Yes / Protein quality control mechanisms are essential for cell health and involve delivery of proteins to specific cellular compartments for recycling or degradation. In particular, stray hydrophobic proteins are captured in the aqueous cytosol by a co-chaperone, the small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA), which facilitates the correct targeting of tail-anchored membrane proteins, as well as the sorting of membrane and secretory proteins that mislocalize to the cytosol and endoplasmic reticulum-associated degradation. Full-length SGTA has an unusual elongated dimeric structure that has, until now, evaded detailed structural analysis. The Cterminal region of SGTA plays a key role in binding a broad range of hydrophobic substrates, yet in contrast to the well-characterized N-terminal and TPR domains, there is a lack of structural information on the C-terminal domain. In this study, we present new insights into the conformation and organization of distinct domains of SGTA and show that the C-terminal domain possesses a conserved region essential for substrate processing in vivo. We show that the C-terminal domain region is characterized by α-helical propensity and an intrinsic ability to dimerize independently of the N-terminal domain. Based on the properties of different regions of SGTA that are revealed using cell biology, NMR, SAXS, Native MS, and EPR, we observe that its C-terminal domain can dimerize in the full-length protein and propose that this reflects a closed conformation of the substrate-binding domain. Our results provide novel insights into the structural complexity of SGTA and provide a new basis for mechanistic studies of substrate binding and release at the C-terminal region. / MRC New Investigator Research Grant: G0900936; BBSRC grants: BB/L006952/1 and BB/L006510/1; BBSRC grant: BB/N006267/1; Wellcome Trust Investigator Award in Science: 204957/Z/16/Z; BBSRC grant: BB/J014567/1

Haematological cancers like leukemia and lymphoma are characterized by genetic abnormalities, specifically chromosomal translocations. Analyses of the translocation breakpoint regions in patients have shown that some loci in the genome are more susceptible to breakage than others. However, very little is known about the mechanism of generation of many such chromosomal translocations. In the present study, we have attempted to understand the mechanism of fragility of three regions, which are prone to breaks during translocations in follicular lymphoma (FL) and T-cell leukemia. The t(14;18) translocation in FL is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3’ UTR of the BCL2 gene and are broadly classified into three clusters, namely major breakpoint region (mbr), minor breakpoint cluster region (mcr) and the intermediate cluster region (icr). The RAG complex has been shown to cleave BCL2 mbr by recognizing an altered DNA structure. In the present study, by using a gel based assay, nature of the non-B DNA structure at BCL2 mbr was identified as parallel intramolecular G-quadruplex. Various studies including circular dichroism (CD), mutagenesis, DMS modification assay and 1H NMR showed the presence of three guanine tetrads in the structure. Further, evidence was also found for the formation of such a G-quadruplex structure within mammalian cells. In an effort to characterize the mechanism of fragility of mcr, a unique pattern of RAG cleavage was observed in a sequence dependent manner. Three independent nicks of equal efficiency were generated by RAGs at the cryptic sequence, “CCACCTCT”, at mcr and at a cytosine upstream of it, unlike a single specific nick at the 5’ of heptamer during V(D)J rearrangement. Interestingly, RAG nicking at mcr occured in the presence of both Mg2+ and Mn2+. Using recombination assay, followed by sequencing of the junctions, we find that mcr can recombine with standard RSS in vivo, albeit at a very low frequency. Mutations to this novel motif abolish recombination at the mcr within the cells. In order to determine the prevalence of t(14;18) translocation in the healthy Indian population, nested PCR approach followed by Southern hybridization was used. Results showed 34% prevalence of t(14;18) translocation in the Indian population. Although, no gender based difference was observed, an age dependent increase was found in adults. Further, presence of the t(14;18) transcripts was also detected. The mechanism underlying the fragility of the t(10;14) translocation involving HOX11 gene in T-cell leukemia is not known. Using primer extension assays on a plasmid DNA containing HOX11 breakpoint region, presence of consistent pause sites corresponding to two G-quadruplex forming regions, flanking the patient breakpoints, were detected. These replication blocks were dependent on K+ ions. Native gel shift assays, mutation analysis, S1 nuclease and CD, further revealed formation of intermolecular G-quadruplexes, unlike the BCL2 mbr. Further, sodium bisulfite modification assay indicated the presence of such structures in the genomic DNA within cells. Hence, we propose that two independent G-quadruplex structures formed in the HOX11 gene could interact with each other, thereby resulting in fragility of the intervening sequences, where majority of the patient breakpoints are mapped. Overall, this study has attempted to understand the role of both sequence and structure of DNA, in generating chromosomal fragility during t(14;18) translocation in FL and t(10;14) translocation in T-cell leukemia. These results may facilitate future studies in unraveling the mechanism leading to genomic instability in other lymphoid cancers.

