Nucleolar p19ARF unlike mitochondrial smARF is incapable of inducing p53-independent autophagy

ARF mRNA encodes two distinct proteins, the nucleolar p19ARF and ashorter mitochondrial isoform, named smARF. Inappropriate proliferative signals generated by proto-oncogenes, such as c-Myc and E2F1, can elevate both p19ARF and smARF proteins. The two ARF isoforms differ not only in their localization but also in their functions. Nucleolar p19ARF inhibits cell growth mainly by activating p53 or by inhibiting ribosomal biogenesis. In contrast, mitochondrial smARF caninduce dissipation of the mitochondrial membrane potential and autophagy in a p53 independent manner. Recently, it was proposed by Abida et. al., that similar to smARF, the nucleolar p19ARF can also induce p53 independent autophagy, but in contrast to smARF it does so from within the nucleolus. Our current work shown here indicates however, that if the ectopic expression of p19ARF is restricted to thenucleolus it cannot induce autophagic vesicle formation. Only upon extreme overexpression, when p19ARF is localized to extra nuclear compartments, it can trigger p53-independent autophagic vesicle formation. Thus, our experiments indicate that the nucleolar p19ARF is incapable to induce autophagy from within the nucleolus.     

Nucleolar p19ARF, unlike mitochondrial smARF, is incapable of inducing p53-independent autophagy

ARF mRNA encodes two distinct proteins, the nucleolar p19(ARF), and a shorter mitochondrial isoform, named smARF. Inappropriate proliferative signals generated by proto-oncogenes, such as c-Myc and E2F1, can elevate both p19(ARF) and smARF proteins. The two ARF isoforms differ not only in their localization but also in their functions. Nucleolar p19(ARF) inhibits cell growth mainly by activating p53 or by inhibiting ribosomal biogenesis. In contrast, mitochondrial smARF can induce dissipation of the mitochondrial membrane potential and autophagy in a p53 independent manner. Recently, it was proposed by Abida et al., that similar to smARF, the nucleolar p19(ARF) can also induce p53 independent autophagy, but in contrast to smARF it does so from within the nucleolus. Our current work shown here indicates, however, that if the ectopic expression of p19(ARF) is restricted to the nucleolus it cannot induce autophagic vesicle formation. Only upon extreme overexpression, when p19(ARF) is localized to extra nuclear compartments, can it trigger p53-independent autophagic vesicle formation. Thus, our experiments indicate that the nucleolar p19(ARF) is incapable of inducing autophagy from within the nucleolus.     

E2F-1 has properties of a radiosensitizer and its regulation by cyclin A kinase is required for cell survival of fibrosarcoma cells lacking p53.

Negative regulation of E2F-1 DNA binding function by cyclin A kinase represents part of an S-phase checkpoint control system that, when activated, leads to apoptosis. In this study, we examined the cellular sensitivity and resistance of isogenic mouse fibrosarcoma cell lines, differing primarily in their p53 status, to ectopic expression of wild-type (wt) E2F-1 and cyclin A kinase binding-defective mutants of it. We found that E2F-1 (wt) potently affected the survival of p53+/+ tumor cells but not that of p53-/- cells. In contrast, expression of cyclin A kinase binding-defective E2F-1 species interfered with cell survival of fibrosarcoma cells irrespective of their p53 status. Finally, expression of E2F-1 (wt) in p53-/- fibrosarcoma cells enhanced the cytotoxic effect of ionizing radiation in vitro and in vivo in a mouse tumor model. These results suggest that E2F-1-dependent activation of an S-phase checkpoint is p53 independent and that E2F-1 possesses radiosensitizing properties in the absence of p53.     

The ARF Tumor Suppressor: Keeping Myc on a Leash

The ARF tumor suppressor protein acts in a checkpoint that guards against unscheduled cellular proliferation in response to oncogenic signaling. Deregulated expression of c-Myc induces ARF expression and apoptosis through the ARF-Mdm2-p53 axis. Our recent study reveals a new direct role for ARF in controlling c-Myc’s oncogenic activity that is independent of p53. ARF binds to and selectively impairs the transactivation ability of c-Myc while leaving its transrepression ability intact. Biologically, ARF prevents hyperproliferation and transformation caused by c-Myc and enhances c-Myc-induced apoptosis independently of p53. These new findings may be especially relevant for therapeutic strategies targeting c-Myc-induced cancers.     

