p53 is a nodal tumor suppressor protein that acts as a major defense against cancers. Approximately 50% of human tumours have mutations in p53 gene. Among its myriad features, the most distinctive is the ability to elicit both apoptotic death and cell cycle arrest. p53 has several isoforms. Most of them are produced by either internal promoter activity of the gene or alternate splicing of the pre-mRNA. Apart from these mechanisms, p53 mRNA has also been shown to be translated into two isoforms, the full-length p53 (FL-p53) and a truncated isoform ΔN-p53, which acts as a dominant-negative inhibitor of FL-p53. Under conditions of cellular stress, the canonical mode of translation initiation is compromised. To maintain the synthesis of proteins important for cell survival and cell-fate decisions, a subset of cellular mRNAs utilizes a non-canonical mode of translation initiation. The 5’ untranslated region of these mRNAs are highly structured and function as Internal Ribosome Entry Site (IRES). Previously, from our laboratory it has been shown that translation of p53 and its N-terminally truncated isoform ΔN-p53 can be initiated by IRES mediated mechanism. IRES mediated translation of ΔNp53 was maximum at G1-S phase but that of FL-p53 was maximum at the G2-M phase. Interestingly in case of a human genetic disorder X-linked dyskeratosis congenita (X-DC), aberrant IRES mediated p53 translation has been reported. It has also been reported that during oncogenic induced senescence (OIS) a switch between cap-dependent to IRES meditated translation occurs in p53 mRNA. From our laboratory, we have also demonstrated that polypyrimidine tract binding protein (PTB) positively regulates the IRES activities of both the p53 isoforms by shuttling from nucleus to the cytoplasm during genotoxic stress conditions. It is very important to understand how these two isoforms are regulated and in turn control the cellular functions. In the first part of the thesis, to investigate the importance of the structural integrity of the cis acting elements within p53 RNA, we have compared the secondary structure of the wild-type RNA with cancer-derived silent mutant p53 RNAs having mutations in the IRES elements such as L22L (CTA to CTG) a natural cancer mutation and Triple Silent Mutation (mutations were present at the wobble position of codon 17, 18, 19). These mutations result in the conformational alterations of p53 IRES RNA that abrogates the IRES function ex vivo significantly. It appears that these mutant RNAs failed to bind some trans-acting factors (p37, p41/44 etc) which might be critical for the IRES function. By super-shift assay using anti hnRNPC1/C2 antibody, we have demonstrated that the TSM mutant showed reduced binding to this protein factor. Partial knockdown of hnRNP C1/C2 showed significant decrease in p53 IRES activity and reduced synthesis of ΔN-p53. Also we have showed that introducing compensatory mutations in TSM mutant RNA rescued the secondary structure as well as function of p53 IRES. Further, the role of another silent point mutation in the coding sequence of p53 was investigated. Silent mutation (CCG to CCA) at codon 36 (P36P) showed decreased IRES activity. The mutation also resulted in differential binding of cellular proteins. Taken together, our observations suggest pivotal role of some specific trans acting factors in regulating the p53-IRES function, which in turn influences the synthesis of different p53 isoforms. In the second part of the thesis, p53 IRES RNA interacting proteins were identified using RNA affinity approach. Annexin A2 and PTB associated Splicing Factor (PSF/SFPQ) were identified and their interaction with p53 IRES RNA in vitro and ex vivo was studied. Interestingly, in the presence of Ca2+ ions Annexin A2 showed increased binding with p53 IRES. By competition UV crosslinking we have showed Annexin A2 and PSF interact specifically with p53 IRES. Toe printing assay results showed the putative contact points of Annexin A2 and PSF proteins on p53 IRES RNA. Interestingly, both proteins showed extensive toe-prints in the neighbourhood of the initiator AUG region of p53. Further, competition