p33(ING1) enhances UVB-induced apoptosis in melanoma cells

The biological functions of the tumor suppressor ING1 have been studied extensively in the past few years since it was cloned. It shares many biological functions with p53 and has been reported to mediate growth arrest, senescence, apoptosis, anchorage-dependent growth, chemosensitivity, and DNA repair. Some of these functions, such as cell cycle arrest and apoptosis, have been shown to be dependent on the activity of both ING1 and p53 proteins. Two recent reports by Scott and colleagues demonstrate that p33(ING1) (one of the ING1 isoforms) translocates to the nucleus and binds to PCNA upon UV irradiation. Here we report that p33(ING1) mediates UV-induced cell death in melanoma cells. We found that overexpression of p33(ING1) increased while the introduction of an antisense p33(ING1) plasmid reduced the apoptosis rate in melanoma cells after UVB irradiation. We also demonstrated that enhancement of UV-induced apoptosis by p33(ING1) required the presence of p53. Moreover, we found that p33(ING1) enhanced the expression of endogenous Bax and altered the mitochondrial membrane potential. Taken together, these observations strongly suggest that p33(ING1) cooperates with p53 in UVB-induced apoptosis via the mitochondrial cell death pathway in melanoma cells.     

p33 Enhances UVB-Induced Apoptosis in Melanoma Cells

The biological functions of the tumor suppressor ING1 have been studied extensively in the past few years since it was cloned. It shares many biological functions with p53 and has been reported to mediate growth arrest, senescence, apoptosis, anchorage-dependent growth, chemosensitivity, and DNA repair. Some of these functions, such as cell cycle arrest and apoptosis, have been shown to be dependent on the activity of both ING1 and p53 proteins. Two recent reports by Scott and colleagues demonstrate that p33^ING1 (one of the ING1 isoforms) translocates to the nucleus and binds to PCNA upon UV irradiation. Here we report that p33^ING1 mediates UV-induced cell death in melanoma cells. We found that overexpression of p33^ING1 increased while the introduction of an antisense p33^ING1 plasmid reduced the apoptosis rate in melanoma cells after UVB irradiation. We also demonstrated that enhancement of UV-induced apoptosis by p33^ING1 required the presence of p53. Moreover, we found that p33^ING1 enhanced the expression of endogenous Bax and altered the mitochondrial membrane potential. Taken together, these observations strongly suggest that p33^ING1 cooperates with p53 in UVB-induced apoptosis via the mitochondrial cell death pathway in melanoma cells.     

The p33ING1b tumor suppressor cooperates with p53 to induce apoptosis in response to etoposide in human osteosarcoma cells

p33ING1b induces cell cycle arrest and stimulates DNA repair, apoptosis and chemosensitivity. The magnitude of some p33ING1b effects may be due to activation of the tumor suppressor p53. To investigate if the p33ING1b protein affected chemosensitivity of osteosarcoma cells, we overexpressed p33ING1b in p53+/+ U2OS cells or in p53-mutant MG63 cells, and then assessed for growth arrest and apoptosis after treatment with etoposide. p33ING1b increased etoposide-induced growth inhibition and apoptosis to a much greater degree in p53+/+ U2OS cells than in p53-mutant MG63 cells. Moreover, ectopic expression of p33ING1b markedly upregulated p53, p21WAF1 and bax protein levels and activated caspase-3 protein kinase in etoposide-treated U2OS cells. Together, our data indicate that p33ING1b prominently enhances etoposide-induced apoptosis through p53-dependent pathways in human osteosarcoma cells. p33ING1b may be an important marker and/or therapeutic target in the prevention and treatment of metastatic osteosarcoma.     

A novel ING2 isoform, ING2b, synergizes with ING2a to prevent cell cycle arrest and apoptosis

We identified a novel inhibitor of growth family member 2 (ING2) isoform, ING2b, which shares exon 2 with ING2a, but lacks the N-terminal p53 binding region. Contrary to ING2a, ING2b’s promoter has no p53 binding sites. Consistently, activation of p53 led to suppression of ING2a, leaving ING2b unaffected. Through isoform-specific targeting, we showed that ING2a knockdown suppressed cell growth only in the presence of p53, ING2b knockdown had no effect on cell growth, and knockdown of both induced cell cycle arrest and apoptosis independently of p53. ING2a and ING2b have compensatory roles that protect cells from cell cycle arrest and apoptosis and may be involved in development of chemotherapeutic resistance.     

