Identification of a cross-talk between EGFR and Wnt/beta-catenin signaling pathways in HepG2 liver cancer cells.

EGFRis a transmembrane receptor tyrosine kinase involved in regulating cell proliferation, differentiation and survival. EGFR is actively pursued as a therapeutic target because its aberrant expression or activity has been reported in several cancers. Several studies have reported the nuclear localization of the EGFR in various cell types, however, its exact nuclear functions are not clear yet. In this study, we have generated GFP fusion constructs of EGFR and its mutants to analyze their subcellular localizationin normal and cancer cells and impact of its sub-cellular location on its various activities using immunoblotting, confocal microscopy, reporter assays, loss-of-function EGFR mutants, and EGFR specific small molecule inhibitors. We show that EGFR is involved in modulating TCF dependent ß-catenin transcriptional activity in HepG2 cells in a similar fashion as IGF1R tyrosine kinase. Moreover, we show that cytoplasmic and nuclear functions are two independent activities of EGFR.

Prevalence and prognostic relevance of BrafV600E mutation in colorectal carcinomas from Kashmir (North India) valley.

Molecular studies have implicated mutant B-type Raf kinase (BRAFMut) of MAP-kinase signalling pathway in the pathogenesis of several cancers including colorectal cancer. Recently, the prognostic and therapeutic relevance of the most frequent BRAFV600E mutation also has been highlighted in colorectal carcinomas (CRC). Thus, the aim of this study was to investigate the prevalence of BRAFV600E mutation and to determine the correlation between this mutation and indicators of poor prognosis and outcome in patients with CRCs from Kashmir, North India. Here, we developed a highly sensitive technique, mutation allele-specific multiplex PCR (MASMP), for detection of BRAFV600E/BRAFc.1799T>A mutation, the results of which were confirmed by sequencing the product and compared to direct DNA sequencing. In total, BRAFV600E mutation status was analyzed in 57 colorectal tumour samples and an equal number of adjacent normal tissues. A high frequency of BRAFV600E mutation 21% (12/57) was identified in tumour tissues by MASMP compared to only 5.2% by direct DNA sequencing. Statistical analysis indicated that compared to BRAF-negative colorectal tumours, patients with BRAFV600E colorectal tumours were more likely to be >50 years old (61%) (P < 0.03). These tumours were more likely to be of clinical tumour stages III and IV (63%) (P < 0.04) with lymph node metastasis (52%) (P < 0.02) and characterised by a high-grade histology (63%) (P < 0.04). Colorectal patients harbouring BRAFV600E mutation experience more relapse/recurrence (52%) (P < 0.02). We, therefore, conclude that BRAFV600E mutation can be used as an indicator of poor prognosis to predict the outcome for CRC patients from Kashmir. MASMP proved to be a simple, sensitive and reliable technique for screening patients for BRAFV600E mutation. Testing for this mutation may be useful for selecting initial therapy mode and for follow-up monitoring in CRC patients.

Identification and characterisation of a novel heat shock protein 90 inhibitor ONO4140.

