A COMPARATIVE ANALYSIS OF METHYLATION STATUS OF TUMOR SUPPRESSOR GENES IN PAIRED BIOPSY AND SERUM SAMPLES FROM CERVICAL CANCER PATIENTS AMONG NORTH INDIAN POPULATION.

Tumor-specific genetic or epigenetic alterations have been detected in serum DNA in case of various types of cancers. In breast cancer, the detection of tumor suppressor gene hypermethylation has been reported in several body fluids. Promoter hypermethylation of some genes like MYOD1, CALCA, hTERT etc. has also been detected in serum samples from cervical cancer. The present study is the first report on the comparison of promoter hypermethylation of tumor suppressor genes likep14, p15, p16, p21, p27, p57, p53, p73, RARß2, FHIT, DAPK, STAT1 and-RB1 genes in paired biopsy and serum samples from cervical cancer patients among north Indian population. This is also the first report on the hypermethylation of these genes in serum samples from cervical cancer patients among north Indian population. According to the results of the present study, promoter hypermethylation of these genes can also be detected in serum samples of cervical cancer patients. The sensitivity of detection of promoter hypermethylation in serum samples of cervical cancer patients as compared to paired biopsy samples was found to be around 83.3%. It was observed that promoter hypermethylation was mainly observed in the serum samples in the higher stages and very rarely in the lower stages. The present study clearly showed that serum of patients with cervical cancer can also be used to study methylated genes as biomarkers.

Ellagic acid derivatives from Terminalia chebula Retz. increase the susceptibility of Pseudomonas aeruginosa to stress by inhibiting polyphosphate kinase.

AIM: Polyphosphate kinase 1 (PPK1) plays an important role in virulence, antibiotic resistance and survival under stress conditions and, therefore, is an attractive therapeutic target to control infections caused by multiple drug resistant Pseudomonas aeruginosa. This study explores the PPK1 inhibiting activity of ellagic acid derivatives (EADs) from Terminalia chebula Retz. that could increase the susceptibility of Ps. aeruginosa to in vitro stress conditions. METHODS AND RESULTS: EADs reduced ppk1 gene expression by 93% (P < 0·05) and completely inhibited its activity (P < 0·01) at 0·5 mg ml(-1) . EADs-treated Ps. aeruginosa showed marked reduction in polyphosphate granules in cytosol. Expression of rpoS, the downstream master stress response regulator, was reduced by 94% (P < 0·05) and the sensitivity of Ps. aeruginosa increased many fold to desiccation, oxidative (H2 O2 ) and antibiotic (piperacillin) stresses. PPK-regulated swimming, swarming and twitching motilities and biofilm formation were also reduced significantly (P ≤ 0·05) in MPAO1 and the clinical strains of Ps. aeruginosa. CONCLUSION: EADs from T. chebula inhibited PPK1 expression and its activity and increased the sensitivity of Ps. aeruginosa to desiccation and oxidative stress while reducing tolerance to piperacillin. SIGNIFICANCE AND IMPACT OF THE STUDY: The study underlines the potential of EADs as therapeutic agent against Ps. aeruginosa.

Enzymatic quorum quenching increases antibiotic susceptibility of multidrug resistant Pseudomonas aeruginosa.

BACKGROUND AND OBJECTIVES: There is increasing emergence of multidrug resistant Pseudomonas aeruginosa (MDRPA) strains and drug resistance is positively-correlated with biofilm-forming ability. Since about 10% of P. aeruginosa genome is controlled by quorum sensing (QS), alteration in its antibiotic susceptibility by targeting QS was the focus of the present study. MATERIALS AND METHODS: One day biofilms of PAO1 and three urinary tract infection MDRPA isolates (PA2, PA8 and PA18) were formed in 96-well microtiter plate. Biofilms were exposed to concentration gradient of ciprofloxacin and gentamicin to obtain Minimum Biofilm Eradication Concentration (MBEC) by direct enumeration method. Susceptibility of 24 h biofilms was evaluated by treatment with ciprofloxacin and gentamicin per se and in combination with lactonase. The effect was also examined on 72 h biofilms by Scanning Electron Microscopy. RESULTS: Lactonase treatment did not have any effect on growth of the selected strains but 73.42, 69.1, 77.34 and 72.5% reduction of biofilm was observed after lactonase (1 unit) treatment, respectively. Antibiotics in combination with lactonase (0.3 units) resulted in an increased susceptibility of the biofilm forms by>3.3, 4, 5 and 1.5 folds of MBEC, for ciprofloxacin and>6.67, 12.5, 6 and>2.5 folds, for gentamicin respectively, which could be due to the disruption of biofilm by lactonase treatment as shown by scanning electron microscopy. Also there was significant reduction (p<0.001) in virulence factor production by the strains. CONCLUSION: Lactonase treatment increased antibiotic susceptibility of the biofilms of MDRPA isolates underscoring the potential of quorum quenching in antimicrobial therapeutics.