Treatment with a BH3 mimetic overcomes the resistance of latency III EBV (+) cells to p53-mediated apoptosis

P53 inactivation is often observed in Burkitt''s lymphoma (BL) cells due to mutations in the p53 gene or overexpression of its negative regulator, murine double minute-2 (MDM2). This event is now considered an essential part of the oncogenic process. Epstein–Barr virus (EBV) is strongly associated with BL and is a cofactor in its development. We previously showed that nutlin-3, an antagonist of MDM2, activates the p53 pathway in BL cell lines harboring wild-type p53. However, nutlin-3 strongly induced apoptosis in EBV (−) or latency I EBV (+) cells, whereas latency III EBV (+) cells were much more resistant. We show here that this resistance to apoptosis is also observed in latency III EBV (+) lymphoblastoid cell lines. We also show that, in latency III EBV (+) cells, B-cell lymphona 2 (Bcl-2) is selectively overproduced and interacts with Bcl-2-associated X protein (Bax), preventing its activation. The treatment of these cells with the Bcl-2-homology domain 3 mimetic ABT-737 disrupts Bax/Bcl-2 interaction and allows Bax activation by nutlin-3. Furthermore, treatment with these two compounds strongly induces apoptosis. Thus, a combination of Mdm2 and Bcl-2 inhibitors might be a useful anti-cancer strategy for diseases linked to EBV infection.     

p21 promotes ceramide-induced apoptosis and antagonizes the antideath effect of Bcl-2 in human hepatocarcinoma cells

p21, a potent cyclin-dependent kinase inhibitor, has been known to induce cell cycle arrest in response to DNA-damaging agents. Although p21 has been reported to play an important role in the regulation of apoptosis, the postulated role for p21 in apoptosis is still controversial. Previously, we reported that p21 was induced in a p53-independent manner during ceramide-induced apoptosis in human hepatocarcinoma cell lines. In the present study, we investigated the precise role of p21 in ceramide-induced apoptosis in human hepatocarcinoma cells by using a tetracycline-inducible expression system. Overexpression of p21 by itself did not induce apoptosis in p53-deficient Hep3B cells. However, Hep3B/p21 cells were more sensitive to ceramide-induced apoptosis. In these cells, p21 overexpression did not result in G1 arrest. The expression level of Bax was increased in Hep3B/p21 cells treated with ceramide and its expression was more accelerated under the p21-overexpressed condition compared to that of the p21-repressed condition. Overexpression of Bax induced apoptosis in Hep3B cells. On the other hand, the levels of p21 and Bax protein were increased by ceramide in another hepatocarcinoma cell line, SK-Hep-1, while the Bcl-2 protein level was not changed. Overexpression of Bcl-2 not only suppressed apoptosis but also completely prevented induction of p21 and Bax caused by ceramide in SK-Hep-1 cells. Furthermore, overexpression of p21 antagonized the death-protective function of Bcl-2 and upregulated expression of Bax protein. These results suggest that p21 promotes ceramide-induced apoptosis by enhancing the expression of Bax, thereby modulating the molecular ratio of Bcl-2:Bax in human hepatocarcinoma cells.     

N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome C release and apoptotic cell death

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase-activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N-terminus, generating a potent proapoptotic 18-kDa fragment (Bax/p18). Both the calpain-mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane-enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and fragmentation of DNA. Unlike the full-length Bax, Bax/p18 did not interact with the antiapoptotic Bcl-2 protein in the mitochondrial fraction of drug-treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and caspase-3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase-3-mediated apoptosis that was not blocked by overexpression of Bcl-2 protein. Therefore, Bax/p18 has a cytochrome c-releasing activity that promotes cell death independent of Bcl-2. Finally, Bcl-2 overexpression inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution.     

High-mobility group A1 protein inhibits p53-mediated intrinsic apoptosis by interacting with Bcl-2 at mitochondria

The high-mobility group A (HMGA) proteins are a family of non-histone chromatin factors, encoded by the HMGA1 and HMGA2 genes. Several studies demonstrate that HMGA proteins have a critical role in neoplastic transformation, and their overexpression is mainly associated with a highly malignant phenotype, also representing a poor prognostic index. Even though a cytoplasmic localization of these proteins has been previously reported in some highly malignant neoplasias, a clear role for this localization has not been defined. Here, we first confirm the localization of the HMGA1 proteins in the cytoplasm of cancer cells, and then we report a novel mechanism through which HMGA1 inhibits p53-mitochondrial apoptosis by counteracting the binding of p53 to the anti-apoptotic factor Bcl-2. Indeed, we demonstrate a physical and functional interaction between HMGA1 and Bcl-2 proteins. This interaction occurs at mitochondria interfering with the ability of p53 protein to bind Bcl-2, thus counteracting p53-mediated mitochondrial apoptosis. This effect is associated with the inhibition of cytochrome c release and activation of caspases. Consistent with this mechanism, a strong correlation between HMGA1 cytoplasmic localization and a more aggressive histotype of thyroid, breast and colon carcinomas has been observed. Therefore, cytoplasmic localization of HMGA1 proteins in malignant tissues is a novel mechanism of inactivation of p53 apoptotic function.     

