<p>Apoptosis is a cellular suicide program that can be initiated by various genotoxic and cytotoxic stimuli. In many cases, such cell damaging agents promote cell death through the intrinsic apoptotic pathway by triggering mitochondrial cytochrome <italic>c</italic> release and subsequent caspase activation. Cytosolic cytochrome <italic>c</italic> is directly responsible for initiating formation of the caspase-activating apoptosome, which plays a crucial role in the apoptotic process. Given the importance of cellular fate, apoptosis is tightly controlled by a balance between survival and death signals. It has been shown that activated cell survival pathways, including the mitogen-activated protein kinase (MAPK) cascade and the PI3K/Akt signaling, enhance cell viability by conferring resistance to apoptotic cell death. However, the underlying mechanism(s) that lead to inhibition of functional apoptosome formation (and caspase activation) has yet to be elucidated. In the studies that are described in this dissertation, I have investigated the regulation of apoptosis downstream of mitochondrial cytochrome <italic>c</italic> release with the goal of understanding how survival signaling can alter the apoptotic program, contributing to human malignancies. </p><p>First, we describe a mechanism for the inhibition of cytochrome <italic>c</italic>-induced caspase activation by MAPK signaling, identifying a novel mode of apoptotic regulation exerted through Apaf-1 phosphorylation by the 90-kDa ribosomal S6 kinase (Rsk). We have found that recruitment of 14-3-3&epsilon; to phosphorylated Ser268 impedes the ability of cytochrome <italic>c</italic> to nucleate apoptosome formation and activate downstream caspases. High endogenous levels of Rsk in PC3 prostate cancer cells or Rsk activation in other cell types promoted 14-3-3&epsilon; binding to Apaf-1 and rendered the cells insensitive to cytochrome <italic>c</italic>, suggesting a role for Rsk signaling in apoptotic resistance of prostate cancers and other cancers with elevated Rsk activity. These results identify a novel locus of apoptosomal regulation wherein MAPK signaling promotes Rsk-catalyzed Apaf-1 phosphorylation and consequent binding of 14-3-3&epsilon;, resulting in decreased cellular responsiveness to cytochrome <italic>c</italic>. </p><p>In the second part, we examine how apoptosis is inhibited by oncogenic tyrosine kinase signaling by using leukemogenic tyrosine kinase-induced leukemia model systems. We have demonstrated that protein phosphatase 5 (PP5) is responsible for Hsp90&beta; hypophosphorylation, which can contribute to impaired cell death in leukemia expressing oncogenic tyrosine kinases. Loss of PP5 results in an increase of Hsp90&beta; phosphorylation, raising leukemic cells' responsiveness to imatinib, a BCR-ABL kinase inhibitor. Further we have discovered that acetylation regulates PP5 activity on Hsp90&beta;. Mutational study showed that K144 acetylation on PP5, which was diminished in leukemic conditions, inhibited PP5 binding to Hsp90&beta;, causing Hsp90&beta; hyperphosphorylation and subsequently potentiating cells to apoptosis. These studies reveal a molecular mechanism by which agents enhancing PP5 acetylation may be a potential treatment for leukemias. Collectively, this work provides new insight into mechanisms of regulation of apoptosome formation/function, helping us understand how the evasion of apoptotic cell death contributes to cancer cell survival. Further, this finding implicates cytochrome <italic>c</italic>-induced apoptotic signaling in the context of cancer cell responsiveness to chemotherapeutic treatments.</p> / Dissertation

XIAP and Bcl-xL are critical anti-apoptotic molecules that directly inhibit caspases and block mitochondrial membrane permeabilization, respectively. In addition to preventing apoptosis, both XIAP and Bcl-xL can be generated by cap-independent translation via the utilization of an IRES in the 5'-UTR of their mRNAs. In recent years it has been shown that activation of S6K2 induces the translational upregulation of these two apoptotic regulators. Here I have determined that activation of S6K2 enhances IRES-mediated translation of XIAP and Bcl-xL by inducing the degradation of PDCD4, which I have identified as a novel regulator of XIAP and Bcl-xL IRES elements. Furthermore, I have shown that PDCD4 is a positive modulator of the Apaf-1 IRES element. The concurrent regulation of XIAP, Bcl-xL and Apaf-1 by PDCD4 suggests a model in which the level of PDCD4 expression alters the apoptotic threshold by specifically impacting IRES-mediated translation of the XIAP, Bcl-xL and Apaf-1 mRNAs.

