Efeitos toxicogenéticos e toxicogenômicos do Isotiocianato de Alila (óleo de mostarda) em linhagens celulares de carcinoma de bexiga

Pós-graduação em Patologia - FMB

Estudos citogenéticos de mutações na transformação de neoplasias benignas em malignas do sistema musculoesquelético

O câncer tem sido alvo de incessantes pesquisas sobre sua etiologia, desenvolvimento, progresso e tratamentos. Sua importância no campo científico se dá pela sua alta taxa de mortalidade e morbidade. O fato de que seja uma doença genética, que pode ter interferência ambiental e dietética, está cada vez mais elucidado, porém ainda existem muitos mecanismos a serem desvendados. As neoplasias ósseas, benignas ou malignas, e processos inflamatórios ósseos acometem desde crianças até adultos e idosos, podendo causar danos físicos, incapacidade motora e até a morte. Portanto, presumir o potencial de transformação maligna das lesões benignas, agressividade tumoral, capacidade de invasão tecidual, probabilidade de recidiva, propensão ao desenvolvimento de metástases e resposta ao tratamento, é um valioso expediente na escolha da proposta terapêutica. Estudos genéticos e citogenéticos têm ajudado a aumentar o entendimento sobre a carcinogênese, progressão tumoral, prognósticos e diagnósticos. Portanto, este estudo teve como objetivo detectar mutações e marcadores cromossômicos consistentes e recorrentes na transformação de tumores benignos em malignos no sistema musculoesquelético, através de análises com citogenética clássica, e relacionar estes achados com o prognóstico e diagnóstico dos pacientes. Dentre os casos coletados e analisados citogeneticamente, foram selecionados três casos de lesões ósseas benignas e três lesões ósseas malignas tidas como a progressão tumoral dos respectivos casos benignos, para discussão e relato de caso. Os achados citogenéticos relataram consistentes alterações numéricas e estruturais clonais em regiões cromossômicas com genes importantes envolvidos na progressão tumoral. Pode-se citar a perda da região contendo o gene TP53, supressor tumoral, tanto em lesões benignas como em maligna, como um dos achados mais relevantes neste estudo. Dessa forma, os resultados ...

Avaliação da expressão proteica de PTEN, MDM2, P53 e AR em lesões proliferativas da próstata canina

Pós-graduação em Medicina Veterinária - FCAV

Inhibition of bladder cancer cell proliferation by allyl isothiocyanate (mustard essential oil)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genomic alterations in patients showing multiple primary tumors and family history of cancer

Multiple primary tumors (MPT) are a major cause of mortality and morbidity among patients that have survived after the treatment of a first cancer. It has been proposed that after the first primary tumor, high risk of a subsequent tumor could be associated with radiotherapy used as treatment for the first cancer. Other potential risk factors include unhealthy lifestyle, genetic predisposition, aging, environmental determinants or an interaction between these factors. However, an association between the presence of MPT and family history of cancer in cases without clinical and molecular evidence of a known hereditary cancer syndrome is rarely described. Genomic DNA from 12 patients with at least two primary tumors and without mutations on TP53 was evaluated by CytoScan HD Array (Affymetrix). Chromosome Analysis Suite (ChAS) software v.2.0.1 was used considering at least 50 markers for gains; 25 for losses and a minimum of 5Mb for cnLOHs. Data from 1038 phenotypically healthy individuals (Affymetrix) and from Database of Genomic Variants were used as reference. Only alterations found in <1% (rare) or never described (new rare) in the reference population were considered. All cases, except one, presented a family history of cancer. Five cases developed MTP after radiotherapy and only one was located in the same treated area. It was detected 67 rare and 15 new rare genomic alterations encompassing 5.906 genes: 17 losses, 29 gains, and 36 cnLOH. X chromosome presented the higher number of alterations. Two patients with breast cancer presented a large deletion/cnLOH on 7q21. Enrichment analysis revealed 1275 genes associated with breast cancer (p= 0.001), which was diagnosed in 6 patients and their family members (all negative for BRCA1/2 or TP53 mutations). cnLOHs accounted for 44% of all the alterations. A significant proportion of cases (11/12) presented family history of cancer and the patients were not submitted to radiotherapy (7/12). We demonstrated the presence of rare genomic alterations in patients with MPT suggesting their involvement in the MPT development. cnLOH may arise as a new mechanism associated with the risk to develop MPT. All authors have declared no conflicts of interest.