UV-crosslinking reveals the interplay of these two proteins. Annexin A2 and PSF appear to compete each other for binding with p53 IRES. PSF is known to interact with PTB protein. Since PTB also interacts with p53 IRES and positively regulates the translation, we wanted to study the interplay between PTB and PSF proteins binding with p53 IRES. To address this, we have performed competition UV crosslinking experiment and showed that increasing concentrations of PTB decreases PSF and p53 IRES interaction. However, increasing concentrations of PSF does not decrease or increase in PTB p53 IRES interaction. Results suggest that both Annexin A2 and PSF proteins play important role in regulation of p53 IRES activity. To address the physiological role of Annexin A2 and PSF proteins on p53 IRES activity, these proteins were partially knocked down in cellulo. This in turn showed decrease in p53 IRES activity in dual luciferase assays as well as in the steady state levels of both the p53 isoforms in transient transfection experiments. Heightened or continued expression of p53 protein is very important under stress where IRES-dependent translation supersedes normal cap-dependent translation. Results showed that expression of Annexin A2 under doxorubicin and thapsigargin induced stress are important for maintenance of both p53 IRES activity and steady state levels of p53 isoforms. Earlier from our laboratory we have showed that the IRES responsible for ∆N-p53 translation is active at G1/S phase while the IRES responsible for full length p53 translation is active at G2/M phase. Subcellular localization of the trans-acting factors plays a pivotal role in regulation of IRES activity of cellular mRNA. In this context we wanted to study the nuclear and cytoplasm localization of Annexin A2 under different cell cycle stages. We have seen Annexin A2 protein is dispersed in nucleus and cytoplasm at G1/S boundary, but post-G2 phase it moved from nucleus to cytoplasm. Further we wanted to investigate the effect of Annexin A2 and PSF on expression of p53 transactivated genes. Partial knock down of Annexin A2 and PSF proteins showed decrease in p21 luciferase activity. By real-time PCR analysis, we have also showed decrease in expression of different p53 targets upon silencing of Annexin A2 protein. Taken together, our observations suggest pivotal role of cis acting and trans-acting factors in regulating the p53-IRES function, which in turn influences the synthesis of p53 isoforms.

A síndrome de Li-Fraumeni (LFS) e sua variante like (LFL) são associadas a mutações germinativas no gene TP53 e predispõe ao alto risco para múltiplos tumores em idade jovem. Analisamos 91 famílias LFS/LFL do sul/sudeste do Brasil para mutações germinativas e haplótipos de TP53 (PIN2, PIN3 e PEX4) e MDM2 (309T-G). A mutação R337H ocorreu em 44,4% das famílias avaliadas. Em 750 controles da região a freqüência populacional da mutação foi 0,3%. A genotipagem de oito indivíduos não relacionados R337H-positivos para 29 TAG SNPs intragênicos demonstrou o mesmo haplótipo raro estabelecendo efeito fundador para R337H. O alelo duplicado no PIN3 apresenta impacto modificador e retardo de 17,1 anos na ocorrência de tumores em famílias com mutação no TP53, enquanto o SNP309 MDM2 modula a idade dos sarcomas de partes moles. / Li-Fraumeni syndrome (LFS) and its variant like (LFL) are associated with germline mutations in the TP53 gene and predispose to a variety of cancers at an earlier age. We analyzed 91 LFS/LFL families from southern Brazil for germline mutations in TP53 and polymorphisms in TP53 (PIN2, PIN3, PEX4) and MDM2 (309T-G). The germline TP53 mutation R337H was found in 44.4% of all families included. In 750 controls from the same region, mutation prevalence was 0.3%. Genotyping of eight unrelated R337H-positive individuals for 29 intragenic TAG SNPs showed that they all shared the same rare haplotype confirming the founder effect for the mutation. Duplication of PIN3 had a modifier effect on the age of tumor onset (delay of 17.1 years) in TP53 mutation carriers whereas MDM2 SNP309 modulated age of onset for soft-tissue sarcomas.

