M. Radhakrishna Pillai

Polymorphisms in DNA repair gene XRCC1 and increased genetic susceptibility to breast cancer.

X-ray repair cross-complementing 1 (XRCC1) gene encodes for a scaffolding protein, which plays an important role in base excision DNA repair by bringing together DNA polymerase beta, DNA ligase III and poly(ADP-Ribose) polymerase (PARP) at the site of DNA damage. Three polymorphisms of the XRCC1 gene at codons 194, 280 and 399 leading to a mino acid changes at evolutionary conserved regions are found to alter the efficiency of the resulting protein and may therefore constitute potential breast cancer risk. In the present study we sought to determine whether these genetic variants of the XRCC1 gene was associated with any increased risk of breast cancer among the South Indian women in a hospital based case control study using PCR-RFLP and DNA sequencing techniques. Our data showed a positive association between the polymorphisms of codons 194 (OR=1.98, 95% CI=1.13--3.48 for Trp allele) and 399 (OR=2.14, 95% CI=1.29--3.58 for Gln allele) and breast cancer risk. However, XRCC1 codon 280 genotype analysis showed no evidence for an association with increased risk of breast cancer. A combined analysis of the effect of XRCC1 codon 194 and 399 revealed the highest risk (OR=3.64, 95% CI=1.57--8.46) for carriers of the polymorphic alleles in both these codons. In conclusion, the present study suggested involvement of XRCC1 codon 194 and 399 polymorphisms in the genetic predisposition to breast cancer among South Indian women. Our preliminary results based on the analysis of functionally relevant polymorphisms in XRCC1 low penetrance gene may provide a better model that would exhibit additive effects on individual susceptibility to breast cancer.

Drug-induced senescence generates chemoresistant stemlike cells with low reactive oxygen species.

Background: Tumor stem cells contribute to tumor recurrence after chemotherapy. Results: Reactivation of antioxidant enzymes through Nrf2-p21 signaling contributes to stem cell enrichment. Conclusion: Nrf2 stabilization through reduced 26 S proteasome activity generates cells with tumor stem cell-like properties. Significance: Nrf2, 26 S proteasome, and p21 are the key players in the emergence of drug-resistant tumor stem cells after chemotherapy. Tumor recurrence after chemotherapy or radiation remains a major obstacle to successful cancer treatment. A subset of cancer cells, termed cancer stem cells, can elude conventional treatments and eventually regenerate a tumor that is more aggressive. Despite the large number of studies, molecular events that govern the emergence of aggressive therapy-resistant cells with stem cell properties after chemotherapy are poorly defined. The present study provides evidence for the rare escape of tumor cells from drug-induced cell death, after an intermediate stay in a non-cycling senescent stage followed by unstable multiplication characterized by spontaneous cell death. However, some cells appear to escape and generate stable colonies with an aggressive tumor stem cell-like phenotype. These cells displayed higher CD133 and Oct-4 expression. Notably, the drug-selected cells that contained low levels of reactive oxygen species (ROS) also showed an increase in antioxidant enzymes. Consistent with this in vitro experimental data, we observed lower levels of ROS in breast tumors obtained after neoadjuvant chemotherapy compared with samples that did not receive preoperative chemotherapy. These latter tissues also expressed enhanced levels of ROS defenses with enhanced expression of superoxide dismutase. Higher levels of Oct-4 and CD133 were also observed in tumors obtained after neoadjuvant chemotherapy. Further studies provided evidence for the stabilization of Nrf2 due to reduced 26 S proteasome activity and increased p21 association as the driving signaling event that contributes to the transition from a high ROS quiescent state to a low ROS proliferating stage in drug-induced tumor stem cell enrichment.

Biology of PPARγ in Cancer: A Critical Review on Existing Lacunae

Peroxisomal proliferator activated receptor γ(PPARγ) belongs to the family of nuclear hormone receptors (NHRs), which directly regulate transcription of target genes. The regulatory role of this receptor on lipid metabolism and insulin sensitization is well established. Recently, the overexpression of this receptor in many human cancers has been identified and understanding its biological significance forms the current theme. PPARγ activation by specific agonists leads to growth inhibition, apoptosis and differentiation of tumor cells. PPARγ possess evident tumor promoting properties but the receptor independent effects of its ligands compound the understanding of its biology in cancers. This review highlights the multifaceted role of PPARγ in cancer progression with specific reference to colon, breast, gastric, lung and urological cancers. Molecular events as well as the mediators involved are analyzed in detail along with PPARγ independent effects of ligands under each cancer type. The crucial cross talk that exists between Wnt and PPARγ signaling is also summarized. An attempt has been made to identify the existing lacunae in understanding the biology of PPARγ in cancers along with suggestions for possible rectification.

meso-Tetrakis(p-sulfonatophenyl)N-Confused Porphyrin Tetrasodium Salt: A Potential Sensitizer for Photodynamic Therapy

A water-soluble derivative of N-confused porphyrin (NCP) was synthesized, and the photodynamic therapeutic (PDT) application was investigated by photophysical and in vitro studies. High singlet oxygen quantum yield in water at longer wavelength and promising IC(50) values in a panel of cancer cell lines ensure the potential candidacy of the sensitizer as a PDT drug. Reactive oxygen species (ROS) generation on PDT in MDA-MB 231 cells and the apoptotic pathway of cell death was illustrated using different techniques.