Heat shock protein (Hsp) 90 is a key component of the super-chaperone complex that maintains functionally active conformation of various client proteins. Many of these client proteins regulate important nodal points in multiple signalling pathways that promote cancer cell growth and survival. Inhibitors of Hsp90, therefore, have the potential of functioning as anti-cancer agents with pleiotropic effects. Identification of novel Hsp90 inhibitors with more favourable pharmacological properties is a priority in cancer therapy. To achieve this goal, we screened a compound library using a biochemical assay based on refolding of denatured firefly luciferase. The assay revealed high sensitivity, reliability and reproducibility with a Z-factor of 0.81 ± 0.17. Six Hsp90 inhibitory compounds identified by this screening with IC50 values between 1.0 and 6 µM were further characterised for anti-proliferative activity by Cell Titer-Blue Cell Viability Assay using multiple tumour cell lines. Of particular interest was ONO4140 with lowest GI50 values in three different cancer cell lines viz; DU-145, BT-474 and K562 cell lines. This study also revealed that short-term exposure of tumour cells with ONO4140 is sufficient to inhibit the catalytic activity of Hsp90, evaluated through disruption of Hsp90-p23 association by immunoprecipitation. This short term exposure appears to initiate events like degradation of Hsp90 client proteins such as ErbB2/Her-2 and Akt with concomitant inhibition of survival signalling leading to the apoptotic death of tumour cells as seen by western blotting and Caspase Glow-3,7 assay. The study also reveals that apoptosis following Hsp90 inhibition with ONO4140 occurs via Caspase9-Caspase3 intrinsic apoptotic pathway, a process that is likely triggered by inactivation of Akt. In conclusion, we have identified a novel class of synthetic compounds which show potent Hsp90 inhibitory action in preclinical studies. The discovery of this novel class of synthetic Hsp90 inhibitors with simple chemical backbone allows us to conduct further structural modifications to improve their potency and pharmacokinetic properties for use in cancer therapy.

Comparison and agreement between venous and arterial gas analysis in cardiopulmonary patients in Kashmir valley of the Indian subcontinent.

BACKGROUND: Arterial blood gas (ABG) analysis is routinely performed for sick patients but is fraught with complications, is painful, and is technically demanding. OBJECTIVE: To ascertain agreement between the arterial and peripheral venous measurement of pH, pCO(2), pO(2), and bicarbonate levels in sick patients with cardiopulmonary disorders in the valley of Kashmir in the Indian subcontinent, so as to use venous gas analysis instead of arterial for assessment of patients. SETTING: Sher-i-Kashmir Institute of Medical Sciences, Srinagar, Kashmir, a 650-bedded tertiary care hospital in North India located at an altitude of 1584 m. METHODS: One hundred patients who required ABG analysis were admitted. Peripheral venous blood was drawn within 5 min of an ABG measurement, and the samples analyzed immediately on a point of care automated ABG analyzer. Finger pulse oximetry was used to obtain oxygen (SpO(2)) saturation. Data were analyzed using Pearson correlation and bias (Bland Altman) methods. RESULTS: The venous measurements of pH, pCO(2), pO(2) and bicarbonate, and the digital oxygen saturation were highly correlated with their corresponding arterial measurements. Bland Altman plots demonstrated a high degree of agreement between the two corresponding sets of measurements with clinically acceptable differences. The difference in pO(2) measurements was, however, higher (-22.34 ± 15.23) although the arterial saturation and finger oximetry revealed a good degree of agreement with clinically acceptable bias. CONCLUSION: Peripheral venous blood gas assessment in conjunction with finger pulse oximetry can obviate the routine use of arterial puncture in patients requiring ABG analysis.

Improved diagnosis of central nervous system tuberculosis by MPB64-Target PCR / Diagnóstico da tuberculose do sistema nervoso central por MPB64-Target PCR

Central nervous system (CNS) tuberculosis is a serious clinical problem, the treatment of which is sometimes hampered by delayed diagnosis. Clearly, prompt laboratory diagnosis is of vital importance as the spectrum of disease is wideand abnormalities of the cerebrospinal fluid (CSF) are incredibly variable. Since delayed hypersensitivity is the underlying immune response, bacterial load is very low. The conventional bacteriological methods rarely detect Mycobacterium tuberculosis in CSF and are of limited use in diagnosis of tuberculous meningitis (TBM). This double blind study was, therefore, directed to the molecular analysis of CNS tuberculosis by an in-house-developed PCR targeted for amplification of a 240bp nucleotidesequence coding for MPB64 protein specific for Mycobacterium tuberculosis. Based on the clinical criteria, 47 patients with CNS tuberculosis and a control group of 10 patients having non-tubercular lesions of the CNS were included in the study. Analyses were done in three groups; one group consisting of 27 patients of TBM, a second group of 20 patients with intracranial tuberculomas and a third group of 10 patients having non-tubercular lesions of the CNS acted as control. There were no false positive results by PCR and the specificity worked out to be 100 percent. In the three study groups, routine CSF analysis (cells and chemistry), CSF for AFB smear and culture were negative in all cases. PCR was positive for 21/27 patients (77.7 percent sensitivity) of the first group of TBM patients, 6/20 patients (30 percent sensitivity) of the second group with intracranial tuberculomas were positive by PCR and none was PCR-positive (100 percent specificity) in the third group. Thus, PCR was found to be more sensitive than any other conventional method in the diagnosis of clinically suspected tubercular meningitis.

