tested.
Cell Biology: Stevens et al. The first two digits of the colony name (Table 1) refer to the treatment group number, and the final letter refers to the individual colony derived from that group. Tumorigenic Transformation of NIH 3T3 Cells with Genomic DNA from Anchorage-Independent Human Fibroblasts. DNA from each of the 17 agar colony populations was independently cotransfected with pSV2neo into NIH 3T3 cells. After selection in G418, all of the G418-resistant colonies (>4000 colonies per group; see ref. 28) from each NIH 3T3 transfection group were pooled, and the cells were injected subcutaneously into the flanks of 5-week-old male nude mice. Where possible, one injection of 5 x 106 cells and one injection of 10 x 106 cells was done in opposite flanks of each animal (three animals). As shown in Table 1, injection of cells transfected with either oncogene-containing plasmids or cellular DNA from human tumors produced tumors in nude mice at 23 of 24 injection sites. All of the positive control DNAs produced tumors with latencies shorter than 20 days (Fig. 1). Transfection of DNA from untreated human fibroblasts along with pSV2neo yielded G418-resistant NIH 3T3 cells that failed to produce tumors at 8 of 8 injection sites through 100 days of postinjection observation. The DNAs isolated from anti-diol-epoxide-induced anchorage-independent human cell colonies showed a range of transforming potentials (Fig. 1); the tumor growth rates were nearly the same in all groups, irrespective of the tumor latency or multiplicity. In aggregate, 11 of 17 DNAs from anti-diolepoxide-induced anchorage-independent human cell populations contain transforming sequences that, after transfection into NIH 3T3 cells, induced at least one tumor. The observation of no tumors in the negative control group (at eight of eight injection sites) increases the likelihood that those single tumors observed in some of the experimental DNA groups were in fact induced by the transfected human DNA. Detection of Human RAS Genes in DNA from NIH 3T3 Tumor Cells. To date, most of the oncogenes detected by NIH 3T3 focus or tumorigenicity assays have been members of the RAS gene family. In addition, the mutagen we used, anti-diol-epoxide, has been shown to preferentially induce GC -- TA transversions in the Escherichia coli lacI gene (29), mutations identical to those originally observed in codon 12 of the human EJ/T24 HRAS oncogene (26). Because of these
prior observations, we screened DNAs from each of the NIH 3T3 tumor cell groups with both the BLUR-8 probe to identify the presence of human DNA and also with human RAS family probes to identify human RAS genes that may have induced the observed NIH 3T3 tumors. In Fig. 2B, the expected 6.6-kb human HRAS fragment was observed in the three human DNA controls (25sk, T24, T24/3T3). Two NIH 3T3 tumor DNAs (groups 20H and 21A) containing DNA from anchorage-independent colonies also showed the characteristic human 6.6-kb hybridizing fragment. None of the characteristic human KRAS (Fig. 2C) or NRAS (Fig. 2D) bands were detectable in the NIH 3T3 tumor cell DNA digests. When RAS p21 proteins from the NIH 3T3 tumor
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