Expression of programmed cell death regulatory p53 and bcl-2 proteins in oral lesions

With the ultimate goal of characterizing the molecular pathogenesis of oral cancer, the most predominant malignancy in India, immunocytochemical evaluation of p53 and bcl-2 proteins was carried out in hypeplastic oral mucosa, dysplastic oral mucosa and invasive oral cancer. All subjects gave a similar and almost uniform history of prolonged use of betal quid and tobacco. Expression of p53 was insignificant while bcl-2 was absent in hyperplastic leukoplakia lesions. Both proteins were however expressed in leukoplakia with apparent dysplasia. Almost all invasive cancer lesions showed high levels of both p53 and bcl-2. Good correlation was therefore evident between expression of these two proteins and increasing histologic abnormality. Moreover relative risk evaluation revealed that lesions expressing p53 and bcl-2 had a high probability of having a histology of dysplasia or worse. Since it has been previously shown that wild type p53 regulates the expression of bcl-2, it may be presumed that the protein detected in the dysplastic and malignant oral tissue is of the mutant type. It is also known that p53 is a positive regulator of programmed cell death or apoptosis while bcl-2 is an anti-apoptotic protein. This suggests the possibility that alterations in p53 followed by over-expression of bcl-2 occur early in oral carcinogenesis resulting in defective apoptosis and subsequent tumor progression.

Decreased programmed cell death in the uterine cervix associated with high risk human papillomavirus infection

The relationship between apoptosis, apoptosis regulatory proteins, cell proliferation and human papillomavirus infection during various phases of tumor progression in the uterine cervix was studied. Apoptosis was defined by morphological criteria and the TUNEL assay. Expression of p53, bcl-2, bax, cyclin D1, Ki 67 and E6 protein was evaluated by immunocytochemistry. Presence of mutant p53 was detected using a mutant specific ELISA. Type of HPV infection was determined by PCR using type specific primers. Apoptosis showed significant negative correlation with increasing histological abnormality (p=0.0005). Higher tumor cell proliferation was associated with increasing histological abnormality (p=0.001 for Ki 67 and cyclin D1). There was significant correlation between histological grade and immunoreactivity of p53 (p=0.0001 ) and bcl-2 (p=0.0002). However, mutant p53 was expressed by only 12 of the 230 samples. Expression of bax and the bax/bcl-2 ratio showed an inverse correlation to histological grade (p=0.0003 and 0.0001, respectively). There was also an inverse correlation between extent of apoptosis and immunoreactivity of p53 (p=0.0001) and bcl-2 (p=0.0001). A significant positive correlation between expression of the bax protein and apoptosis was evident (p=0.0001). HPV infection significantly correlated to the extent of histological abnormality (p=0.0001). High risk HPV-E6 protein also showed this significant correlation (p=0.0002). There was an inverse correlation between apoptosis and HPV infection (p=0.0002). High risk HPV infection was associated with decreased apoptosis and also increased human cell proliferation. Lowest levels of bax/bcl-2 ratio was also associated with HPV 16 and 18 infection (p=0.0001). Modulation of apoptosis and apoptotic regulatory proteins by high risk HPV infection may be an important factor in the development of cervical cancer.

Tumour-proliferative fraction and growth factor expression as markers of tumour response to radiotherapy in cancer of the uterine cervix

Abstract This study seeks to define the role of pre-treatment of evaluation of tumour growth fraction in cervical cancer and its relationship to the clinical course of the disease. In addition, it also seeks to explain whether cell kinetics and growth factor expression have an association with tumour response to radiotherapy and hence could be of value in the management of patients. All pre-treatment biopsies were analysed for the tumour-proliferative compartment by evaluation of Ki67 antigen expression and argyrophilic nucleolar organiser region (AgNOR) counts. Growth factor analysis was done by analysing for expression of epidermal growth factor (EGF), epidermal growth factor receptor (EGF-R) and transforming growth factors α and β (TGFα, TGFβ). A total of 152 patients were evaluated and a correlation obtained between pre-treatment status of the tumour-growth-fraction-associated markers and clinical outcome following radiotherapy. Such patients were either disease-free (group 1, nu2009=u2009106) or with residual/recurrent disease (group 2, nu2009=u200946) at a 16-month follow-up. Pre-treatment analysis of AgNOR significantly correlated to disease status after treatment (ru2009=u2009−0.517, Pu2009=u20090.0000). This may be due to an effect of cell proliferation. Lower AgNOR counts were significantly associated with recurrent/residual tumours, suggesting that increased proliferative activity may be a positive prognostic indicator. Similar results were also obtained for the other proliferation-associated marker Ki67 (ru2009=u2009−0.443, Pu2009=u20090.0000). Expression of EGF and EGF-R also showed significant pre-treatment correlations with the final disease outcome (ru2009=u20090.248, Pu2009=u20090.031 and ru2009=u20090.503, Pu2009=u20090.0000 respectively). Both these markers were expressed more by patients belonging to group 2. The opposite was the case for TGFα, where patients belonging to group 1 showed higher values (ru2009=u20090.417, Pu2009=u20090.0001). The other growth factor investigated, TGFβ, also showed a conspicuous differential expression in the two groups of patients (ru2009=u2009−0.604, Pu2009=u20090.0000). Group 1 patients showed mostly mild to moderate expression while most group 2 patients were negative for the growth factor. It therefore appears that tumours with high AgNOR counts and Ki67 index, along with expression of the two types of transforming growth factor (α and β), responded better to radiotherapy.