As proteínas p53, MDM-2, c-Kit, ErbB-2, PCNA e p-glicoproteína foram estudadas em 42 amostras de osteossarcoma ao diagnóstico, através da técnica de imunohistoquímica, e foram correlacionados estes achados com o prognóstico destes pacientes. O p-53 foi positivo em 23,1% (9/39), PCNA em 71,4% (25/35), p-glicoproteína em 40,5% (15/37), MDM-2 em 34,8% (8/23), c-kit em 67,6% (25/37) e ErbB-2 em 17,9% (7/39). Na análise univariada, a presença de metástases ao diagnóstico, a positividade de p53 e ErbB-2 influenciaram o prognóstico individualmente, entretanto, na análise multivariada, a presença de metástase ao diagnóstico revelou-se o único fator de prognóstico estatisticamente significante / We study, by imunohistochemistry technique, the proteins p53, MDM-2, c-Kit, ErbB-2, PCNA and p-glycoprotein in samples of osteosarcoma tumors at diagnosis and its correlation with the prognosis of this patients. The p-53 was positive in 23,1% (9/39), PCNA in 71,4% (25/35), p-glycoprotein in 40,5% (15/37), MDM-2 in 34,8% (8/23), c-kit in 67,8%(25/37) and ErbB-2 in 17,9% (7/39) of the samples. In the univariate analysis, the presence of metastasis at diagnosis, the positivity of p-53 and ErbB-2 influenced the prognosis individually, otherwise, in the multivariate analysis, the presence of metastasis at diagnosis was the only prgnostic factor statistically significant

O melanoma cutâneo é a neoplasia de pele de maior mortalidade. A imprevisibilidade de sua evolução é uma de suas características principais, o tratamento do tumor primário é, atualmente, de pouca morbidade e, na doença disseminada, as opções terapêuticas são pouco eficazes. É fundamental a pesquisa de marcadores tumorais que permitam a previsão da evolução, melhor compreensão da patogênese do melanoma e possibilitem a descoberta de alvos moleculares. Nesse contexto, estudos genéticos mostraram a importância da regulação do ciclo celular, especialmente a passagem da fase G1-S. Importantes fatores envolvidos compõem a cascata da proteína Rb (p16, ciclina D1, CDK4 e pRb) e da proteína p53 (p53 e p21). Objetivo: verificar a frequência da expressão de p16, ciclina D1,CDK4, pRb, p53 e p21 em melanomas cutâneos de cabeça, pescoço e tronco e sua relação com prognóstico. Métodos: Estudo retrospectivo envolvendo 46 pacientes (sendo 67,3% homens, idade média 57,7 ± 15,8 anos) com melanoma cutâneo de cabeça, pescoço e tronco que foram tratados pela mesma equipe com seguimento mínimo de dois anos. Foram estudados fatores clínicos (topografia do tumor primário, tempo de seguimento, ocorrência de metástases e óbito relacionado), histopatológicos (tipo histológico, índice de Clark, índice de Breslow) e análise imuno-histoquímica pela técnica de micro-array para as proteínas reguladoras do ciclo celular p16, ciclina D1, CDK4, pRb, p53 e p21. Resultados: Houve proporção igualitária entre as topografias (23 casos em tronco, 23 em cabeça e pescoço). Treze pacientes com Clark I (29,5%), cinco com II (11,3%), 16 com III (36,5%), 10 com IV (22,7%) e nenhum com Clark V. A média das medidas de Breslow foi 0,96 (DP=1,01). O seguimento médio foi de 77 meses (DP=47). Oito dos 46 pacientes (17,3%) tiveram evolução desfavorável, com seis óbitos relacionados. A idade foi mais elevada no grupo com evolução desfavorável (p=0,04). Houve expressão de p16 em 80%, ciclina D1 em 58,9%, CDK4 em 43,5%, pRb em 58,5%, p53 em 53,6% e p21 em 52,3% dos melanomas. Em análise univariada, a expressão do p21 foi relacionada com evolução desfavorável (p=0,04), o que não