Loss of transcription factor IRF-1 affects tumor susceptibility in mice carrying the Ha-ras transgene or nullizygosity for p53

Abstract

The transcription factor IRF-1 has been implicated in tumor suppression: IRF-1 suppresses cell transformation and mediates apoptosis in vitro. Here we show that the loss of IRF-1 alleles per se has no effect on spontaneous tumor development in the mouse but dramatically exacerbates previous tumor predispositions caused by the c-Ha-ras transgene or by nullizygosity for p53. Grossly altered tumor spectrum, as compared to p53-null mice, was also observed in mice lacking both IRF-1 and p53, and cells from these mice show significantly higher mutation rate. Our results suggest that IRF-1 is a new member of the tumor susceptibility genes.

Figure 1

Survival rate, cause of death, and histology of tumors in subject mice. (A) Survival curves of wild-type, IRF-1^−/−, p53^−/−, and IRF-1^−/−p53^−/− mice followed up to 200 days by the Kaplan–Meier method. Mice were sacrificed upon becoming apparently moribund. (n) Total number of mice in each genotype. At 200 days, 2% of IRF-1^−/− mice, 56% of p53^−/− mice, and 96% of IRF-1^−/−p53^−/− mice developed tumors; 0.2% of wild-type mice died before 200 days, but no tumors were found. (B) The first 100 mice of each genotype were monitored for up to 200 days and autopsied to assess for the presence and number of tumors. (C) Raw numbers of histological types of tumors in p53^−/− and IRF-1^−/−p53^−/− mice. Data shown are from the same cohort of 100 mice as in B. The bar patterns for tumor types are shown in B and C.

Figure 2

Histological analysis of representative tumors obtained from IRF-1^−/− and IRF-1^−/−p53^−/− mice (hematoxylin and eosin staining). (A) Malignant fibrous histiocytoma-like sarcoma characteristically found in IRF-1^−/− mice. Tumor chiefly consists of atypical spindle-shaped cells. (B) Angiosarcoma in an IRF-1^−/−p53^−/− mouse. Atypical tumor cells surround individual or groups of erythrocytes and also form neoplastic blood vessels. (C) Immature teratoma in testis of a male IRF-1^−/−p53^−/− mouse. Tumor consists of immature adenomatous, adipose-like, and chondroid tissue components. (D) Ganglioneuroblastoma in an IRF-1^−/−p53^−/− mouse. The spinal cord (right) is invaded by neuron-like tumor cells with enlarged nuclei and prominent nucleoli (left). This type of tumor was not observed in singly null mice. Original magnifications: (A,C,D) 500×; (B) 600×.

Figure 3

Cellular abnormalities in IRF-1^−/−p53^−/− MEFs. (A,B) Mutation frequency in MEFs treated with cisplatin (0.05 μg/ml for 72 hr; A) or MNNG (5 μm for 3 hr; B). The numbers of Oua^r colonies/10⁵ MEF cells were plotted. Plating efficiencies of p53^−/− and IRF-1^−/−p53^−/− MEFs were similar (∼90%). (C) Representative growth curves of MEFs. Cells were plated at a density of 10⁵ cells/35-mm dish at passage 4, and cell number counted (bars indicate s.d.). Experiments performed on at least four clones of each genotype showed the results to be essentially reproducible. Mean doubling times in log phase for wild-type and IRF-1^−/− MEFs were similar (66.6 ± 39.8 and 56.9 ± 18.6 hr, respectively). p53^−/− and IRF-1^−/−p53^−/− MEFs showed similar growth rates (mean doubling time, 22.9 ± 2.5 and 25.0 ± 4.3 hr, respectively). Saturation density of MEFs of each genotype was 11.0 ± 2.5 × 10⁵ (wild-type, ○), 8.6 ± 1.9 × 10⁵ (IRF-1^−/−, ●), 26.8 ± 4.4 × 10⁵ (p53^−/−, □), and 35.4 ± 3.4 × 10⁵ cells (IRF-1^−/− p53^−/−, █).