XIAP and Bcl-xL are critical anti-apoptotic molecules that directly inhibit caspases and block mitochondrial membrane permeabilization, respectively. In addition to preventing apoptosis, both XIAP and Bcl-xL can be generated by cap-independent translation via the utilization of an IRES in the 5'-UTR of their mRNAs. In recent years it has been shown that activation of S6K2 induces the translational upregulation of these two apoptotic regulators. Here I have determined that activation of S6K2 enhances IRES-mediated translation of XIAP and Bcl-xL by inducing the degradation of PDCD4, which I have identified as a novel regulator of XIAP and Bcl-xL IRES elements. Furthermore, I have shown that PDCD4 is a positive modulator of the Apaf-1 IRES element. The concurrent regulation of XIAP, Bcl-xL and Apaf-1 by PDCD4 suggests a model in which the level of PDCD4 expression alters the apoptotic threshold by specifically impacting IRES-mediated translation of the XIAP, Bcl-xL and Apaf-1 mRNAs.

XIAP and Bcl-xL are critical anti-apoptotic molecules that directly inhibit caspases and block mitochondrial membrane permeabilization, respectively. In addition to preventing apoptosis, both XIAP and Bcl-xL can be generated by cap-independent translation via the utilization of an IRES in the 5'-UTR of their mRNAs. In recent years it has been shown that activation of S6K2 induces the translational upregulation of these two apoptotic regulators. Here I have determined that activation of S6K2 enhances IRES-mediated translation of XIAP and Bcl-xL by inducing the degradation of PDCD4, which I have identified as a novel regulator of XIAP and Bcl-xL IRES elements. Furthermore, I have shown that PDCD4 is a positive modulator of the Apaf-1 IRES element. The concurrent regulation of XIAP, Bcl-xL and Apaf-1 by PDCD4 suggests a model in which the level of PDCD4 expression alters the apoptotic threshold by specifically impacting IRES-mediated translation of the XIAP, Bcl-xL and Apaf-1 mRNAs.

XIAP and Bcl-xL are critical anti-apoptotic molecules that directly inhibit caspases and block mitochondrial membrane permeabilization, respectively. In addition to preventing apoptosis, both XIAP and Bcl-xL can be generated by cap-independent translation via the utilization of an IRES in the 5'-UTR of their mRNAs. In recent years it has been shown that activation of S6K2 induces the translational upregulation of these two apoptotic regulators. Here I have determined that activation of S6K2 enhances IRES-mediated translation of XIAP and Bcl-xL by inducing the degradation of PDCD4, which I have identified as a novel regulator of XIAP and Bcl-xL IRES elements. Furthermore, I have shown that PDCD4 is a positive modulator of the Apaf-1 IRES element. The concurrent regulation of XIAP, Bcl-xL and Apaf-1 by PDCD4 suggests a model in which the level of PDCD4 expression alters the apoptotic threshold by specifically impacting IRES-mediated translation of the XIAP, Bcl-xL and Apaf-1 mRNAs.

