Histone deacetylase inhibitors (HDIs) are a group of chemo-preventive and chemo-therapeutic agents that have generated significant attention in clinical trials, given their ability to selectively induce cell cycle arrest, differentiation and/or apoptosis of tumor cells. Presently, these agents are proposed to function by altering gene expression levels, primarily by promoting histone hyperacetylation and gene transcription. However, in this thesis, HDIs are reported to control the expression of genes from the c-Src kinase family and p21WAF1 by means other than transcriptional activation. <p>Overexpression and activation of c-Src, a 60kDa non-receptor tyrosine kinase, has been implicated in the development, growth, progression, and metastasis of several human cancers, especially those of the colon. Butyrate and the more specific histone deacetylase inhibitor trichostatin A (TSA) were both found to effectively inhibit the expression of c-Src mRNA and protein in a number of tumor cell lines, including those of the colon, liver and breast. Expression of the SRC oncogene is alternatively regulated by the SRC1A and SRC1 promoters. HDIs were shown to repress c-Src expression by inhibiting transcription of both of these promoters, independent of any new protein synthesis. Furthermore, butyrate and TSA similarly regulated the expression of the c-Src family kinase (SFK) members Yes, Fyn, Lyn and Lck in human colon cancer cell lines. In addition, TATA binding protein (TBP) associated factor 1 (TAF1) was shown to be necessary for basal transcription of the SRC1A, YES and LYN promoters, but was not required for HDI mediated repression. <p>Induction of the potent cyclin dependent kinase inhibitor p21WAF1 has been identified to be a key feature of HDI mediated cell cycle arrest. The level of p21WAF1 expression has been extensively reported to be directly upregulated by HDIs in a p53 independent manner that requires Sp family binding sites in the p21WAF1 proximal promoter to induce transcription. However, HDIs were shown to be capable of inducing p21WAF1 gene expression, dependent on new protein synthesis, by increasing mRNA stability. To date, p21WAF1 mRNA stability has been extensively studied and a number of cis-acting elements in the 3 untranslated region (UTR) of the p21WAF1 mRNA have been implicated in the regulation of mRNA stability, such as AU rich elements (AREs) and a 42 nucleotide HuD/Elav binding element. Similarly, in this work, two novel cis-acting elements were identified in the 3 UTR of p21WAF1 and were shown to facilitate basal and HDI induced post-transcriptional regulation of p21WAF1 mRNA stability in HepG2 cells. Collectively, these studies highlight the intricacy of HDI mediated effects and challenge the preconceptions regarding the molecular mechanism of these anti-tumor agents.

Histone deacetylase inhibitors (HDIs) are a group of chemo-preventive and chemo-therapeutic agents that have generated significant attention in clinical trials, given their ability to selectively induce cell cycle arrest, differentiation and/or apoptosis of tumor cells. Presently, these agents are proposed to function by altering gene expression levels, primarily by promoting histone hyperacetylation and gene transcription. However, in this thesis, HDIs are reported to control the expression of genes from the c-Src kinase family and p21WAF1 by means other than transcriptional activation. <p>Overexpression and activation of c-Src, a 60kDa non-receptor tyrosine kinase, has been implicated in the development, growth, progression, and metastasis of several human cancers, especially those of the colon. Butyrate and the more specific histone deacetylase inhibitor trichostatin A (TSA) were both found to effectively inhibit the expression of c-Src mRNA and protein in a number of tumor cell lines, including those of the colon, liver and breast. Expression of the SRC oncogene is alternatively regulated by the SRC1A and SRC1 promoters. HDIs were shown to repress c-Src expression by inhibiting transcription of both of these promoters, independent of any new protein synthesis. Furthermore, butyrate and TSA similarly regulated the expression of the c-Src family kinase (SFK) members Yes, Fyn, Lyn and Lck in human colon cancer cell lines. In addition, TATA binding protein (TBP) associated factor 1 (TAF1) was shown to be necessary for basal transcription of the SRC1A, YES and LYN promoters, but was not required for HDI mediated repression. <p>Induction of the potent cyclin dependent kinase inhibitor p21WAF1 has been identified to be a key feature of HDI mediated cell cycle arrest. The level of p21WAF1 expression has been extensively reported to be directly upregulated by HDIs in a p53 independent manner that requires Sp family binding sites in the p21WAF1 proximal promoter to induce transcription. However, HDIs were shown to be capable of inducing p21WAF1 gene expression, dependent on new protein synthesis, by increasing mRNA stability. To date, p21WAF1 mRNA stability has been extensively studied and a number of cis-acting elements in the 3 untranslated region (UTR) of the p21WAF1 mRNA have been implicated in the regulation of mRNA stability, such as AU rich elements (AREs) and a 42 nucleotide HuD/Elav binding element. Similarly, in this work, two novel cis-acting elements were identified in the 3 UTR of p21WAF1 and were shown to facilitate basal and HDI induced post-transcriptional regulation of p21WAF1 mRNA stability in HepG2 cells. Collectively, these studies highlight the intricacy of HDI mediated effects and challenge the preconceptions regarding the molecular mechanism of these anti-tumor agents.