A tuberculose do sistema nervoso central (CNS) é um problema clínico sério, cujo tratamento é dificultado pelo diagnóstico tardio. O diagnóstico laboratorial rápido é de importância vital considerando que o espectro da doença é amplo e as anormalidades do liquor são muito variáveis. Considerando que a hipersensibilidade tardia é a resposta imune fundamental, a carga bacteriana é muito baixa. Os métodos bacteriológicos convencionais raramente detectam Mycobacterium tuberculosis no liquor e são de uso limitado para diagnóstico da meningite tuberculosa (TBM). O presente estudo duplo-cego objetivou a análise molecular da tuberculose do CNS através de um PCR desenvolvido in-house direcionado para a amplificação de uma seqüência de nucleotídios de 240pb que codificam a proteína MPB64 especifica de Mycobacterium tuberculosis. Baseando-se em critérios clínicos, selecionou-se 47 pacientes com tuberculose do CNS e um grupo controle de 10 pacientes com lesões não-tuberculosas no CNS. As análises foram divididas em três grupos: um grupo de 27 pacientes com TBM, um segundo grupo com 20 pacientes com tuberculomas intracraniais e um terceiro grupo de 10 pacientes com lesões não-tuberculosas no CNS (controles). O PCR não forneceu nenhum resultado falso-positivo, com 100 por cento de especificidade. Em todos os três grupos de estudo, os resultados das análises de rotina do liquor por histologia, química e baciloscopia e também cultura foram negativos em todos os casos. No primeiro grupo de pacientes com TBM, PCR foi positivo em 21/27 pacientes (sensibilidade de 77,7 por cento). No segundo grupo de pacientes com tuberculomas intracraniais, 6/20 foram positivos (sensibilidade de 30 por cento). Nenhum dos pacientes do grupo controle foi positivo (100 por cento de especificidade). Dessa forma, o PCR mostrou-se mais sensível que os métodos convencionais no diagnóstico de casos suspeitos de meningite tuberculosa.

Improved diagnosis of central nervous system tuberculosis by MPB64-Target PCR.

Central nervous system (CNS) tuberculosis is a serious clinical problem, the treatment of which is sometimes hampered by delayed diagnosis. Clearly, prompt laboratory diagnosis is of vital importance as the spectrum of disease is wide and abnormalities of the cerebrospinal fluid (CSF) are incredibly variable. Since delayed hypersensitivity is the underlying immune response, bacterial load is very low. The conventional bacteriological methods rarely detect Mycobacterium tuberculosis in CSF and are of limited use in diagnosis of tuberculous meningitis (TBM). This double blind study was, therefore, directed to the molecular analysis of CNS tuberculosis by an in-house-developed PCR targeted for amplification of a 240bp nucleotide sequence coding for MPB64 protein specific for Mycobacterium tuberculosis. Based on the clinical criteria, 47 patients with CNS tuberculosis and a control group of 10 patients having non-tubercular lesions of the CNS were included in the study. Analyses were done in three groups; one group consisting of 27 patients of TBM, a second group of 20 patients with intracranial tuberculomas and a third group of 10 patients having nontubercular lesions of the CNS acted as control. There were no false positive results by PCR and the specificity worked out to be 100%. In the three study groups, routine CSF analysis (cells and chemistry), CSF for AFB smear and culture were negative in all cases. PCR was positive for 21/27 patients (77.7% sensitivity) of the first group of TBM patients, 6/20 patients (30% sensitivity) of the second group with intracranial tuberculomas were positive by PCR and none was PCR-positive (100% specificity) in the third group. Thus, PCR was found to be more sensitive than any other conventional method in the diagnosis of clinically suspected tubercular meningitis.