No description available.

O linfoma difuso de grandes células B (LDGCB) é o subtipo de linfoma mais comum em países em desenvolvimento. Entretanto, apesar de sua prevalência e importância, ainda existem poucas publicações com dados epidemiológicos para a população brasileira. Conhecer os fatores de prognóstico é imperativo para identificar os pacientes que responderão melhor ao tratamento, além de permitir sua individualização terapêutica. Alguns estudos demonstraram que pacientes com o mesmo Índice Internacional de Prognóstico (IPI) podem apresentar diferentes sobrevidas, justificando a necessidade de identificar novos marcadores biológicos de prognóstico. O objetivo deste estudo foi avaliar o impacto prognóstico da expressão dos genes BCL2, MDR1 e OCT1, de suas respectivas proteínas e da translocação t(14;18), nos desfechos de resposta completa (RC), sobrevida global (SG), sobrevida livre de doença (SLD) e sobrevida livre de progressão (SLP) em pacientes com LDGCB. Foram avaliados de forma retrospectiva 98 pacientes com LDGCB de novo tratados com R-CHOP. A expressão gênica foi avaliada por PCR em Tempo Real com RNA extraído de amostras parafinadas. A expressão proteica foi avaliada pelo método de imuno-histoquímica. A mediana de idade foi de 54,5 anos e 49 pacientes (50%) eram do sexo masculino. Sessenta e quatro pacientes (85,3%) obtiveram RC, com uma mediana de acompanhamento de 2,66 anos. A expressão mediana de BCL2, MDR1 e OCT1 foi 6,27; 0 e 24,49, respectivamente. Não encontramos impacto prognóstico da expressão do gene BCL2 na RC (p=0,277), SG (p=0,068) e SLD (p=0,860). Porém, a expressão de BCL2 >= à mediana associou-se à menor SLP (p=0,040). Encontramos associação entre expressão do gene OCT1 >= à mediana e pior prognóstico para SG (p=0,010) e SLP (p=0,016). Porém, não observamos impacto prognóstico da expressão de OCT1 para RC (p=0,464) e SLD (p=0,717). Não houve associação entre expressão do gene MDR1 e das proteínas BCL-2, Pg-p e OCT-1 com o prognóstico dos pacientes em relação à RC, SG, SLD e SLP. O número de sítios extralinfonodais (p=0,004 e p=0,005), estádio clínico (p < 0,001 para ambas), IPI (p < 0,001 para ambas) e nível de DHL (p=0,010 e p=0,008) apresentaram impacto prognóstico na SG e SLP, respectivamente. Quando os pacientes foram estratificados pelo estádio, IPI e idade, o grupo com expressão de OCT1 >= à mediana e IPI intermediário-alto ou alto risco apresentou pior SG (p=0,048) e o grupo com idade >= 60 anos e expressão de OCT1 >= à mediana apresentou pior prognóstico para SG e SLP (p=0,025 para ambas). Em conclusão, a expressão de MDR1 não apresentou impacto no prognóstico de portadores de LDGCB, porém, a expressão do gene BCL2 >= à mediana foi associada a menor SLP. Além disso, a hiperexpressão de OCT1 apresentou valor preditivo de prognóstico para a SG e SLP em pacientes com LDGCB tratados com R-CHOP / The diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype in developing countries. However, despite its prevalence and importance, there are still few publications showing the epidemiological data of the Brazilian population. Knowing the prognostic factors is imperative to identify patients supposed to better respond to treatment, as well as to allow their therapeutic individualization. Some studies have shown that patients with the same International Prognostic Index (IPI) may present different survival rates, thus justifying the need to identify new prognosis biomarkers. The aim of this study was to assess the prognostic impact of the genes BCL2, MDR1 and OCT1 and their respective proteins and t (14; 18) translocation on the complete response (CR), overall survival (OS), disease-free survival (DFS) and progression-free survival (PFS) of patients with DLBCL. We retrospectively assessed 98 patients with de novo DLBCL treated with R-CHOP. The gene expression