Increased expression of Bcl-xL and c-Myc is associated with transformation by Abelson murine leukemia virus

Transformation mediated by the v-Abl oncoprotein, a tyrosine kinase encoded by the Abelson murine leukemia virus, is a multi-step process requiring genetic alterations in addition to expression of v-Abl. Loss of p53 or p19ARF was previously shown to be required for Abelson murine leukemia virus transformation of primary mouse embryonic fibroblasts (MEFs). By comparing gene expression patterns in primary p53-/- MEFs acutely infected with the v-Abl retrovirus, v-Abl-transformed MEF clones, and v-Abl-transformed MEF clones treated with Abl kinase inhibitor STI 571, we have identified additional genetic alterations associated with v-Abl transformation. Bcl-xL mRNA was elevated in three of five v-Abl-transformed MEF clones. In addition, elevated expression of c-Myc mRNA, caused either by c-myc gene amplification or by enhanced signaling via STAT3, was observed in five v-Abl-transformed MEF clones. The data suggest that increases in cell survival associated with Bcl-xL and increases in cell growth associated with c-Myc facilitate the transformation process dependent on constitutive mitogenic signaling by v-Abl.     

p19ARF-induced p53-independent apoptosis largely occurs through BAX

Combined disruption of the ARF gene and the p53 gene causes mouse predisposition to tumors of a wider variety and at a higher frequency than disruption of the p53 gene, indicating that the ARF gene has p53-independent anti-tumor function in addition to p53-dependent function. Coincidentally with this notion, ectopic expression of the p19(ARF) induces apoptosis for wild-type mouse embryo fibroblasts which have been immortalized by introduction of the SV40 virus genome (SV40-MEFs). The protein expression levels of p53, p21(Cip1), and Bax were not upregulated by ectopic expression of p19(ARF) in SV40-MEFs, indicating that expression of p19(ARF) induced apoptosis through p53-independent pathways in this system. Ectopic expression of p19(ARF) induced prominent apoptosis even in SV40-Bak-/-MEFs. In contrast, expression of p19(ARF) induced only a very low grade of apoptosis in Bax-/- or Bax-/-/Bak-/-SV40-MEFs. Remarkable attenuation of p19(ARF)-induced apoptosis by disruption of the Bax gene thus leads to the conclusion that Bax plays a major role in p53-independent apoptosis induced by p19(ARF).     

Oncogenic ras and p53 cooperate to induce cellular senescence

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.     

Multiple domains of the mouse p19ARF tumor suppressor are involved in p53-independent apoptosis

The ARF (p19ARF for the mouse ARF consisting of 169 amino acids and p14ARF for the human ARF consisting of 132 amino acids) genes upregulate p53 activities to induce cell cycle arrest and sensitize cells to apoptosis by inhibiting Mdm2 activity. p53-independent apoptosis also is induced by ectopic expression of p19ARF. We constructed various deletion mutants of p19ARF with a cre/loxP-regulated adenoviral vector to determine the regions of p19ARF which are responsible for p53-independent apoptosis. Ectopic expression of the C-terminal region (named C40) of p19ARF whose primary sequence is unique to the rodent ARF induced prominent apoptosis in p53-deficient mouse embryo fibroblasts. Relatively low-grade but significant apoptosis also was induced in p53-deficient mouse embryo fibroblasts by ectopic expression of p19ARF1-129, a p19ARF deletion mutant deficient in the C40 region. In contrast, ectopic expression of the wild-type p14ARF did not induce significant apoptosis in human cells. Taken together, we concluded that p53-independent apoptosis was mediated through multiple regions of the mouse ARF including C40, and the ability of the ARF gene to mediate p53-independent apoptosis has been not well conserved during mammalian evolution.     

p53-independent apoptosis is induced by the p19ARF tumor suppressor

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.