ROBO1 deletion as a novel germline alteration in breast and colorectal cancer patients

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sarcoma histiocítico: análise molecular pela técnica de hibridação genômica comparativa, microRNA e imunoistoquímica

Pós-graduação em Patologia - FMB

Potenciais microRNAs circulantes como biomarcadores de Leucemia Mieloide Crônica - Fase Crônica: recém diagnosticada e tratada com mesilato de imatinibe

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

PIK3CA exon 20 mutations are associated with poor prognosis in breast cancer patients

OBJECTIVES: The phosphatidylinositol 3-kinase/AKT axis is an important cell-signaling pathway that mediates cell proliferation and survival, two biological processes that regulate malignant cell growth. The phosphatidylinositol 3-kinase CA gene encodes the p110 alpha subunit of the phosphatidylinositol 3-kinase protein. There are phosphatidylinositol 3-kinase CA mutations in several types of human tumors, and they are frequently observed in breast cancer. However, these mutations have not been investigated in Brazilian breast cancer patients. METHODS: PCR-SSCP and direct DNA sequencing were performed to identify phosphatidylinositol 3-kinaseCA exon 9 and exon 20 mutations in 86 patients with sporadic breast cancer. The relationships between PIK3CA mutations and patient clinicopathological characteristics and survival were analyzed. The presence of the TP53 mutation was also examined. RESULTS: Twenty-three (27%) of the 86 primary breast tumors contained PIK3CA mutations. In exons 9 and 20, we identified the hotspot mutations E542K, E545K, and H1047R, and we identified two new missense mutations (I1022V and L1028S) and one nonsense (R992X) mutation. Phosphatidylinositol 3-kinase CA exon 20 mutations were associated with poor overall survival and TP53 gene mutations. CONCLUSIONS: Phosphatidylinositol 3-kinase CA mutations are common in tumors in Brazilian breast cancer patients, and phosphatidylinositol 3-kinase CA and TP53 mutations are not mutually exclusive. Phosphatidylinositol 3-kinase CA exon 20 mutations are associated with poor survival, and they may be useful biomarkers for identifying breast cancer patients with aggressive tumors and for predicting the response to treatment with PI3K pathway inhibitors.

Li-Fraumeni-like syndrome associated with a large BRCA1 intragenic deletion

Background: Li-Fraumeni (LFS) and Li-Fraumeni-like (LFL) syndromes are associated to germline TP53 mutations, and are characterized by the development of central nervous system tumors, sarcomas, adrenocortical carcinomas, and other early-onset tumors. Due to the high frequency of breast cancer in LFS/LFL families, these syndromes clinically overlap with hereditary breast cancer (HBC). Germline point mutations in BRCA1, BRCA2, and TP53 genes are associated with high risk of breast cancer. Large rearrangements involving these genes are also implicated in the HBC phenotype. Methods: We have screened DNA copy number changes by MLPA on BRCA1, BRCA2, and TP53 genes in 23 breast cancer patients with a clinical diagnosis consistent with LFS/LFL; most of these families also met the clinical criteria for other HBC syndromes. Results: We found no DNA copy number alterations in the BRCA2 and TP53 genes, but we detected in one patient a 36.4 Kb BRCA1 microdeletion, confirmed and further mapped by array-CGH, encompassing exons 9-19. Breakpoints sequencing analysis suggests that this rearrangement was mediated by flanking Alu sequences. Conclusion: This is the first description of a germline intragenic BRCA1 deletion in a breast cancer patient with a family history consistent with both LFL and HBC syndromes. Our results show that large rearrangements in these known cancer predisposition genes occur, but are not a frequent cause of cancer susceptibility.

Differential Proteomic Analysis of Noncardia Gastric Cancer from Individuals of Northern Brazil

Gastric cancer is the second leading cause of cancer-related death worldwide. The identification of new cancer biomarkers is necessary to reduce the mortality rates through the development of new screening assays and early diagnosis, as well as new target therapies. In this study, we performed a proteomic analysis of noncardia gastric neoplasias of individuals from Northern Brazil. The proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. For the identification of differentially expressed proteins, we used statistical tests with bootstrapping resampling to control the type I error in the multiple comparison analyses. We identified 111 proteins involved in gastric carcinogenesis. The computational analysis revealed several proteins involved in the energy production processes and reinforced the Warburg effect in gastric cancer. ENO1 and HSPB1 expression were further evaluated. ENO1 was selected due to its role in aerobic glycolysis that may contribute to the Warburg effect. Although we observed two up-regulated spots of ENO1 in the proteomic analysis, the mean expression of ENO1 was reduced in gastric tumors by western blot. However, mean ENO1 expression seems to increase in more invasive tumors. This lack of correlation between proteomic and western blot analyses may be due to the presence of other ENO1 spots that present a slightly reduced expression, but with a high impact in the mean protein expression. In neoplasias, HSPB1 is induced by cellular stress to protect cells against apoptosis. In the present study, HSPB1 presented an elevated protein and mRNA expression in a subset of gastric cancer samples. However, no association was observed between HSPB1 expression and clinicopathological characteristics. Here, we identified several possible biomarkers of gastric cancer in individuals from Northern Brazil. These biomarkers may be useful for the assessment of prognosis and stratification for therapy if validated in larger clinical study sets.

Oncogenic potential diverge among human papillomavirus type 16 natural variants

We compared E6/E7 protein properties of three different HPV-16 variants: AA. E-P and E-350G. Primary human foreskin keratinocytes (PHFK) were transduced with HPV-16 E6 and E7 and evaluated for proliferation and ability to grow in soft agar. E-P infected keratinocytes presented the lowest efficiency in colony formation. AA and E-350G keratinocytes attained higher capacity for in vitro transformation. We observed similar degradation of TP53 among HPV-16 variants. Furthermore, we accessed the expression profile in early (p5) and late passage (p30) transduced cells of 84 genes commonly involved in carcinogenesis. Most differences could be attributed to HPV-16 E6/E7 expression. In particular, we detected different expression of ITGA2 and CHEK2 in keratinocytes infected with AA and AA/E-350G late passage cells, respectively, and higher expression of MAP2K1 in E-350G transduced keratinocytes. Our results indicate differences among HPV-16 variants that could explain, at least in part, differences in oncogenic potential attributed to these variants. (C) 2012 Elsevier Inc. All rights reserved.