was assessed through real-time PCR using RNA extracted from paraffin samples. The protein expression was assessed through the immunohistochemistry method. The median age was 54.5 years; 49 patients (50%) were men. Sixty-four patients (85.3%) had CR and median follow-up 2.66 years. The median expression of BCL2, MDR1 and OCT1 was 6.27; 0 and 24.49, respectively. We did not find the prognostic impact of the BCL2 gene expression on CR (p = 0.277), OS (p = 0.068) and on DFS (p = 0.860). However, the expression of BCL2 >= the median was associated with the lower PFS (p = 0.040). We found association between OCT1 gene expression >= the median and worse prognosis for OS (p = 0.010) and PFS (p = 0.016). However, we did not find the prognostic impact of OCT1 expression on CR (p = 0.464) and DFS (p = 0.717). There was no association between the MDR1 gene expression and the BCL-2, Pg-p and OCT-1 proteins, and the patients\' prognosis regarding CR, OS, DFS and PFS. The number of extranodal sites (p = 0.004 and p = 0.005), the clinical status (p <0.001 for both), the IPI (p < 0.001 for both) and DHL levels (p = 0.010 and p = 0.008) presented PFS, respectively. When the patients were stratified by stage, IPI and age, the group with OCT1 expression at the median and intermediate-to-high or high-risk IPI had worse OS results (p = 0.048) and the patients in the age group >= 60 years and expression of OCT1 >= the median presented worse prognosis for OS and PFS (p = 0.025 for both). Therefore, the MDR1 expression had no impact on the prognosis of DLBCL carriers. However, the expression of the BCL2 gene >= the median was associated with lower PFS. In addition, the OCT1 hyperexpression presented a predictive prognosis value for OS and PFS in patients with DLBCL treated with R-CHOP

O linfoma difuso de grandes células B (LDGCB) é o subtipo de linfoma mais comum em países em desenvolvimento. Entretanto, apesar de sua prevalência e importância, ainda existem poucas publicações com dados epidemiológicos para a população brasileira. Conhecer os fatores de prognóstico é imperativo para identificar os pacientes que responderão melhor ao tratamento, além de permitir sua individualização terapêutica. Alguns estudos demonstraram que pacientes com o mesmo Índice Internacional de Prognóstico (IPI) podem apresentar diferentes sobrevidas, justificando a necessidade de identificar novos marcadores biológicos de prognóstico. O objetivo deste estudo foi avaliar o impacto prognóstico da expressão dos genes BCL2, MDR1 e OCT1, de suas respectivas proteínas e da translocação t(14;18), nos desfechos de resposta completa (RC), sobrevida global (SG), sobrevida livre de doença (SLD) e sobrevida livre de progressão (SLP) em pacientes com LDGCB. Foram avaliados de forma retrospectiva 98 pacientes com LDGCB de novo tratados com R-CHOP. A expressão gênica foi avaliada por PCR em Tempo Real com RNA extraído de amostras parafinadas. A expressão proteica foi avaliada pelo método de imuno-histoquímica. A mediana de idade foi de 54,5 anos e 49 pacientes (50%) eram do sexo masculino. Sessenta e quatro pacientes (85,3%) obtiveram RC, com uma mediana de acompanhamento de 2,66 anos. A expressão mediana de BCL2, MDR1 e OCT1 foi 6,27; 0 e 24,49, respectivamente. Não encontramos impacto prognóstico da expressão do gene BCL2 na RC (p=0,277), SG (p=0,068) e SLD (p=0,860). Porém, a expressão de BCL2 >= à mediana associou-se à menor SLP (p=0,040). Encontramos associação entre expressão do gene OCT1 >= à mediana e pior prognóstico para SG (p=0,010) e SLP (p=0,016). Porém, não observamos impacto prognóstico da expressão de OCT1 para RC (p=0,464) e SLD (p=0,717). Não houve associação entre expressão do gene MDR1 e das proteínas BCL-2, Pg-p e OCT-1 com o prognóstico dos pacientes em relação à RC, SG, SLD e SLP. O número de sítios extralinfonodais (p=0,004 