Melanoma cutâneo ainda constitui a principal causa de morte por câncer de pele nos países desenvolvidos. A variabilidade do comportamento clínico dessa neoplasia tem sido apenas parcialmente explicada pelos aspectos clínicos e histológicos, e a identificação de variáveis biológicas pode vir a ser importante na determinação de grupos de risco específicos. Foram estudados 69 pacientes com melanoma cutâneo primário de diversos graus de gravidade, tratados entre 1990 e 2007, com o intuito de verificar aspectos clínicos e epidemiológicos, preparar Tissue microarray (TMA) para estudo dos melanomas cutâneos primários com espessura maior que 1,0 mm, avaliar por análise imuno-histoquímica a expressão das proteínas da apoptose celular Bcl2, Bax, Bak, Apaf-1, Caspase 3, Caspase 9, Caspase 8, FasL e Fas em nevos-controle e em melanomas primários com espessuras menores e maiores que 1mm, e correlacionar a expressão dessas proteínas da apoptose com a evolução de melanomas cutâneos primários. Os resultados ratificaram tanto dados epidemiológicos já publicados em relação a sexo, idade e local da lesão, quanto a correlação entre a evolução da doença e os índices de Breslow. A análise dos escores compostos relativos à expressão das proteínas da apoptose revelou que o perfil imuno-histoquímico dessas proteínas parece não ter significado prognóstico, uma vez que não houve diferenças de expressão entre pacientes com e sem doença disseminada. Não foram encontradas alterações na expressão das proteínas da apoptose estudadas que pudessem sugerir o seu envolvimento tanto na gênese quanto na progressão de melanoma primário. O perfil imuno-histoquímico com tendência pró-apoptótica parece indicar que outros fatores seriam responsáveis pelo crescimento e disseminação da neoplasia / Cutaneous melanoma still constitutes the main cause of skin cancer death in developed world. Clinical behavior variability of this neoplasia has been only partially explained by clinical and histological aspects, and identification of biological variables can be important for determining specific risk groups. Sixty nine (69) patients with mild to severe primary cutaneous melanoma treated in 1990-2007 were studied aiming at (a) verifying clinical epidemiological aspects, (b) generating a Tissue microarray (TMA) for characterizing proteins expression of cutaneous melanoma > 1.0 mm, (c) analyzing the immunohistochemical expression of the apoptosis proteins Bcl2, Bax, Bak, Apaf-1, Caspase 3, Caspase 9, Caspase 8, FasL e Fas in 10 control nevi and in primary melanomas with thickness 1mm, (d) and correlating these proteins expression with the disease prognosis. Results have ratified known epidemiological date on gender, age and lesion localization as well as the correlation between the disease prognosis and the Breslow\'s indexes. Analysis of the composite scores relating to apoptosis proteins has revealed that their immunohistochemical profile seems to be not significant for determining the disease prognosis, since no differences in proteins expression were found when compared patients with and without disease dissemination. Alterations in proteins expression suggesting their role in the genesis as well as in the prognosis of primary melanoma were not evidenced. Immunohistochemical profile with pro-apoptosis trend seems to indicate other factor as responsible for the neoplasia growth and dissemination

No description available.

Melanoma cutâneo ainda constitui a principal causa de morte por câncer de pele nos países desenvolvidos. A variabilidade do comportamento clínico dessa neoplasia tem sido apenas parcialmente explicada pelos aspectos clínicos e histológicos, e a identificação de variáveis biológicas pode vir a ser importante na determinação de grupos de risco específicos. Foram estudados 69 pacientes com melanoma cutâneo primário de diversos graus de gravidade, tratados entre 1990 e 2007, com o intuito de verificar aspectos clínicos e epidemiológicos, preparar Tissue microarray (TMA) para estudo dos melanomas cutâneos primários com espessura maior que 1,0 mm, avaliar por análise imuno-histoquímica a expressão das proteínas da apoptose celular Bcl2, Bax, Bak, Apaf-1, Caspase 3, Caspase 9, Caspase 8, FasL e Fas em nevos-controle e em melanomas primários com espessuras menores e maiores que 1mm, e correlacionar a expressão dessas proteínas da apoptose com a evolução de melanomas cutâneos primários. Os resultados ratificaram tanto dados epidemiológicos já publicados em relação a sexo, idade e local da lesão, quanto a correlação entre a evolução da doença e os índices de Breslow. A análise dos escores compostos relativos à expressão das proteínas da apoptose revelou que o perfil imuno-histoquímico dessas proteínas parece não ter significado prognóstico, uma vez que não houve diferenças de expressão entre pacientes com e sem doença disseminada. Não foram encontradas alterações na expressão das proteínas da apoptose estudadas que pudessem sugerir o seu envolvimento tanto na gênese quanto na progressão de melanoma primário. O perfil imuno-histoquímico com tendência pró-apoptótica parece indicar que outros fatores seriam responsáveis pelo crescimento e disseminação da neoplasia / Cutaneous melanoma still constitutes the main cause of skin cancer death in developed world. Clinical behavior variability of this neoplasia has been only partially explained by clinical and histological aspects, and identification of biological variables can be important for determining specific risk groups. Sixty nine (69) patients with mild to severe primary cutaneous melanoma treated in 1990-2007 were studied aiming at (a) verifying clinical epidemiological aspects, (b) generating a Tissue microarray (TMA) for characterizing proteins expression of cutaneous melanoma > 1.0 mm, (c) analyzing the immunohistochemical expression of the apoptosis proteins Bcl2, Bax, Bak, Apaf-1, Caspase 3, Caspase 9, Caspase 8, FasL e Fas in 10 control nevi and in primary melanomas with thickness 1mm, (d) and correlating these proteins expression with the disease prognosis. Results have ratified known epidemiological date on gender, age and lesion localization as well as the correlation between the disease prognosis and the Breslow\'s indexes. Analysis of the composite scores relating to apoptosis proteins has revealed that their immunohistochemical profile seems to be not significant for determining the disease prognosis, since no differences in proteins expression were found when compared patients with and without disease dissemination. Alterations in proteins expression suggesting their role in the genesis as well as in the prognosis of primary melanoma were not evidenced. Immunohistochemical profile with pro-apoptosis trend seems to indicate other factor as responsible for the neoplasia growth and dissemination