Evidence for premature aging due to oxidative stress in iPSCs from Cockayne syndrome

Cockayne syndrome (CS) is a human premature aging disorder associated with neurological and developmental abnormalities, caused by mutations mainly in the CS group B gene (ERCC6). At the molecular level, CS is characterized by a deficiency in the transcription-couple DNA repair pathway. To understand the role of this molecular pathway in a pluripotent cell and the impact of CSB mutation during human cellular development, we generated induced pluripotent stem cells (iPSCs) from CSB skin fibroblasts (CSB-iPSC). Here, we showed that the lack of functional CSB does not represent a barrier to genetic reprogramming. However, iPSCs derived from CSB patients fibroblasts exhibited elevated cell death rate and higher reactive oxygen species (ROS) production. Moreover, these cellular phenotypes were accompanied by an up-regulation of TXNIP and TP53 transcriptional expression. Our findings suggest that CSB modulates cell viability in pluripotent stem cells, regulating the expression of TP53 and TXNIP and ROS production.

SET overexpression decreases cell detoxification efficiency: ALDH2 and GSTP1 are downregulated, DDR is impaired and DNA damage accumulates

Alcohol and tobacco consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Aldehyde dehydrogenase 2 (ALDH2) and glutathione Stransferase pi 1 (GSTP1) are important enzymes for cellular detoxification and low efficiencies are implicated in cancer. We assessed the potential role of SET protein overexpression, a histone acetylation modulator accumulated in HNSCC, in gene regulation and protein activity of ALDH2 and GSTP1. SET was knocked down in HN13, HN12 and Cal27, and overexpressed in HEK293 cells; ethanol and cisplatin were the chemical agents. Cells with SET overexpression (HEK293/SET, HN13 and HN12) showed lower ALDH2 and GSTP1 mRNA levels and trichostatin A increased them (real-time PCR). Ethanol upregulated GSTP1 and ALDH2 mRNAs, whereas cisplatin upregulated GSTP1 in HEK293 cells. SET-chromatin binding revealed SET interaction with ALDH2 and GSTP1 promoters, specifically via SET NAP domain; ethanol and cisplatin abolished SET binding. ALDH2 and GSTP1 efficiency was assessed by enzymatic and comet assay. A lower ALDH2 activity was associated with greater DNA damage (tail intensity) in HEK293/SET compared with HEK293 cells, whereas HN13/siSET showed ALDH2 activity higher than HN13 cells. HN13/siSET cells showed increased tail intensity. Cisplatin-induced DNA damage response showed negative relationship between SET overexpression and BRCA2 recruitment. SET downregulated repair genes ATM, BRCA1 and CHEK2 and upregulated TP53. Cisplatin-induced cell-cycle arrest occurred in G0/G1 and S in HEK293 cells, whereas HEK293/SET showed G2/M stalling. Overall, cisplatin was more cytotoxic for HN13 than HN13/siSET cells. Our data suggest a role for SET in cellular detoxification, DNA damage response and genome integrity.

Morphological alterations and gene and protein expression profiling of bladder tumor cells after treatment with gemcitabine.

Chemical agents used in cancer therapy are associated with cell cycle arrest, activation or deactivation of mechanisms associated to DNA repair and apoptosis. However, due to the complexity of biological systems, the molecular mechanisms responsible for these activities are not fully understood. Thus, studies about gene and protein expression have shown promising results for understanding the mechanisms related to cellular responses and regression of cancer after chemotherapy. This study aimed to evaluate the gene and protein expression profiling in bladder transitional cell carcinoma (TCC) with different TP53 status after gemcitabine (1.56 μM) treatment. The RT4 (grade 1, TP53 wild type), 5637 (grade 2, TP53 mutated) and T24 (grade 3, TP53 mutated) cell lines were used. PCR arrays and mass spectrometry were used to analyze gene and protein expression, respectively. Morphological alterations were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results of PCR array showed that gemcitabine activity was mainly related to CDKN1A, GADD45A and SERTDA1 overexpression, and BAX overexpression only in the wild type TP53 cells. Mass spectrometry demonstrated that gemcitabine modulated the protein expression, especially those from genes related to apoptosis, transport of vesicles and stress response. Analyses using SEM and TEM showed changes in cell morphology independently on the cell line studied. The observed decreased number of microvillus suggests low contact among the cells and between cell and extracellular matrix; irregular forms might indicate actin cytoskeleton deregulation; and the reduction in the amount of organelles and core size might indicate reduced cellular metabolism. In conclusion, independently on TP53 status or grade of bladder tumor, gemcitabine modulated genes related to the cell cycle and apoptosis, that reflected in morphological changes indicative of future cell death.