foi observado com a expressão dos outros marcadores (p>0,05). Conclusão: A expressão da proteína p21 nos melanomas cutâneos de cabeça, pescoço e tronco foi relacionada com evolução desfavorável, o que não ocorreu com outros fatores envolvidos na regulação do ciclo celular / Melanoma is the most lethal skin cancer. The outcome of melanoma is not predictable in most cases. Although the treatment for the primary tumor is well tolerated, there are no effective therapeutic options in disseminated disease. Efforts are being made in the search for tumoral markers that may predict outcome, increase the comprehension of melanoma pathogenesis, and may also help the search for molecular targets. In this issue, genetic studies concerning the regulation of cell cycle, including the G1-S checkpoint, are important. The retinoblastoma protein (pRb) pathway (p16, cyclin D1, CDK4 and pRb) and the p53 pathway (p53 and p21) are part of this regulation. Objectives: to verify the expression of p16, cyclin D1, CDK4, pRb, p53 and p21 in head, neck and trunk melanomas, and its correlation with prognosis. Method: Retrospective study approved by institution ethics committee. Fourtysix head, neck and trunk melanoma patients (67.3% men, mean age 57.7±15.8) treated by a single surgeon with minimum 2-years follow-up were enrolled. Clinical factors (primary tumor location, follow-up period, metastasis or related deaths), pathologic (histological subtype, Clark and Breslow index) and microarray immunohystochemical analysis of the cell cycle proteins p16, cyclin D1, CDK4, pRb, p53 and p21. Results: Location of the primary tumor was equal for head/neck and trunk (50% each). Thirteen patients were classified as Clark I (29.5%), five as Clark II (11.3%), 16 as Clark III (36.5%), 10 as IV (22.7%), none as Clark V. Mean Breslow measure was 0.96±1.01. Mean follow-up was 77±47 months. Eight patients (17.3%) had bad outcome, with six related deaths. Patients with worse outcome had a higher mean age at diagnosis (p=0,04). Expression of p16 was positive in 80%. Cyclin D1 was positive in 58.9%. CDK4 was positive in 43.5%. pRb was positive in 58.5%. p53 was positive in 53.6%. p21 was positive in 52.3%. Univariated analysis showed that p21 expression was related to worse outcome (p=0,04), while the other markers were not (p>0,05). Conclusion: p21 expression in head, neck and trunk melanomas was related to worse outcome. Expression of the other cell cycle regulators proteins was not

Abstract Mutations in the currently known breast cancer susceptibility genes account for only 25–30% of all familial cases. Novel susceptibility genes can be identified by several methods, including candidate gene re-sequencing and genome-wide microarrays. We have applied microarrays for the detection of a new genomic variation class, copy number variants (CNVs), which potentially could disrupt genes in multiple pathways related to breast cancer susceptibility. The aim of the current study was to evaluate the role of the RNF8, UBC13, MMS2 and RAD51C DNA damage response genes in breast cancer susceptibility as well as to study if rare CNVs are associated with the predisposition to this disease. The analysis of 123 familial breast cancer cases revealed altogether nine different changes in the RNF8 and UBC13 candidate genes. However, none of the observed alterations were considered pathogenic. No alterations were observed in MMS2. The obtained results suggest that breast cancer predisposing alterations in RNF8, UBC13 and MMS2 are rare, or even absent. The RAD51C mutation screening of 147 familial breast cancer cases and 232 unselected ovarian cancer cases revealed two deleterious mutations: c.