Tumor promotion by caspase-resistant retinoblastoma protein

Abstract

The retinoblastoma (RB) protein regulates cell proliferation and cell death. RB is cleaved by caspase during apoptosis. A mutation of the caspase-cleavage site in the RB C terminus has been made in the mouse Rb-1 locus; the resulting Rb-MI mice are resistant to endotoxin-induced apoptosis in the intestine. The Rb-MI mice do not exhibit increased tumor incidence, because the MI mutation does not disrupt the Rb tumor suppressor function. In this study, we show that Rb-MI can promote the formation of colonic adenomas in the p53-null genetic background. Consistent with this tumor phenotype, Rb-MI reduces colorectal epithelial apoptosis and ulceration caused by dextran sulfate sodium. By contrast, Rb-MI does not affect the lymphoma phenotype of p53-null mice, in keeping with its inability to protect thymocytes and splenocytes from apoptosis. The Rb-MI protein is expressed and phosphorylated in the tumors, thereby inactivating its growth suppression function. These results suggest that RB tumor suppressor function, i.e., inhibition of proliferation, is inactivated by phosphorylation, whereas RB tumor promoting function, i.e., inhibition of apoptosis, is inactivated by caspase cleavage.

Fig. 1.

Teratomas in Tm(Rb^MI/MIxp53^–/–) mice. (A) Kaplan-Meier survival curve of animals that died with teratoma (P = 0.0377, Log-rank test comparing Rb^+/+xp53^–/– with Rb^MI/MIxp53^–/–). (B and C) Representative photomicrographs of hematoxylin/eosin-stained paraffin sections of differentiated tumor areas. (D and E) Representative photomicrographs of hematoxylin/eosin-stained paraffin sections of less differentiated, highly cellular tumor areas. (F and G) Photomicrographs of normal liver (F) and germ cell tumor expanding into the liver (G, asterisk) in mice with the indicated genotypes. Small arrow in B shows brain-like tissue. Large arrow in C indicates epithelium, and arrowhead indicates laminar keratin structure. (Scale bars: 100 μm.)

Fig. 2.

Increased incidence of colon adenoma in the Tm(Rb^MI/MIxp53^–/–) mice. (A and B) A rare adenocarcinoma of the Tm(Rb^+/+xp53^–/–) genotype. (C and D) Representative adenomas from Tm(Rb^MI/MIxp53^–/–) mice. (E and F) Adenocarcinoma of Tm(Rb^MI/MIxp53^–/–) genotype. Dashed lines delineate tumors. Ac, adenocarcinoma; Ad, adenoma; Co, normal colon. (G) Histogram showing the incidence of adenomas and adenocarcinomas in the colons of mice with the indicated genotypes. The difference in colon tumor incidence is significant (P = 0.0196, Fisher's exact test). (H–K) The Rb-MI protein is phosphorylated in colon tumors. Photomicrographs of sections stained with bisbenzimide (Hoechst, left images) or anti-phosphoSer807/811 (green) and anti-phosphoThr821/826 (red) (right images). (L–O) Photomicrographs of sections stained with bisbenzimide (Hoechst, left images) or processed for TUNEL assay (green) (right images). Arrowheads indicate TUNEL-positive nuclei. Tissue types and genotypes are indicated above each photomicrograph. (Scale bars: 50 μm for A–F and L–O; 30 μm for H–K.)

Fig. 3.

Rb-MI attenuates colonic epithelial cell death and ulceration induced by DSS. (A and B) Representative photomicrographs of colon sections processed for TUNEL assay (red) and counterstained with bisbenzimide (Hoechst-blue) from Tm(Rb^+/+xp53^–/–) (A) and Tm(Rb^MI/MIxp53^–/–) mice (B) treated with 3% DSS for 3 days. (C) Histogram shows the number of TUNEL-positive nuclei with s.e.m. in the distal colon from animals treated with 3% DSS for 3 days with the indicated genotypes. (D–F) Representative photomicrographs of hematoxylin/eosin-stained paraffin sections of the entire colon (D and E) or higher magnification showing ulcers (F) after 7 days of DSS treatment. The area of ulcers is marked in the figures. The mucosa and submucosa were heavily infiltrated with inflammatory cells (F). (G) Histogram shows the mean plus s.e.m. of the total size of ulcers after 7 days of DSS treatment with the indicated genotypes. P values are indicated in each histogram (unpaired t test). (Scale bars: 60 μmin A, B, and F; 240 μmin D and E.)