Checkpoint kinase 1 (Chk1) is a key player in the DNA damage response signalling pathway and the mode of Chk1 activation whereby it undergoes ATRdependent phosphorylation at Ser317 and Ser345 is well characterised. It has been suggested that phosphorylation at the ATR sites relieves the auto-inhibitory action conferred by the C-terminal negative regulatory domain on the catalytic core of Chk1. In this study, we show that Chk1 activity can also be stimulated by docking to an N-terminal region of the growth regulator p21waf1 and this docking domain is necessary for efficient Chk1-dependent phosphorylation of p21 at Ser146. In addition, Chk1 and p21 are shown to form a transient interaction by immunoprecipitation. Interestingly, although the isolated p21 docking domain can activate Chk1 in trans, a mutant where the C-terminal 70 amino acids are truncated is refractory to stimulation whereas mutation of the ATR phosphoacceptor sites does not affect docking dependent activation. Furthermore, when the amino acid sequence of the p21 docking domain was aligned with the sequence of Chk1, homology to the F region on the kinase domain was identified. Mutation of two conserved tryptophan residues within the homology region appears to release the C-terminus from intramolecular interactions rendering it susceptible to cleavage and refractory to allosteric stimulation. Furthermore, small peptides based on this region of Chk1, like the p21 docking domain, are able to activate Chk1 in trans and disrupt interaction between the N-terminal and Cterminal domains. Interestingly, peptide microarray showed that Chk1 stimulated by activating peptide is able to phosphorylate novel peptide substrates which are not observed with unstimulated Chk1. The data suggest that the last C-terminal 70 amino acids of Chk1 play an important role in auto-inhibition through interaction with the F region of the core catalytic domain. Binding to p21 is able to activate Chk1 by inhibiting the auto-inhibitory interaction independent of phosphorylation at the Ser317 and Ser345 sites. Furthermore, activating peptide is able to modulate Chk1 specificity towards other substrates.

A proteína P53 pode estar virtualmente alterada em todos os cânceres humanos e portanto, na ausência de mutação, uma possibilidade para a inativação da p53 é a formação de complexo com outras proteínas, tal como a proteína Mdm2. Estudos prévios realizados em nosso laboratório demonstraram a superexpressão de Mdm2 na ausência de expressão da proteína P53 em adenomas pleomórficos. O objetivo deste estudo foi analisar a expressão das proteínas Mdm2, P53, P21 e Akt em adenoma pleomórfico e o mioepitelioma através das técnicas de imunoistoquímica, western blotting e imunofluorescência. A superexpressão de Mdm2 e Akt foi encontrada na maioria das linhagens e lesões utilizadas neste estudo enquanto as proteínas P53 e P21 não demonstraram expressão nas neoplasias estudadas. As superexpressões das proteínas Mdm2 e Akt estão relacionadas à tumorigênese em neoplasias benignas de glândula salivar. / The P53 protein can be altered in virtually all human cancers and in the absence of mutations, P53 inactivation is possible via complex formation with others proteins, such as the Mdm2. Previous studies from our laboratory showed overexpression of mdm2 and lack of p53 expression in pleomorphic adenomas. The aim of this study was to analyze the expression of Mdm2, P53, P21 and Akt proteins in pleomorphic adenomas and myoepiteliomas by Western blotting, immunohistochemistry and immunofluorescence techniques. Overexpression of Mdm2 and Akt was present in the majority of cell lineages and tumors studied, while the expression of P53 and P21 proteins was considered absent. Overexpression of Mdm2 and Akt are related to the tumorigenesis of benign salivary gland neoplasms.

