p19(ARF) is dispensable for oncogenic stress-induced p53-mediated apoptosis and tumor suppression in vivo

Abstract

Recent studies have shown the p19(ARF) tumor suppressor to be involved in the response to oncogenic stress by regulating the activity of p53. This response is mediated by antagonizing the function of Mdm2, a negative regulator of p53, indicating a pathway for tumor suppression that involves numerous genes altered in human tumors. We previously described a transgenic mouse brain tumor model in which oncogenic stress, provided by cell-specific inactivation of the pRb pathway, triggers a p53-dependent apoptotic response. This response suppresses the growth of developing tumors and thus represents a bona fide in vivo tumor suppressor activity. We further showed that E2F1, a transcription factor known to induce p19(ARF) expression, was required for the response. Here, we use a genetic approach to test whether p19(ARF) functions to transduce the signal from E2F1 to p53 in this tumor suppression pathway. Contrary to the currently accepted hypothesis, we show that a deficiency in p19(ARF) has no impact on p53-mediated apoptosis or tumor suppression in this system. All measures of p53 function, including the level of apoptosis induced by pRb inactivation, the expression of p21 (a p53-responsive gene), and the rate of tumor growth, were comparable in mice with and without a functional p19(ARF) gene. Thus, although p19(ARF) is required in some cell types to transmit an oncogenic response signal to p53, it is dispensable for this function in an in vivo epithelial system. These results underscore the complexity of p53 tumor suppression and further indicate the existence of distinct cell-specific pathways that respond to similar stimuli.

FIG. 1.

p53 tumor suppression in brain epithelium. The diagram depicts previously elucidated steps in the development of choroid plexus tumors in TgT₁₂₁ mice. (A) Cell-specific expression of the T₁₂₁ transgene induces proliferation of normally nondividing epithelial cells by inactivating the pRb family proteins pRb, p107, and p130. p53-dependent apoptosis is then activated, as evidenced by the 85% reduction in apoptosis in TgT₁₂₁;p53^−/− mice. (B) Using a genetic approach, we previously showed that E2F1 acts upstream of p53 to induce apoptosis in response to T₁₂₁. Previously, E2F1 was shown to activate p19^ARF transcription, and p19^ARF was shown to induce p53 activities by regulating Mdm2 in cultured cells (see the introduction). In the current report, we tested whether this pathway is operative to induce p53-dependent apoptosis and suppression of brain epithelial tumors in vivo.

FIG. 2.

p19^ARF transcripts are induced in aberrantly proliferating CP epithelium. RNA in situ hybridization was performed with an antisense p19^ARF RNA probe. (A) Hematoxylin and eosin (H & E)-stained section from the same brain as in panel C shows the morphology of CP in the lateral ventricle and is in an orientation similar to that of dark-field panels B to D. The borders between brain parenchyma (br) and the ventricles (V) are depicted with white dashed lines in panels B to D. No p19^ARF expression above background is detected in the CP of nontransgenic mice (B). (Compare the signal in brain with that in CP.) p19^ARF transcripts are induced in TgT₁₂₁ CP (C). T₁₂₁-induced p19^ARF expression is p53 independent; the level of p19^ARF transcripts is similar in TgT₁₂₁;p53^−/− CP (D) and TgT₁₂₁ CP (C). (A) Bar, 200 μm (magnification is the same in all panels). Sense probe hybridization showed no signal above background (not shown).

FIG. 3.

T₁₂₁-induced apoptosis and cell proliferation do not require p19^ARF. T₁₂₁ expression causes abnormal CP cell proliferation and apoptosis. About 85% of the apoptosis is p53-dependent (27) and requires E2F1 (18). The TUNEL assay was used to detect apoptosis (A). Apoptosis levels and morphology were indistinguishable between TgT₁₂₁;p19^ARF−/− (a) and TgT₁₂₁;p19^ARF+/+ (b) CP. Apoptotic nuclei are stained and appear black; representative apoptotic cells are indicated by arrows. The bar (b) is equal to 100 μm; both panels are of the same magnification. Quantitative analysis of apoptosis (B) and proliferation (C) in the CP of three mice each at 4 and 8 weeks of age was carried out. Levels are compared to that of TgT₁₂₁;p19^ARF+/+ tissue, which is considered to represent 100%. Average relative values for all mice of each age and genotype are shown in the left panels. For these data, error bars indicate the variation among mice. In the right panels, data for each mouse are presented, with error bars indicating the field-to-field variation in counts taken from 10 fields per brain. Different mice were used for apoptosis and proliferation assays so as to avoid any impact of BrdU incorporation on apoptosis levels. There is no significant difference in apoptotic indexes between TgT₁₂₁;p19^ARF+/+ and TgT₁₂₁;p19^ARF−/− mice (P = 0.601). Importantly, the index of TgT₁₂₁;p19^ARF−/− CP was significantly higher than that of TgT₁₂₁;p53^−/− CP (P < 0.05). The level of CP cell proliferation was determined by immunostaining of BrdU incorporated in vivo (C). The data show that p19^ARF deficiency does not significantly alter cell proliferation (P = 0.288).

FIG. 4.

Tumor growth and morphology are unaffected by p19^ARF deficiency. H & E-stained CP tumors from 20-week-old TgT₁₂₁;p19^ARF+/+ (A) and TgT₁₂₁;p19^ARF−/− (B) mice are similar in size and morphology. In contrast, tumors become life-threatening by 4 weeks of age in TgT₁₂₁;p53^−/− mice (D). Leukemia caused by the p19^ARF−/− mutation is frequently observed invading the brains of TgT₁₂₁;p19^ARF−/− mice (C), a pathology that is present only in mice with compound genotypes and is thought to further reduce survival time (Table 1). Representative leukemia-filled vessels in the brain are indicated by arrows. The bar in panel C is equal to 100 μm. The bar in panel D is equal to 200 μm; panels A, B, and D are of the same magnification. br, brain tissue.

FIG. 5.

p53-mediated transactivation of p21 is unaffected by p19^ARF deficiency. Brains were examined from nontransgenic (A and B), TgT₁₂₁;p19^ARF+/+ (C and D), TgT₁₂₁;p19^ARF−/− (E and F), and TgT₁₂₁;p53^−/− (G and H) mice. RNA in situ hybridization was performed on brain sections using an antisense p21 RNA probe and viewed by dark-field microscopy (B, D, F, and H). Adjacent sections were stained with H & E and viewed by bright-field microscopy to show the location and morphology of the CP (A, C, E, and G). No p21 expression is detected in normal CP (B). p21 expression is induced by expression of T₁₂₁ (D) and remains unchanged in the absence of p19^ARF (F). T₁₂₁-induced p21 expression in CP requires functional p53 (H) (18). The bar in panel H is equal to 200 μm; all panels are of the same magnification. br, brain tissue.