Cellular manifestations of human papillomavirus infection in the oral mucosa.

Human papillomavirus infection has been suggested to play a role in the development of epithelial carcinomas, particularly those of the uterine cervix. Less information is available on the role of the virus in oral lesions. It has been proposed that the viral oncoproteins specifically complex with the products of cellular tumor suppressor gene, namely E6 with p53 and E7 with retinoblastoma gene product. Inactivation or mutation in p53 gene is also known to result in loss of control over the cell cycle and increases in tumor proliferation rates. The present study examines the role of HPV infection in relation to p53 and the extent of the tumor proliferative compartment reflected by cyclin D1 and Ki‐67 expression during various phases of tumor progression in the oral epithelium.

Immune function in malignant cervical neoplasia: A multiparameter analysis

Immunological assessment was carried out in 67 patients with various stages and histological grades of squamous cell carcinoma of the uterine cervix and compared with normal controls to discover whether any of these parameters could be used as an indicator of the patients clinical status. The results show a gross impairment of the various lymphocyte subpopulations and anti-tumor immunity. This impairment was more pronounced in patients with high tumor load and having a histology of poorly differentiated squamous cell carcinomas. None of the parameters studied (total leukocyte numbers, lymphocyte subpopulations, leukocyte adherence inhibition test, natural killer cell activity, circulating immune complexes, and blocking effect of autologous serum) could be used as a clinical indicator, since no correlation could be noted between immune impairment and either the lymphocyte subpopulations or the circulating immune complexes.

Mutant p53 protein, Bcl-2/Bax ratios and apoptosis in paediatric acute lymphoblastic leukaemia.

Abstractu2002Purpose: The Bcl-2 family of proteins regulates a late step in the apoptosis pathway. Bcl-2 protein is believed to be involved in imparting resistance to programmed cell death or apoptosis induced by chemotherapeutic agents and radiation. The anti-apoptotic function of the Bcl-2 protein appears to be modulated by its ability to heterodimerize with other members of the gene family, predominantly Bax, a protein favouring induction of apoptosis. Susceptibility to undergoing apoptosis may, therefore, be dependent on the ratio between Bcl-2 and Bax. Both Bax and Bcl-2 are regulated by the tumour-suppressor protein p53. The present study therefore aims to study the significance of the Bcl-2:Bax ratio, p53 expression and apoptosis in paediatric acute lymphoblastic leukaemia (ALL). Methods: Expression of Bax, Bcl-2 and p53 was determined by immunocytochemistry, and apoptosis was evaluated by an enzymatic end-labelling technique using biotin-dUTP and further confirmed by annexin binding. The presence of mutant p53 was determined using a mutant-p53-specific enzyme-linked immunosorbent assay (ELISA). Results: A total of 32 cases and 20 controls were evaluated. Bcl-2 was found to be expressed in 22/32 of the ALL cases. Pretreatment (spontaneous) apoptosis was observed in 23/32 cases. The mean pretreatment apoptotic index was 11.34u2009±u20092.04% with a median value of 7.5%. Conclusions: There was a positive correlation between apoptosis and Bax expression (ru2009=u20090.5044; Pu2009=u2009 0.0038). There was good correlation between the immunoreactivity of p53 and detection of mutant p53 by ELISA (ru2009=u20090.4605; Pu2009=u20090.0079). The apoptosis index showed a negative borderline correlation to the expression of Bcl-2 protein (ru2009=u2009−0.3181; Pu2009=u20090.076). There was an inverse correlation between extent of apoptosis and the presence of mutant p53 protein (ru2009=u2009−0.4732; Pu2009=u20090.006). p53 protein expression also showed a correlation with both Bcl-2 (ru2009=u20090.4647; Pu2009=u20090.007) and Bax (ru2009=u20090.4128; Pu2009=u20090.018). The Bcl-2/Bax ratio, however, showed no significant correlation with apoptosis (ru2009=u2009−0.3131; Pu2009=u20090.08) or with p53 expression. No significant association was evident between clinical and laboratory parameters with the Bcl-2/Bax protein expression except lymphadenopathy (ru2009=u20090.5774; Pu2009=u20090.03). However, Bax expression showed a borderline correlation with the immediate tumour response to chemotherapy (ru2009=u2009−0.338; Pu2009=u20090.0628). These patients are being followed-up to look for any association between clinical outcome, Bcl-2/Bax ratio and apoptosis.