-13_14del27 was observed in a breast cancer case with familial history of ovarian cancer and c.774delT in an ovarian cancer case. Both mutations were absent in the control cohort. The results of the study support the hypothesis that rare variants of RAD51C predispose predominantly to ovarian cancer. A genome-wide scan of CNVs was performed for 103 familial breast cancer cases and 128 controls. The biological networks of the genes disrupted by CNVs were different between the two groups. In familial breast cancer cases, the observed mutations disrupted genes, which were significantly overrepresented in cellular functions related to maintenance of genomic integrity (P=0.0211). Biological network analysis showed that the disrupted genes were closely related to estrogen signaling and TP53-centered tumor suppressor network, and this result was confirmed by the analysis of an independent young breast cancer cohort of 75 cases. These results suggest that rare CNVs represent an alternative source of genetic variation contributing to hereditary risk for breast cancer. / Tiivistelmä Tunnetut rintasyöpäalttiusgeenien mutaatiot selittävät vain 25–30 prosenttia kaikista perinnöllisistä rintasyöpätapauksista. Uusia alttiusgeenejä voidaan tunnistaa useilla eri menetelmillä, kuten kandidaattigeenien mutaatiokartoituksella ja genomin-laajuisilla mikrosirutekniikoilla. Tässä tutkimuksessa sovelsimme mikrosirutekniikkaa uuden geneettisen variaatioluokan, kopiolukuvariaation (CNV), tutkimiseen. CNV:t voivat vaurioittaa lukuisia rintasyöpäalttiuteen liittyviä biokemiallisia reittejä. Tämän tutkimuksen tarkoitus oli arvioida RNF8-, UBC13-, MMS2- ja RAD51C -DNA- vauriovastegeenien sekä harvinaisten CNV:iden yhteyttä rintasyöpä-alttiuteen. 123 familiaalisen rintasyöpätapauksen analyysissä löytyi yhteensä yhdeksän muutosta RNF8- ja UBC13-geeneistä, joista yksikään ei osoittautunut patogeeniseksi. MMS2-geenissä ei havaittu muutoksia. Tulosten perusteella rintasyövälle altistavat muutokset RNF8-, UBC13- ja MMS2- geeneissä ovat joko erittäin harvinaisia tai niitä ei esiinny lainkaan. RAD51C-geenin mutaatiokartoitus 147 familiaalisesta rintasyöpätapauksesta sekä 232 valikoimattomasta munasarjasyöpätapauksesta paljasti kaksi haitallista mutaatiota. c.-13_14del27 havaittiin rintasyöpäpotilaalla, jonka suvussa esiintyi munasarjasyöpää, ja c.774delT todettiin munasarjasyöpäpotilaalta. Kumpaakaan mutaatiota ei havaittu verrokkiaineistossa. Tulokset vahvistavat hypoteesia RAD51C-geenin harvinaisten varianttien yhteydestä pääasiassa munasarjasyöpäriskiin. CNV:iden genomin-laajuinen skannaaminen suoritettiin 103 familiaaliselle rintasyöpätapaukselle ja 128 verrokille. CNV:iden häiritsemien geenien muodostamat biologiset verkostot olivat erilaiset näiden kahden ryhmän välillä. Familiaalisilla rintasyöpätapauksilla havaitut CNV:t vaikuttivat geeneihin, jotka olivat voimakkaasti korostuneita genomin eheyttä ylläpitävissä tehtävissä (P=0.0211). Biologisten verkostojen analyysi paljasti, että CNV:iden vahingoittamat geenit liittyivät läheisesti estrogeenisignalointiin sekä TP53-tuumorisupressoriverkostoon, ja tämä tulos vahvistettiin analysoimalla riippumatonta nuorista rintasyöpäpotilaista koostuvaa kohorttia (N=75). Tutkimuksen tulosten mukaan harvinaiset CNV:t ovat vaihtoehtoinen geneettisen variaation lähde perinnölliseen rintasyöpäalttiuteen.