e p=0,005), estádio clínico (p < 0,001 para ambas), IPI (p < 0,001 para ambas) e nível de DHL (p=0,010 e p=0,008) apresentaram impacto prognóstico na SG e SLP, respectivamente. Quando os pacientes foram estratificados pelo estádio, IPI e idade, o grupo com expressão de OCT1 >= à mediana e IPI intermediário-alto ou alto risco apresentou pior SG (p=0,048) e o grupo com idade >= 60 anos e expressão de OCT1 >= à mediana apresentou pior prognóstico para SG e SLP (p=0,025 para ambas). Em conclusão, a expressão de MDR1 não apresentou impacto no prognóstico de portadores de LDGCB, porém, a expressão do gene BCL2 >= à mediana foi associada a menor SLP. Além disso, a hiperexpressão de OCT1 apresentou valor preditivo de prognóstico para a SG e SLP em pacientes com LDGCB tratados com R-CHOP / The diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype in developing countries. However, despite its prevalence and importance, there are still few publications showing the epidemiological data of the Brazilian population. Knowing the prognostic factors is imperative to identify patients supposed to better respond to treatment, as well as to allow their therapeutic individualization. Some studies have shown that patients with the same International Prognostic Index (IPI) may present different survival rates, thus justifying the need to identify new prognosis biomarkers. The aim of this study was to assess the prognostic impact of the genes BCL2, MDR1 and OCT1 and their respective proteins and t (14; 18) translocation on the complete response (CR), overall survival (OS), disease-free survival (DFS) and progression-free survival (PFS) of patients with DLBCL. We retrospectively assessed 98 patients with de novo DLBCL treated with R-CHOP. The gene expression was assessed through real-time PCR using RNA extracted from paraffin samples. The protein expression was assessed through the immunohistochemistry method. The median age was 54.5 years; 49 patients (50%) were men. Sixty-four patients (85.3%) had CR and median follow-up 2.66 years. The median expression of BCL2, MDR1 and OCT1 was 6.27; 0 and 24.49, respectively. We did not find the prognostic impact of the BCL2 gene expression on CR (p = 0.277), OS (p = 0.068) and on DFS (p = 0.860). However, the expression of BCL2 >= the median was associated with the lower PFS (p = 0.040). We found association between OCT1 gene expression >= the median and worse prognosis for OS (p = 0.010) and PFS (p = 0.016). However, we did not find the prognostic impact of OCT1 expression on CR (p = 0.464) and DFS (p = 0.717). There was no association between the MDR1 gene expression and the BCL-2, Pg-p and OCT-1 proteins, and the patients\' prognosis regarding CR, OS, DFS and PFS. The number of extranodal sites (p = 0.004 and p = 0.005), the clinical status (p <0.001 for both), the IPI (p < 0.001 for both) and DHL levels (p = 0.010 and p = 0.008) presented PFS, respectively. When the patients were stratified by stage, IPI and age, the group with OCT1 expression at the median and intermediate-to-high or high-risk IPI had worse OS results (p = 0.048) and the patients in the age group >= 60 years and expression of OCT1 >= the median presented worse prognosis for OS and PFS (p = 0.025 for both). Therefore, the MDR1 expression had no impact on the prognosis of DLBCL carriers. However, the expression of the BCL2 gene >= the median was associated with lower PFS. In addition, the OCT1 hyperexpression presented a predictive prognosis value for OS and PFS in patients with DLBCL treated with R-CHOP