A proteína P53 pode estar virtualmente alterada em todos os cânceres humanos e portanto, na ausência de mutação, uma possibilidade para a inativação da p53 é a formação de complexo com outras proteínas, tal como a proteína Mdm2. Estudos prévios realizados em nosso laboratório demonstraram a superexpressão de Mdm2 na ausência de expressão da proteína P53 em adenomas pleomórficos. O objetivo deste estudo foi analisar a expressão das proteínas Mdm2, P53, P21 e Akt em adenoma pleomórfico e o mioepitelioma através das técnicas de imunoistoquímica, western blotting e imunofluorescência. A superexpressão de Mdm2 e Akt foi encontrada na maioria das linhagens e lesões utilizadas neste estudo enquanto as proteínas P53 e P21 não demonstraram expressão nas neoplasias estudadas. As superexpressões das proteínas Mdm2 e Akt estão relacionadas à tumorigênese em neoplasias benignas de glândula salivar. / The P53 protein can be altered in virtually all human cancers and in the absence of mutations, P53 inactivation is possible via complex formation with others proteins, such as the Mdm2. Previous studies from our laboratory showed overexpression of mdm2 and lack of p53 expression in pleomorphic adenomas. The aim of this study was to analyze the expression of Mdm2, P53, P21 and Akt proteins in pleomorphic adenomas and myoepiteliomas by Western blotting, immunohistochemistry and immunofluorescence techniques. Overexpression of Mdm2 and Akt was present in the majority of cell lineages and tumors studied, while the expression of P53 and P21 proteins was considered absent. Overexpression of Mdm2 and Akt are related to the tumorigenesis of benign salivary gland neoplasms.

The p53 protein is a primary mediator of apoptosis and growth arrest after exposure to DNA-damaging agents. Previous work has categorised a wild type p53 gene in the majority of childhood acute lymphoblastic leukaemia (ALL) cases, in which instance the p53 protein functions as a modulator of chemotherapy-induced cell death. In contrast, certain p53-induced proteins, such as p21WAF1, can act in an anti-apoptotic manner, and bestow resistance to chemotherapy. Previous studies of the p53 pathway in ALL have utilised cell lines and primary material. In this study a model of ALL was utilised that had previously been developed from a heterogeneous panel of patient biopsies established as xenografts in immune-deficient mice, and are adaptable for short term in vitro culture. A wild-type p53 protein response to etoposide and nutlin-3 exposure was a feature of the whole ALL xenograft panel, irrespective of clinical characteristics and disease biology. While a range of p53 target genes were induced in B-cell precursor (BCP)-ALL and T-ALL xenografts after etoposide exposure, there was negligible induction of p21WAF1 in T- ALL samples. Further work with the histone deacetylase inhibitor vorinostat facilitated p53-independent induction of p21WAF1 in BCP-ALL samples, yet failed to induce p21WAF1 in T- ALL. An association was observed between reduced p21WAF1 expression in the T-ALL samples and decreased histone H3 acetylation in the p21WAF1 promoter together with increased cytosine methylation in the first exon/intron of the p21WAF1 gene. These results suggest that p21WAF1 in T-ALL cells is subject to epigenetic modifications that cause transcriptional silencing. Defective induction of p21WAF1 in T-ALL xenografts was associated with increased sensitivity to the death-inducing effects of drugs, phosphatidylserine (PS) externalisation and caspase-3/-7 activity after drug exposure, indicating that p21WAF1 may exert an anti-apoptotic activity. As proof of principle, p21WAF1 was silenced in Nalm-6 cells by micro-RNA transduction and these cells exhibited increased sensitivity and rapid PS externalisation after drug exposure. A combination of a p21WAF1 inhibitory agent and vorinostat gave some pharmacological evidence to suggest that p21WAF1 inhibition could enhance drug efficacy. Overall, these investigations provide insight into the epigenetic regulation of p21WAF1 and demonstrate an anti-apoptotic role for p21WAF1 in childhood ALL cells.