<p>Many pro-apoptotic signals trigger mitochondrial cytochrome c release, leading to caspase activation and ultimate cellular breakdown. Cell survival pathways, including the mitogen-activated protein kinase (MAPK) cascade, promote cell viability both by impeding mitochondrial cytochrome c release and by inhibiting subsequent activation of caspases. Cytosolic cytochrome c is directly responsible for initiating formation of the caspase-activating apoptosome, which, in many cell types, plays a crucial role in the apoptotic process. Given the important role of cytochrome c in dismantling the dying cell, we wanted to investigate the process of cytochrome c-induced apoptosis with the goal of understanding how this mechanism is altered in certain malignant conditions. </p> <p> First, we examined cytochrome c-induced caspase activation in normal and tumorigenic mammary epithelial cells. Although most tumor types have developed mechanisms for evading apoptosis, we surprisingly discovered that breast cancer cells were hypersensitive to cytochrome c when compared with their normal counterpart. Specifically, breast cancer cells show increased binding of caspase-9 to the Apaf-1 caspase recruitment domain. This altered apoptosome formation is mediated by overexpression of the protein PHAPI in the malignant mammary epithelial cells. Immunoblot analysis demonstrated that protein levels of PHAPI are also elevated in human breast tumors. These results suggest a novel paradigm where breast cancer cells are refractory to cytochrome c release in response to certain stimuli, but they are quite sensitive to apoptosis downstream of the mitochondria. </p> <p> Secondly, we describe a mechanism for the inhibition of cytochrome c-induced caspase activation by MAPK signaling, identifying a novel mode of apoptotic regulation exerted through Apaf-1 phosphorylation by the 90-kDa ribosomal S6 kinase (Rsk). This Apaf-1 phosphorylation results in impaired apoptosome formation, thereby inhibiting caspase activation. The Rsk effect on Apaf-1 is antagonized by protein phosphatase 1 (PP1), which promotes Apaf-1 dephosphorylation. High endogenous levels of Rsk in PC3 prostate cancer cells leads to Apaf-1 phosphorylation and renders them relatively insensitive to cytochrome c, suggesting a role for Rsk signaling in the apoptotic resistance of certain cancers. These results identify a novel locus of apoptosomal regulation wherein MAPK signaling promotes direct Rsk-catalyzed phosphorylation of Apaf-1, resulting in decreased cellular responsiveness to cytochrome c. Collectively, this work provides insight into novel mechanisms of regulation for cytochrome c-induced apoptosis.</p> / Dissertation

A novel marine active polypeptide (PCF), isolated from the gonochoric Chinese scallop, Chlamys farreri, has potential antioxidant and anti-apoptotic activity against ultraviolet irradiation. We investigated whether UVB-induced HaCaT cell apoptosis occurs via the mitochondrial pathways Apaf-1/caspase-9 and Smac/XIAP/caspase-3. We then investigated the molecular mechanisms controlling the anti-apoptotic effect of PCF. Pre-treatment with PCF and caspase-9 inhibitor significantly inhibited UVB-induced apoptosis in HaCaT cells based on a DNA fragmentation assay and Hoechst 33258 staining. The expression of Apaf-1 and the cleavage of procaspase-9 were dose-dependently reduced by 1.42-5.96 mmol/L PCF pretreatment in UVB-irradiated HaCaT cells. This was followed by inhibition of cleavage of procaspase-3, whose activation induced cell apoptosis. Meanwhile, PCF significantly and dose-dependently enhanced the activation of ATPase. Furthermore, we demonstrated that PCF strongly inhibited the release of Smac from the mitochondria to cytosol by reducing the degradation of XIAP dose-dependently. We conclude that the protective effect of PCF against UVB irradiation in HaCaT cells may be attributed to the inhibition of the Apaf-1/caspase-9 and Smac/XIAP/caspase-3 apoptotic signaling pathways.