BRCA1 and TP53 mutation spectrum of breast carcinoma in an ethnic population of Kashmir, an emerging high-risk area.

Breast cancer shows geographical variation in its incidence, even within areas of ethnic homogeneity. Kashmir valley (India), over past few years, witnesses an increase in incidence and occurrence of familial, early onset, and male breast cancer in its unexplored ethnic population. Here, we make a preliminary attempt to estimate the nature and frequency of BRCA1 and TP53 gene mutations of breast cancer patients from Kashmir. PCR-SSCP analysis followed by direct sequencing revealed the presence of only two germline intronic variations (c.199+67T>C and c.5396+187T>C) in BRCA1 gene in only 5.26% (2/38) patients while as 44% (11/25) of sporadic breast cancer patients harboured significant amount of somatic mutations in TP53 (p=0.0074; OR=0.053). The 17 mutations found in TP53 in 11 patients, comprised of 13 substitutions [11 single-base (9 transitions+2 transversions), 1 double-base and 1 complex] and four insertions. The 11 substitutions represent missense mutations, leading to aminoacid substitution while as rest two were silent mutations. The four insertions represented three frame-shifts and one non-sense mutation. The mutation effect data was found to be significant (p=0.0002). Significant amount of mutations were found in exon 6 (p=0.04; OR=0.273) and a combination of exons 6 and 7 (p=0.0145; OR=14.22) of TP53. Comparison of mutation profile with other ethnic populations and regions reflected both differences and similarities indicating co-exposure to a unique set of risk factors. The differences could be due to exposure to particular environmental carcinogens; different lifestyle, reproductive pattern; dietary or cultural practices of Kashmiri women that need further investigations. The infrequent presence of germline BRCA1 mutations in our study agree with the idea that a great proportion of moderate risk breast cancer population could be due to the susceptibility genes distinct from BRCA1. However, high frequency of somatic TP53 gene mutations implicates TP53 as a predominant factor for breast carcinogenesis in moderate risk ethnic Kashmiri population. The study also suggests TP53 as a potential molecular marker and prognostic tool, at least in a subset of sporadic breast tumors.

Evaluation of polymerase chain reaction for rapid diagnosis of clinically suspected tuberculous pleurisy.

Pleural effusion is one of the commonest presentations of tuberculosis, the clinical manifestations being typically abrupt resembling bacterial pneumonia. Since delayed hypersensitivity is the underlying immune response, bacterial load is very low. Owing to these facts, tuberculous pleurisy as an extra-pulmonary disease poses a diagnostic dilemma. The conventional bacteriological methods rarely detect Mycobacterium tuberculosis in pleural fluid and are of limited use in diagnosis of tuberculous pleurisy. We evaluated the efficacy of polymerase chain reaction (PCR) in the diagnosis of tuberculous pleurisy by targeting the gene segment coding for MPB64 protein specific forMycobacterium tuberculosis. Based on the clinical criteria, 82 patients with lymphocytic exudative pleural effusion were included in the study. Patients were analyzed in two groups; one group consisting of 48 patients of tubercular pleural effusion confimed by various diagnostic procedures and another group of 34 patients comprising of non-tubercular pleural effusion. There were no false positive results by PCR and the specificity worked out to be 100%. Twenty two patients tested positive for Mantoux with a sensitivity of 45%. ZN-staining for AFB was found in samples from 15 patients (20% sensitivity). ADA was positive for 28 patients with a sensitivity of 53%. PCR was positive for 32/48 patients (67% sensitivity). Thus, PCR was found to be more sensitive than any other conventional method in diagnosis of clinically suspected tubercular pleurisy.