The p53 protein is a primary mediator of apoptosis and growth arrest after exposure to DNA-damaging agents. Previous work has categorised a wild type p53 gene in the majority of childhood acute lymphoblastic leukaemia (ALL) cases, in which instance the p53 protein functions as a modulator of chemotherapy-induced cell death. In contrast, certain p53-induced proteins, such as p21WAF1, can act in an anti-apoptotic manner, and bestow resistance to chemotherapy. Previous studies of the p53 pathway in ALL have utilised cell lines and primary material. In this study a model of ALL was utilised that had previously been developed from a heterogeneous panel of patient biopsies established as xenografts in immune-deficient mice, and are adaptable for short term in vitro culture. A wild-type p53 protein response to etoposide and nutlin-3 exposure was a feature of the whole ALL xenograft panel, irrespective of clinical characteristics and disease biology. While a range of p53 target genes were induced in B-cell precursor (BCP)-ALL and T-ALL xenografts after etoposide exposure, there was negligible induction of p21WAF1 in T- ALL samples. Further work with the histone deacetylase inhibitor vorinostat facilitated p53-independent induction of p21WAF1 in BCP-ALL samples, yet failed to induce p21WAF1 in T- ALL. An association was observed between reduced p21WAF1 expression in the T-ALL samples and decreased histone H3 acetylation in the p21WAF1 promoter together with increased cytosine methylation in the first exon/intron of the p21WAF1 gene. These results suggest that p21WAF1 in T-ALL cells is subject to epigenetic modifications that cause transcriptional silencing. Defective induction of p21WAF1 in T-ALL xenografts was associated with increased sensitivity to the death-inducing effects of drugs, phosphatidylserine (PS) externalisation and caspase-3/-7 activity after drug exposure, indicating that p21WAF1 may exert an anti-apoptotic activity. As proof of principle, p21WAF1 was silenced in Nalm-6 cells by micro-RNA transduction and these cells exhibited increased sensitivity and rapid PS externalisation after drug exposure. A combination of a p21WAF1 inhibitory agent and vorinostat gave some pharmacological evidence to suggest that p21WAF1 inhibition could enhance drug efficacy. Overall, these investigations provide insight into the epigenetic regulation of p21WAF1 and demonstrate an anti-apoptotic role for p21WAF1 in childhood ALL cells.

As proteínas MDM2, P53, P21 e pAKT estão entre as muitas já identificadas que visam, por meio do equilíbrio direto e indireto entre si, manter o balanço entre morte e proliferação celular. Existe um leque aberto de hipóteses sobre a via de atuação dessas proteínas na tumorigênese de glândulas salivares, porém não há dados conclusivos. O objetivo deste estudo foi avaliar a expressão das proteínas MDM2, P53, P21 e pAkt em carcinoma adenóide cístico e adenocarcinoma não-específico de glândula salivar, através das técnicas de imunoistoquímica, em casos fixados em parafina; e imunofluorescência e western-blotting, em linhagens celulares provenientes dessas lesões. Os estudos imunoistoquímicos mostraram expressão de MDM2 e pAKT na maioria dos carcinomas adenóides císticos (CAC) avaliados e nos 3 casos de adenocarcinoma não-específicos (ANE). As linhagens Cac2 (proveniente de carcinoma adenóide cístico) e HSG (derivada de adenocarcinoma não-específico) também exibiram expressão das proteínas MDM2 e pAKT tanto no núcleo quanto no citoplasma celular. A proteína P53 mostrou expressão variável entre os diferentes CAC analisados e os 3 casos de ANE estudados mostraram marcação positiva para a proteína P53. Com relação às linhagens celulares, a Cac2 não expressou a proteína P53, enquanto a HSG apresentou expressão nuclear e citoplasmática dessa proteína. As glândulas salivares normais não exibiram marcação imunoistoquímica para as proteínas MDM2, P53, P21 e pAKT. Os resultados deste estudo sugerem que as proteínas pAKT e MDM2 estão envolvidas na tumorigênese e/ou progressão tumoral de carcinoma adenóide cístico e adenocarcinoma não especifico. / MDM2, P53, P21 and pAKT are proteins co-related to the balance between cell death and survival. There are many hypothesis on the role of these proteins in salivary gland tumorigenesis, however, no conclusive data have been published. Theaim of this study was to evaluate the expression of MDM2, P53, P21 and pAKT proteins on adenoid cystic carcinomas (ACC) and adenocarcinoma not otherwise specified (ANOS) through immunohistochemistry, immunofluorescence and westernblotting techniques. The immunostaining studies showed MDM2 and pAKT expression in most cases of adenoid cystic carcinoma and in all adenocarcinoma not-otherwise specified. The cell lines CAC2 (derived from adenoid cystic carcinoma) and HSG (derived from adenocarcinoma not otherwise specified) also showed nuclear and cytoplasmic expression of MDM2 and pAKT. The expression of P53 was variable in the different ACCs analyzed and was absent on CAC2 cells. However, P53 was strongly positive in all ANOS, and HSG cells also showed nuclear and cytoplasmic staining. No MDM2, P53, P21 and pAKT expression was found in normal salivary glands. Therefore, MDM2 and pAKT may participate in the tumorigenesis and/or progression of adenoid cystic carcinoma and adenocarcinoma not otherwise specified.

