Defects in transforming growth factor-beta signaling cooperate with a Ras oncogene to cause rapid aneuploidy and malignant transformation of mouse keratinocytes

Abstract

Genetic inactivation of the transforming growth factor-beta (TGF-beta) signaling pathway can accelerate tumor progression in the mouse epidermal model of multistage carcinogenesis. By using an in vitro model of keratinocyte transformation that parallels in vivo malignant conversion to squamous cell carcinoma, we show that v-ras(Ha) transduced primary TGF-beta1-/- keratinocytes and keratinocytes expressing a TGF-beta type II dominant-negative receptor transgene have significantly higher frequencies of spontaneous transformation than control genotypes. Malignant transformation in the TGF-beta1-/- keratinocytes is preceded by aneuploidy and accumulation of chromosomal aberrations. Similarly, transient inactivation of TGF-beta signaling with a type II dominant-negative receptor adenovirus causes rapid changes in ploidy. Exogenous TGF-beta1 can suppress aneuploidy, chromosome breaks, and malignant transformation of the TGF-beta1-/- keratinocytes at concentrations that do not significantly arrest cell proliferation. These results point to genomic instability as a mechanism by which defects in TGF-beta signaling could accelerate tumor progression in mouse multistage carcinogenesis.

Figure 1

v-ras^Ha-transduced TGF-β−/− and AM3 dominant negative TβRII transgenic keratinocytes exhibit increased frequency of in vitro malignant conversion. The indicated cell types were infected with the v-ras^Ha retrovirus on day 3 and allowed to proliferate for 15 days before selection in 0.5 mM calcium as described in Materials and Methods. (A) Calcium-resistant foci per dish generated by TGF-β1+/+, TGF-β1+/−, and TGF-β1−/− keratinocytes. Each bar represents the mean of nine independent experiments each with 7–10 dishes per genotype ±SEM. *, Significantly different from +/+ (P = 0.0027) and +/− (P = 0.0083). (B) Calcium-resistant foci per dish generated by FVB/n control (NT), AM3 keratinocytes (DN), and AM3 keratinocytes cultured in the presence of ZnCl₂ (DN+Zn). Each bar represents the average of six dishes ±SEM. Two independent experiments are shown. ZnCl₂ did not alter the frequency of foci in the NT cultures. *, **, ***, Significantly different from NT (P = 0.024, 0.0009, 0.0001, respectively). (C) The truncated TβRII is induced by ZnCl₂ in the v-ras^Ha-transduced AM3 keratinocytes. ¹²⁵I-labeled TGF-β1 was crosslinked to monolayers of NT or AM3 cells cultured in the presence (+) or absence (−) of 25 mM ZnCl₂ for 24 hr. The crosslinked products were electrophoresed through a precast 4–12% polyacrylamide gel (NOVEX, San Diego). The truncated TβRII runs at a lower molecular mass than the native TβRII. The + in the TGF-β1 row indicates crosslinking in the presence of 100 nM unlabeled TGF-β1. Kd, kilodaltons.

Figure 2

TGF-β1−/− calcium-resistant cells are aneuploid. Foci of calcium-resistant cells were pooled and expanded in 0.05 mM calcium medium for five passages before analysis of chromosome number as described in Materials and Methods. The histogram represents the percentage of the total metaphases analyzed (n = 60) with a specific chromosome number.

Figure 3

Rapid aneuploidy in TGF-β1−/− keratinocytes after transduction with v-ras^Ha. Percentages of metaphases with specific chromosome numbers in TGF-β1+/+ and TGF-β1−/− keratinocytes at the indicated times after infection with the v-ras^Ha retrovirus. Each histogram represents the sum of three independent chromosome harvests. Between 50 and 100 metaphases were counted for each genotype at a specific time point, and the percentage of total mitoses with a specific chromosome number was plotted. Metaphase spreads with an extremely high chromosome number were excluded from the histogram for uniformity. (A–C) TGF-β1+/+ keratinocytes. (D–F) TGF-β1−/− keratinocytes. Results are for 4 days (A and D), 8 days (B and E), and 15 days (C and F) after infection of TGF-β1+/+ and TGF-β1−/− keratinocytes with v-ras^Ha.

Figure 4

v-ras^Ha-transduced TGF-β1−/− keratinocytes have a higher frequency of chromosomal aberrations. Giemsa-stained chromosomes were isolated from keratinocyte cultures 15 days after v-ras^Ha transduction and analyzed for chromosomal abnormalities. n = 70 for TGF-β1−/− cells; n = 40 for TGF-β1+/+ cells.

Figure 5

Transient inactivation of TGF-β type II receptor causes rapid changes in ploidy. Percentage of cells in each phase of the cell cycle in normal and v-ras^Ha-infected BALB/c keratinocytes 4 and 8 days after superinfection with AdTGFβTR and 4 days after superinfection with Adβgal. The percentage of cells in each cell-cycle compartment was determined by two-color flow cytometric analysis as described in Materials and Methods. The S1 population represents the diploid S phase, and the S2 population represents the tetraploid S phase. Each bar is the average of two independent experiments.

Figure 6

Suppression of malignant conversion and aneuploidy by exogenous TGF-β1. (A) TGF-β1 suppresses the frequency of calcium-resistant foci in TGF-β1−/− keratinocytes. The indicated doses of TGF-β1 were added to keratinocytes 3 days after infection with v-ras^Ha and maintained through the course of the experiment. Each histogram represents the average of five to seven dishes. *, Significantly different from untreated keratinocytes (P < 0.0001). (B) The development of aneuploidy in TGF-β1−/− v-ras^Ha keratinocytes is suppressed by treatment with TGF-β1. Histogram shows the percentage of total mitoses with specific chromosome numbers. Treatment with TGF-β1 (50 pg/ml) was begun 3 days after infection with v-ras^Ha and continued for 15 days. A total of 90 metaphases from 2 independent experiments were counted. (C) Dose response for inhibition of DNA synthesis by exogenous TGF-β1 in v-ras^Ha-transduced TGF-β1−/− keratinocytes. DNA synthesis was measured by the incorporation of [³H]thymidine (³H-TdR).