Activated oncogenes promote and cooperate with chromosomal instability for neoplastic transformation

Abstract

Most cancer cells are aneuploid. The chromosomal instability hypothesis contends that aneuploidy is the catalyst for transformation, whereas the gene mutation hypothesis asserts that cancer is driven by mutations to proto-oncogenes and tumor-suppressor genes, with the aneuploidy a side effect of tumorigenesis. Because genotoxic stress induced by "culture shock" can obscure the transforming potential of exogenous genes, we cultured wild-type and p53(-/-) mouse embryo fibroblasts in a more physiological (serum-free) environment. Under these conditions, the cells were immortal and, more importantly, chromosomally stable. Expression of oncogenic H-RasV12 did not induce senescence, but sensitized these cells to p53-dependent apoptosis. In addition, H-RasV12 induced chromosomal instability, as well as accumulation and phosphorylation of p53. Significantly, whereas cells grown under standard conditions could be transformed by coexpression of H-RasV12 and E1A, the chromosomally stable cells were refractory to transformation, as measured by anchorage-independent growth and tumor formation in nude mice. These oncogenes required a third genetic alteration that abolished the p53 pathway to create a permissive environment that promotes rapid chromosomal instability and transformation. Oncogene-induced chromosomal instability and transformation was attenuated by antioxidants. These data indicate that chromosomal instability could be a catalyst for oncogenic transformation, and bring together aspects of the chromosomal instability hypothesis and the gene mutation hypothesis for tumorigenesis.

Figure 1.

Wild-type and p53^-/- SF-MEFs are immortal. (A) Representative growth curves of wild-type (WT) or p53^-/- mouse embryo cultures passaged under 3T3 conditions. Mouse embryo fibroblasts were grown in either serum-containing media (MEFs) or in serum-free media (SF-MEFs). Fibroblasts derived from 15-16-d embryos were cultured by passaging 10⁶ cells per 100-mm dish every 3 d as described in Materials and Methods. Some plates of wild-type SF-MEFs were supplemented with 10% FBS at day 39. (B) MEFs enter senescence in serum-containing but not serum-free conditions. Wild-type MEFs and wild-type SF-MEFs at population doubling (PD) 3 and 15 were stained for SA-β-gal (pH 5.5) activity. Photographs are at the same magnification. (C) Increased expression of p53 and p21^CIP1/WAF1 in serum-containing but not serum-free conditions. Immunoblot of cell lysates from wild-type MEFs and wild-type SF-MEFs for p53 and p21^CIP1/WAF1 at the indicated PD. Equal loading of lysates was confirmed by immunoblotting for tubulin. The postcrisis (PC) wild-type MEFs represents a sample taken from cells at ∼15 PD after the cells had bypassed senescence and became spontaneously immortalized.

Figure 2.

Wild-type and p53^-/- SF-MEFs have stable karyotypes. Embryo fibroblasts at the indicated PD were harvested for karyotype analysis. (A) Wild-type MEFs at PD 3. (B) Wild-type MEFs at PD 15. (C) Postcrisis wild-type MEFs at PD 30 (these cells had doubled ∼15 times from the first splitting of a near-confluent flask of spontaneously immortalized cells that escaped senescence). (D) Wild-type SF-MEFs at PD 60. (E) p53^-/- MEFs at PD 3. (F) p53^-/- MEFs at PD 30. (G) p53^-/- SF-MEFs at PD 60. (H) p53^-/- SF-MEFs at PD 60 were supplemented with 10% FBS for 48 h and then prepared for metaphase spreads as outlined in Materials and Methods. Approximately 50 metaphases from two representative cultures were counted in each case. The exception was the wild-type MEFs at PD 15, where a total of 54 metaphases were counted from two representative cultures. These cells were approaching senescence, which limited the number of available metaphases.

Figure 3.

Oncogenic Ras fails to induce senescence in wild-type SF-MEFs. Wild-type MEFs and wild-type SF-MEFs at PD 3 were transduced with H-RasV12 or empty viral vector. Day 0 represents the point when infected cell populations were split for the first time onto three plates (∼48 h after initial exposure to the retroviruses). (A) Growth curves for the wild-type MEFs and wild-type SF-MEFs transduced with H-RasV12 or empty vector. Each time point represents the average from the triplicate plates, and each curve was performed three times. Error bars represent the standard error of the mean from the three independent experiments. (B) Representative photographs of the indicated cells stained for SA-β-gal (pH 5.5) activity at day 4. Photographs are at the same magnification.

Figure 4.

Oncogenic Ras promotes p53-dependent apoptosis in SF-MEFs. Wild-type or p53^-/- SF-MEFs were infected with retroviruses expressing empty viral vector, E1A, or H-RasV12. The indicated cells were simultaneously treated with 5 mM NAC. (A) The infected cells were either untreated (Control) or exposed to 5 Gy of ionizing radiation (IR). The cells were fixed 16 h after IR, stained with DAPI, and analyzed for the morphological characteristics associated with apoptosis. Apoptotic values were calculated as the number of apoptotic cells relative to the total number of cells. The data represent the average of at least three independent experiments ± S.E.M. (>100 cells counted per experiment). (B) Representative fluorescent images of DAPI-stained cells. Arrows identify cells with heterochromatin aggregation typical of apoptotic cells.

Figure 5.

Expression of oncogenic Ras elicits a DNA-damage-like response leading to the induction and phosphorylation of p53. Wild-type SF-MEFs were infected with vector control or H-RasV12-expressing retrovirus. The indicated cells were simultaneously treated with 5 mM NAC. Cells were harvested ∼24 h after infection. Total cellular lysates were immunoblotted for total p53 and Ser 18-phosphorylated p53. Uniform loading of cell lysates was confirmed by immunoblotting for tubulin.

Figure 6.

Activated oncogenes H-RasV12 and E1A promote chromosomal instability. Wild-type or p53^-/- MEFs or SF-MEFs were infected with (A) vector control, (B) H-RasV12, or (C) E1A and H-RasV12 retroviruses. At 24 h after infection, the cells were treated with nocodazole to induce metaphase arrest and then harvested for karyotype analysis. Between 30 and 40 metaphases from three independent infections were counted in each case. The indicated cultures were preincubated with 5 mM NAC before retroviral infection; these cells were maintained in NAC until the samples were harvested for metaphase spreads.

Figure 7.

Loss of p53 promotes anchorage-independent growth of SF-MEFs transduced with E1A and H-RasV12. Wild-type or p53^-/- MEFs or SF-MEFs infected with retroviruses expressing E1A and H-RasV12 were analyzed for anchorage-independent growth as described in Materials and Methods. The photographs are of wild-type MEFs (A), wild-type SF-MEFs (B), p53^-/- MEFs (C), or p53^-/- SF-MEFs (D). Colonies were counted (see Table 1) and photographs were taken after 14 d.

Figure 8.

Loss of p53 promotes the tumor-forming potential of SF-MEFs transduced with E1A and H-RasV12. Wild-type or p53^-/- MEFs or SF-MEFs were transduced with E1A and H-RasV12. Cells (2 × 10⁶) in 100 μL of PBS were injected subcutaneously into immunocompromised nude mice. The examples shown are mice injected with wild-type MEFs (A), wild-type SF-MEFs (B), p53^-/- MEFs (C), or p53^-/- SF-MEFs (D).