As proteínas MDM2, P53, P21 e pAKT estão entre as muitas já identificadas que visam, por meio do equilíbrio direto e indireto entre si, manter o balanço entre morte e proliferação celular. Existe um leque aberto de hipóteses sobre a via de atuação dessas proteínas na tumorigênese de glândulas salivares, porém não há dados conclusivos. O objetivo deste estudo foi avaliar a expressão das proteínas MDM2, P53, P21 e pAkt em carcinoma adenóide cístico e adenocarcinoma não-específico de glândula salivar, através das técnicas de imunoistoquímica, em casos fixados em parafina; e imunofluorescência e western-blotting, em linhagens celulares provenientes dessas lesões. Os estudos imunoistoquímicos mostraram expressão de MDM2 e pAKT na maioria dos carcinomas adenóides císticos (CAC) avaliados e nos 3 casos de adenocarcinoma não-específicos (ANE). As linhagens Cac2 (proveniente de carcinoma adenóide cístico) e HSG (derivada de adenocarcinoma não-específico) também exibiram expressão das proteínas MDM2 e pAKT tanto no núcleo quanto no citoplasma celular. A proteína P53 mostrou expressão variável entre os diferentes CAC analisados e os 3 casos de ANE estudados mostraram marcação positiva para a proteína P53. Com relação às linhagens celulares, a Cac2 não expressou a proteína P53, enquanto a HSG apresentou expressão nuclear e citoplasmática dessa proteína. As glândulas salivares normais não exibiram marcação imunoistoquímica para as proteínas MDM2, P53, P21 e pAKT. Os resultados deste estudo sugerem que as proteínas pAKT e MDM2 estão envolvidas na tumorigênese e/ou progressão tumoral de carcinoma adenóide cístico e adenocarcinoma não especifico. / MDM2, P53, P21 and pAKT are proteins co-related to the balance between cell death and survival. There are many hypothesis on the role of these proteins in salivary gland tumorigenesis, however, no conclusive data have been published. Theaim of this study was to evaluate the expression of MDM2, P53, P21 and pAKT proteins on adenoid cystic carcinomas (ACC) and adenocarcinoma not otherwise specified (ANOS) through immunohistochemistry, immunofluorescence and westernblotting techniques. The immunostaining studies showed MDM2 and pAKT expression in most cases of adenoid cystic carcinoma and in all adenocarcinoma not-otherwise specified. The cell lines CAC2 (derived from adenoid cystic carcinoma) and HSG (derived from adenocarcinoma not otherwise specified) also showed nuclear and cytoplasmic expression of MDM2 and pAKT. The expression of P53 was variable in the different ACCs analyzed and was absent on CAC2 cells. However, P53 was strongly positive in all ANOS, and HSG cells also showed nuclear and cytoplasmic staining. No MDM2, P53, P21 and pAKT expression was found in normal salivary glands. Therefore, MDM2 and pAKT may participate in the tumorigenesis and/or progression of adenoid cystic carcinoma and adenocarcinoma not otherwise specified.

La sénescence induite par l'oncogène, ou OIS (Oncogene-Induced Senescence), est un mécanisme permettant d'empêcher la prolifération anarchique des cellules lors du stress oncogénique. Lors de la tumorigenèse, ce processus est inhibé par l'inactivation des gènes suppresseurs de tumeurs pour permettre la prolifération et la transformation cellulaire. L'OIS est un processus conservé dans les cellules primaires, cependant, nous l'avons observé dans des cellules transformées à la suite de l'expression de H-RasV12. Nous avons étudié les acteurs de cette réponse dans une lignée d'adénome colorectal ayant déjà inactivé les suppresseurs de tumeurs p16Ink4a et p53. Nous avons observé que l'oncogène Ras induisait l'expression de p21Waf1 et conduisait à la mise en place de la sénescence. Dans ces conditions, nous avons vu que p21Waf1 inhibait la transcription des gènes du cycle cellulaire Cdc25A et Plk1 pour permettre l'arrêt de la prolifération. Au cours de ce travail nous avons remarqué que certaines cellules échappaient à cet arrêt. Par l'étude de la présence de SAHF (Senescence- Associated Heterochromatine Foci), nous avons observé que ces cellules ne présentaient plus ce phénotype caractéristique de la sénescence, mais des marqueurs de cellules mésenchymateuses, et que certaines d'entre elles avaient subi une dédifférenciation. Les résultats que nous avons obtenus indiquent que lors de l'expression de l'oncogène Ras, certaines cellules échappent à l'OIS et que cet échappement est associé à une EMT (Epithelial-Mesenchymal Transition) et à l'émergence de cellules dédifférenciées. L'EMT contribuerait à l'échappement des cellules à l'arrêt du cycle cellulaire.