Inactivation of p21WAF1 sensitizes cells to apoptosis via an increase of both p14ARF and p53 levels and an alteration of the Bax/Bcl-2 ratio

p21(WAF1) appears to be a major determinant of the cell fate in response to anticancer therapy. It was shown previously that HCT116 human colon cancer cells growing in vitro enter a stable arrest upon DNA damage, whereas cells with a defective p21(WAF1) response undergo apoptosis. Here we report that the enhanced sensitivity of HCT116/p21(-/-) cells to chemotherapeutic drug-induced apoptosis correlates with an increased expression of p53 and a modification of their Bax/Bcl-2 ratio in favor of the pro-apoptotic protein Bax. Treatment of HCT116/p21(-/-) cells with daunomycin resulted in a reduction of the mitochondrial membrane potential and in activation of caspase-9, whereas no such changes were observed in HCT116/p21(+/+) cells, providing evidence that p21(WAF1) exerts an antagonistic effect on the mitochondrial pathway of apoptosis. Moreover, the role of p53 in activation of this pathway was demonstrated by the fact that inhibition of p53 activity by pifithrin-alpha reduced the sensitivity of HCT116/p21(-/-) cells to daunomycin-induced apoptosis and restored a Bax/Bcl-2 ratio similar to that observed in HCT116p21(+/+) cells. Enhancement of p53 expression after disruption of p21(WAF1) resulted from a stabilization of p53, which correlated with an increased expression of the tumor suppressor p14(ARF), an inhibitor of the ubiquitin ligase activity of Mdm2. In accordance with the role of p14(ARF) in p53 stabilization, overexpression of p14(ARF) in HCT116/p21(+/+) cells resulted in a strong increase in p53 activity. Our results identify a novel mechanism for the anti-apoptotic effect of p21(WAF1) consisting in maintenance of mitochondrial homeostasis that occurs in consequence of a negative control of p14(ARF) expression.     

Nucleolar protein CSIG is required for p33ING1 function in UV-induced apoptosis

Cellular senescence-inhibited gene (CSIG) protein, a nucleolar protein with a ribosomal L1 domain in its N-terminus, can exert non-ribosomal functions to regulate biological processes, such as cellular senescence. Here, we describe a previously unknown function for CSIG: promotion of apoptosis in response to ultraviolet (UV) irradiation-induced CSIG upregulation. We identified p33ING1 as a binding partner that interacts with CSIG. After UV irradiation, p33ING1 increases its protein expression, translocates into the nucleolus and binds CSIG. p33ING1 requires its nucleolar targeting sequence region to interact with CSIG and enhance CSIG protein stability, which is essential for activation of downstream effectors, Bcl-2-associated X protein, to promote apoptosis. Thus, our data imply that p33ING1–CSIG axis functions as a novel pro-apoptotic regulator in response to DNA damage.     

p14(ARF)-induced apoptosis in p53 protein-deficient cells is mediated by BH3-only protein-independent derepression of Bak protein through down-regulation of Mcl-1 and Bcl-xL proteins

The p14(ARF) tumor suppressor plays a central role in regulating cell cycle arrest and apoptosis. We reported previously that p14(ARF) is capable of triggering apoptosis in a p53-independent manner. However, the mechanism remained unclear. Here we demonstrate that the p53-independent activation of the mitochondrial apoptosis pathway by p14(ARF) is primarily mediated by the pro-apoptotic Bax-homolog Bak. Expression of p14(ARF) exclusively triggers a N-terminal conformational switch of Bak, but not Bax, which allows for mitochondrial permeability shift, release of cytochrome c, activation of caspases, and subsequent fragmentation of genomic DNA. Although forced expression of Bak markedly sensitizes toward p14(ARF)-induced apoptosis, re-expression of Bax has no effect. Vice versa, knockdown of Bak by RNA interference attenuates p14(ARF)-induced apoptosis, whereas down-regulation of Bax has no effect. Bak activation coincides with a prominent, caspase-independent deprivation of the endogenous Bak inhibitors Mcl-1 and Bcl-x(L). In turn, mitochondrial apoptosis is fully blocked by overexpression of either Mcl-1 or Bcl-x(L). Taken together, these data indicate that in the absence of functional p53 and Bax, p14(ARF) triggers mitochondrial apoptosis signaling by activating Bak, which is facilitated by down-regulating anti-apoptotic Mcl-1 and Bcl-x(L). Moreover, our data suggest that the simultaneous inhibition of two central endogenous Bak inhibitors, i.e. Mcl-1 and Bcl-x(L), may be sufficient to activate mitochondrial apoptosis in the absence of BH3-only protein regulation.     

ARF tumor suppressor induces mitochondria-dependent apoptosis by modulation of mitochondrial Bcl-2 family proteins

A tumor suppressor gene product, ARF, sensitizes cells to apoptosis in the presence of appropriate collateral signals. In this study, we analyzed the mechanism of ARF-dependent apoptosis and demonstrated that ARF induces mitochondria-dependent apoptosis in p53 wild-type, ARF/p16-null cells. We also found that ARF evokes cytochrome c release from mitochondria, decreases mitochondrial membrane potential, and activates pro-caspase-9 to induce apoptosis. Our findings suggest that this apoptotic cellular modulation is brought about by up-regulation of the proapoptotic Bcl-2 family proteins Bax and Bim and down-regulation of antiapoptotic Bcl-2 in mitochondrial fractions. Additionally, ARF seems to down-regulate Bcl-2 in a p53-dependent manner while up-regulating Bax/Bim via a p53-independent pathway.