Mitochondrial protein homeostasis is crucial for cellular function and integrity. It is ensured by many specific mitochondrial proteases with possible chaperone functions located across the various mitochondrial subcompartments. In the first part, we have focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 and YME1L of the mitochondrial inner membrane complexes m- and i-AAA in HEK293 cells. The double AFG3L2/YME1L knockdown cells showed severe alteration in OPA1 protein processing, marked elevation in OMA1 protease and severe reduction in SPG7. Our results reveal cooperative and partly redundant involvement of AFG3L2 and YME1L in the maintenance of mitochondrial protein homeostasis and further emphasize their importance for mitochondrial and cellular function and integrity. The aim of the second part was to characterize the cellular function of LACE1 (lactation elevated 1) in mitochondrial protein homeostasis. LACE1 protein is a human homologue of yeast Afg1 (ATPase family gene 1) ATPase. We show that LACE1 is a mitochondrial integral membrane protein that exists as a part of three complexes of approximately 140, 400 and 500 kDa. We demonstrate that LACE1 mediates degradation of nuclear-encoded complex IV subunits COX4, COX5A and COX6A. Using affinity...

Yes / Topoisomerase inhibitors are in common use as chemotherapeutic agents although they can display reduced efficacy in chemotherapy-resistant tumours, which have inactivated DNA damage response (DDR) genes, such as ATM and TP53. Here, we characterise the cellular response to the dual-acting agent, Alchemix (ALX), which is a modified anthraquinone that functions as a topoisomerase inhibitor as well as an alkylating agent. We show that ALX induces a robust DDR at nano-molar concentrations and this is mediated primarily through ATR- and DNA-PK- but not ATM-dependent pathways, despite DNA double strand breaks being generated after prolonged exposure to the drug. Interestingly, exposure of epithelial tumour cell lines to ALX in vitro resulted in potent activation of the G2/M checkpoint, which after a prolonged arrest, was bypassed allowing cells to progress into mitosis where they ultimately died by mitotic catastrophe. We also observed effective killing of lymphoid tumour cell lines in vitro following exposure to ALX, although, in contrast, this tended to occur via activation of a p53-independent apoptotic pathway. Lastly, we validate the effectiveness of ALX as a chemotherapeutic agent in vivo by demonstrating its ability to cause a significant reduction in tumour cell growth, irrespective of TP53 status, using a mouse leukaemia xenograft model. Taken together, these data demonstrate that ALX, through its dual action as an alkylating agent and topoisomerase inhibitor, represents a novel anti-cancer agent that could be potentially used clinically to treat refractory or relapsed tumours, particularly those harbouring mutations in DDR genes.

No / Vitiligo is characterized by a patchy loss of inherited skin color affecting approximately 0.5% of individuals of all races. Despite the absence of the protecting pigment and the overwhelming evidence for hydrogen peroxide (H(2)O(2))-induced oxidative stress in the entire epidermis of these patients, there is neither increased photodamage/skin aging nor a higher incidence for sun-induced nonmelanoma skin cancer. Here we demonstrate for the first time increased DNA damage via 8-oxoguanine in the skin and plasma in association with epidermal up-regulated phosphorylated/acetylated p53 and high levels of the p53 antagonist p76(MDM2). Short-patch base-excision repair via hOgg1, APE1, and polymerasebeta DNA repair is up-regulated. Overexpression of Bcl-2 and low caspase 3 and cytochrome c levels argue against increased apoptosis in this disease. Moreover, we show the presence of high epidermal peroxynitrite (ONOO(-)) levels via nitrotyrosine together with high nitrated p53 levels. We demonstrate by EMSA that nitration of p53 by ONOO(-) (300 x 10(-6) M) abrogates DNA binding, while H(2)O(2)-oxidized p53 (10(-3) M) enhances DNA binding capacity and prevents ONOO(-)-induced abrogation of DNA binding. Taken together, we add a novel reactive oxygen species to the list of oxidative stress inducers in vitiligo. Moreover, we propose up-regulated wild-type p53 together with p76(MDM2) as major players in the control of DNA damage/repair and prevention of photodamage and nonmelanoma skin cancer in vitiligo.