Acquired cellular resistance to traditional chemotherapeutics is a common obstacle in the treatment of most cancer cell types. This resistance occurs as a result of changes in the underlying molecular mechanisms of disease progression. The development of novel chemotherapeutic approaches designed to enhance the efficacy of protypical anti-cancer drugs is important in order to overcome this issue. Such approaches will aid in understanding the biomolecular phenomena responsible for drug resistance and disease progression. Combining signaling pathway inhibitors has become an effective strategy for enhancing tumor cell death by targeting multiple pathways known to regulate cell survival. Pemetrexed, an FDA-approved anti-folate drug, targets thymidylate synthase (TS) and a secondary folate-dependent enzyme, 5’ aminoimidazole-carboximide ribonucleotide formyltransferase (AICART); both important for DNA synthesis. Studies performed by our collaborator demonstrated that TS inhibition causes intracellular accumulation of ZMP+ and activation of AMPK which is known to induce autophagy in mammalian cells. Previous studies from our lab and others showed that sorafenib, a multi-kinase inhibitor of Raf-1 and class III receptor tyrosine kinases, was able to induce a cytotoxic form of autophagy in a variety of tumor cell types. Combination treatment using pemetrexed and sorafenib in these cancer cells resulted in an enhancement of autophagy and cell lethality beyond that of individual drugs alone. Inhibition of autophagy suppressed the toxic interactions of these drugs in all cell types examined. Pemetrexed/sorafenib cotherapy also proved to be an effective treatment for triple negative breast cancer cells having advanced to a stage of estrogen independence. Fulvestrant-resistant MCF7 cells were more sensitive to the drug combination than parental, estrogen-dependent MCF7 cells. Breast cancer cells cotreated with pemetrexed and sorafenib exhibited enhanced MEK/ERK signaling, Src activation that was dependent on platelet-derived growth factor β (PDGFRβ) downregulation, elevated protein phosphatase 2A (PP2A) activity, and increased de novo ceramide synthesis. Studies using a mouse model of experimentally-induced breast cancer validated drug combination effectiveness through inhibition of tumor growth, while no deleterious effects on normal tissues were observed. The data presented demonstrates that pemetrexed/sorafenib cotreatment augments chemosensitivity in both in vitro and in vivo systems. Based upon these findings, a Phase I clinical trial involving pemetrexed and sorafenib in breast cancer patients with solid, recurrent tumors was begun in 2011. In conclusion, this work strongly supports a promising therapeutic utility for the pemetrexed/sorafenib combination in treatment of various cancer cell types.

DNA damage response is a complex system responsible for protection of a cell against internal and external DNA damaging agents and in maintaining genome integrity. Many of genes participating in DNA damage response pathways are polymorphic. Genetic polymorphisms in coding and regulatory regions may have impact on the function of proteins encoded by the genes. Phenotypic effect of single nucleotide polymorphisms (SNPs) is subject of investigation in connection with the ability of a cell to manage genotoxic stress and subsequently, in relation to cancer susceptibility. The aim of this thesis was to evaluate the association between SNPs in DNA repair genes (hOGG1, XRCC1, XPC) and cell cycle genes (TP53, p21CDKN1A , BCL2 and BAX) and their mRNA expression in peripheral blood lymphocytes from individuals occupationally exposed to styrene and control individuals. The aim was extended to analyses of relationships between mRNA expression levels of the above-mentioned genes and markers of exposure to styrene (concentration of styrene in blood and in air), markers of DNA damage (single strand breaks - SSBs, and endonuclease III specific sites - Endo III sites) and the base excision repair (BER) capacity, by means of γ-irradiation specific DNA repair rates and oxidative repair. Study on the group of healthy...