Reversal of hypermethylation and reactivation of the RARß2 gene by natural compounds in cervical cancer cell lines.

Reactivation of tumour suppressor genes that have been silenced by promoter methylation is a very attractive molecular target for cancer therapy. The treatment of a squamous cervical cancer cell line, SiHa, with 20 µM curcumin and genistein resulted in demethylation of promoter of the RARß2 gene and led to the reactivation of the gene. The degree of methylation as observed by MSP decreased as the time period of treatment was increased from 72 h to 6 days. In HeLa cells (an adenocarcinoma cervical cancer cell line) there was also reversal of hypermethylation of the RARß2 gene after six days of treatment with 20 µM curcumin. However, allyl sulphide treatment (20 µM) did not cause the reversal of hypermethylation until 72 h of treatment in the SiHa cell line. This is the first report to show the reversal of hypermethylation of the RARß2 gene by genistein and curcumin in cervical cancer cell lines. Furthermore, these compounds acted as doublepronged agents as they caused apoptosis in the treated cervical cancer cell lines in addition to reversal of promoter hypermethylation.

Telomerase activation and incidence of HPV in human gastrointestinal tumors in North Indian population.

The present study was initiated with the objective of finding out the role of possible factors in the etiology of gastrointestinal tract cancers. HPV-DNA was detected in 62.5% (25/40) of the patients by PCR. Telomerase activity as shown by TRAP-ELISA assay was detected in 82.5% (33/40) of the tumor samples and absent in 85.7% (30/35) of the normal samples taken from the same patients. As many as 53.6% (15/28) of the invasive cases were positive both for telomerase activity and for HPV, while 39.3% (11/28) of them, although indicating telomerase expression, showed no signal for HPV. This suggests that activation of telomerase could be by a pathway independent of HPV activation, although both parameters could act as diagnostic and prognostic markers for gastrointestinal tract cancers.

Epidemiology of cervical cancer--a case control study on north Indian population.

The present case control study on married women with cervical cancer and controls (100 each) revealed the association of age at marriage, socioeconomic status, education status and parity with cervical cancer but young age at marriage (rr 3.79) and low socioeconomic status (rr -3.81) emerged as independent predictors of disease status.

Incidence of human papilloma virus in patients with invasive cervical carcinoma.

The dot-blot hybridization of biotin-dUTP-labeled HPV-16 DNA with genomic DNA extracted from biopsies taken from patients with invasive carcinoma and abnormal cytology showed the presence of HPV-DNA in 88% and 80% cases under relaxed conditions and 40% and 20% cases under stringent conditions of hybridization, respectively. Southern blot revealed the HPV-DNA in randomly integrated form in two cases and in episomal form in the other two.

Spontaneous genomic fragility and cell cycle progression in lymphocytes of patients with cervical carcinoma.

Thirty patients with invasive carcinoma of the cervix, along with 15 age-matched healthy females as controls, were studied to examine the frequency of spontaneous SCEs, chromosomal aberrations (CAs), and cell cycle progression in lymphocytes. The frequency of SCEs and TCAs was statistically significant in the patients over the control group. The correlation of SCEs and TCAs with the stage of cancer, age, and number of pregnancies was also made. The cell cycle of lymphocytes showed its prolongation in the patients, as is evident from the higher proportion of cells at M1 (metaphase of first cell division after 72 hours).

Biodegradation of textile azo-dyes byPhanerochaete chrysosporium.

Of 18 commercially used textile dyes, eight were degraded by the white rot fungus,Phanerochaete chrysosporium, by 40 to 73% based on decrease of colour. Both the lignin-degrading enzyme system ofP. chrysosporium and adsorption to its cell mass were involved in the degradation of the diazo dye, Reactofix Gold Yellow. Degradation was best achieved by adding the dye to the medium and then inoculating with pre-grown mycelium; inoculation with spores resulted mainly in dye adsorption.

Use of a modified cupric acetate method for the detection and quantitation of xylanolytic activities: a comparative study with the congo red method.

A modified cupric acetate method for the screening and quantitation of xylanolytic activities was comparable with the more widely used congo red method with respect to sensitivity and ease of use and was shown to have points of merit over the latter. The use of a non-linear correction, in comparison to the conventional linear one, for the effect of dilution on the quantitative plate assay was evaluated.