Rb+/−;ARF−/− compound mutant mice exhibit significantly decreased survival relative to Rb+/− and Rb+/−;ARF+/− mice. Survival curves of Rb+/− (n = 37), Rb+/−;ARF−/− (n = 32), and Rb+/−;ARF+/− (n = 13) mice show that Rb+/−;ARF−/− mice had a significantly decreased median survival of 168.5 days vs. 277 days for Rb+/− controls (P < 0.0001). The Rb+/−; ARF+/− mice (n = 13) exhibited a median survival of 262 days, comparable to that of Rb+/− mice (P = 0.34) and significantly longer than that of Rb+/−; ARF−/− mice (P < 0.0001).

EAPs appear in the pituitaries of Rb+/−;ARF−/− compound mutants earlier than those in Rb+/− and Rb+/−; p53−/− compound mutant mice and are larger by PND 30. Comparison of hematoxylin-stained EAPs from Rb+/−, Rb+/−;ARF−/−, and Rb+/−; p53−/− compound mutant mice. (A) EAP of a PND 30 Rb+/−;ARF−/− compound mutant mouse (×1,000) showing dense groups of abnormal cells in the intermediate lobe (I) containing coarse chromatin, and large, irregular nuclei at the border with the posterior (neural) lobe. (B) EAP of a PND 60 Rb+/− mouse at the intermediate-neural lobe border (×1,000). Arrows indicate abnormal cells. Note the size of this EAP relative to the lesion in A. (C) EAP of a PND 60 Rb+/−;p53−/− compound mutant mouse showing a large lesion with densely packed abnormal cells in the intermediate lobe (×1,000). (D) Low-power view of an EAP of a PND 60 Rb+/−;ARF−/− compound mutant mouse (×400) showing typical large cluster of intermediate lobe (I) tumor cells near the neural lobe (N). (E) Higher-power (×1,000) view of one of the same lesions (D) demonstrating typical morphology of tumor cells. Note the presence of an apoptotic cell (arrow). (F) LOH at the Rb locus is observed in PND 60 EAPs from Rb+/−;ARF−/− mice. PCR analysis of DNA from laser capture-dissected samples shows retention of mutant allele with absence of WT allele in captured EAPs, while both alleles are intact in adjacent normal pituitary (N). (Calibration bar: 10 μm for A–C and E; 25 μm for D.)

Comparison of tumors of similar stage from Rb+/−;ARF−/− and Rb+/− mice shows higher levels of proliferation in Rb+/−;ARF−/− samples. Midsagittal sections of pituitary tumors (P) at comparable stages of development were stained with hematoxylin and eosin (A and B) or used for BrdUrd immunohistochemistry (C and D). Samples from a Rb+/− (6.5 months) mouse (A) and a Rb+/−;ARF−/− (4.5 months) compound mutant mouse (B) are shown. These animals were injected with BrdUrd (10 μg/kg body weight) 1 h before death, and analysis of BrdUrd incorporation demonstrated a significantly higher S-phase fraction (P < 0.05 for three matched pairs) in the Rb+/−; ARF−/− sample (D) relative to the Rb+/− control (C). (Calibration bar: 100 μm for A and B; 50 μm for C and D.)

Tumors isolated from Rb+/−;ARF+/− animals exhibit alterations at the ARF locus. (A) DNA from normal adjacent cerebellum (con) confirms that the animals were heterozygous for Rb, and all samples of pituitary (pit) tumors showed LOH for Rb with only the 5.2-kB lower band associated with the mutant allele remaining. (B) DNA from normal adjacent cerebellum (con) confirmed that all of the animals were heterozygous for ARF. Of four pituitary samples that also showed this pattern of equivalent hybridization, two are shown here (pit 8566 and 8239). Two pituitary samples exhibited alterations at the ARF locus. These samples demonstrated enrichment for the lower 6.0-kB band associated with mutant allele (pit 8904 and 8249).