Birendranath Banerjee

Specific Role of Chk1 Phosphorylations in Cell Survival and Checkpoint Activation

ABSTRACT Chk1 is a multifunctional protein kinase that plays essential roles in cell survival and cell cycle checkpoints. Chk1 is phosphorylated at multiple sites by several protein kinases, but the precise effects of these phosphorylations are largely unknown. Using a knockout-knockin system, we examined the abilities of Chk1 mutants to reverse the defects of Chk1-null cells. Wild-type Chk1 could rescue all the defects of Chk1-null cells. Like endogenous Chk1, wild-type Chk1 localized in both the cytoplasm and the nucleus, and its centrosomal association was enhanced by DNA damage. The mutation at S345 resulted in mitotic catastrophe, impaired checkpoints, and loss of the ability to localize in the cytoplasm, but the mutant retained the ability to be released from chromatin upon encountering genotoxic stressors. In contrast, the mutation at S317 resulted in impaired checkpoints and loss of chromatin release upon encountering genotoxic stressors, but its mutant retained the abilities to prevent mitotic catastrophes and to localize in the cytoplasm, suggesting the distinct effects of these phosphorylations. The forced immobilization of S317A/S345A in centrosomes resulted in the prevention of apoptosis in the presence or absence of DNA damage. Thus, two-step phosphorylation of Chk1 at S317 and S345 appeared to be required for proper localization of Chk1 to centrosomes.

Effects of an integrated yoga program in modulating psychological stress and radiation-induced genotoxic stress in breast cancer patients undergoing radiotherapy.

Effects of an integrated yoga program in modulating perceived stress levels, anxiety, as well as depression levels and radiation-induced DNA damage were studied in 68 breast cancer patients undergoing radiotherapy. Two psychological questionnaires—Hospital Anxiety and Depression Scale (HADS) and Perceived Stress Scale (PSS)—and DNA damage assay were used in the study. There was a significant decrease in the HADS scores in the yoga intervention group, whereas the control group displayed an increase in these scores. Mean PSS was decreased in the yoga group, whereas the control group did not show any change pre- and postradiotherapy. Radiation-induced DNA damage was significantly elevated in both the yoga and control groups after radiotherapy, but the postradiotherapy DNA damage in the yoga group was slightly less when compared to the control group. An integrated approach of yoga intervention modulates the stress and DNA damage levels in breast cancer patients during radiotherapy.

Cationic Antimicrobial Peptides and Biogenic Silver Nanoparticles Kill Mycobacteria without Eliciting DNA Damage and Cytotoxicity in Mouse Macrophages

ABSTRACT With the emergence of multidrug-resistant mycobacterial strains, better therapeutic strategies are required for the successful treatment of the infection. Although antimicrobial peptides (AMPs) and silver nanoparticles (AgNPs) are becoming one of the popular antibacterial agents, their antimycobacterial potential is not fully evaluated. In this study, we synthesized biogenic-silver nanoparticles using bacterial, fungal, and plant biomasses and analyzed their antibacterial activities in combination with AMPs against mycobacteria. Mycobacterium smegmatis was found to be more susceptible to AgNPs compared to M. marinum. We found that NK-2 showed enhanced killing effect with NP-1 and NP-2 biogenic nanoparticles at a 0.5-ppm concentration, whereas LLKKK-18 showed antibacterial activity only with NP-2 at 0.5-ppm dose against M. smegmatis. In case of M. marinum NK-2 did not show any additive activity with NP-1 and NP-2 and LLKKK-18 alone completely inhibited the bacterial growth. Both NP-1 and NP-2 also showed increased killing of M. smegmatis in combination with the antituberculosis drug rifampin. The sizes and shapes of the AgNPs were determined by transmission electron microscopy and dynamic light scattering. AgNPs showed no cytotoxic or DNA damage effects on macrophages at the mycobactericidal dose, whereas treatment with higher doses of AgNPs caused toxicity and micronuclei formation in cytokinesis blocked cells. Macrophages actively endocytosed fluorescein isothiocyanate-labeled AgNPs resulting in nitric oxide independent intracellular killing of M. smegmatis. Apoptosis and cell cycle studies showed that treatment with higher dose of AgNPs arrested macrophages at the G1-phase. In summary, our data suggest the combined effect of biogenic-AgNPs and antimicrobial peptides as a promising antimycobacterial template.

Ribosomal protein S27-like, a p53-inducible modulator of cell fate in response to genotoxic stress.

Activation of the p53 tumor suppressor upon DNA damage elicits either cell cycle arrest or apoptosis, and the precise mechanism governing cell fate after p53 response has not been well defined. Through genomic analysis, we have identified the ribosomal protein S27-like (RPS27L) as a novel p53 transcriptional target gene. Although RPS27L mRNA levels were consistently induced after diverse p53 activating signals, its change in protein level was stimuli-dependent: it was up-regulated when cells were arrested in response to DNA-damaging agents Adriamycin or VP16 but was down-regulated when cells underwent apoptosis in response to antimetabolite agent 5-fluorouracil. RPS27L is a nuclear protein that forms nuclear foci upon DNA damage. Depletion of RPS27L resulted in deficiency in DNA damage checkpoints, leading to conversion of DNA damage-induced p53 response from cell cycle arrest to apoptosis. We further show that RPS27L positively regulates p21 protein expression. Through this mechanism, RPS27L induction by p53 facilitates p21-mediated cell cycle arrest and protects against DNA damage-induced apoptosis. Thus, RPS27L modulates DNA damage response and functions as a part of the control switch to determine cell fate to DNA damage-p53 response.

Cooperative functions of Chk1 and Chk2 reduce tumour susceptibility in vivo

Although the linkage of Chk1 and Chk2 to important cancer signalling suggests that these kinases have functions as tumour suppressors, neither Chk1+/− nor Chk2−/− mice show a predisposition to cancer under unperturbed conditions. We show here that Chk1+/−Chk2−/− and Chk1+/−Chk2+/− mice have a progressive cancer‐prone phenotype. Deletion of a single Chk1 allele compromises G2/M checkpoint function that is not further affected by Chk2 depletion, whereas Chk1 and Chk2 cooperatively affect G1/S and intra‐S phase checkpoints. Either or both of the kinases are required for DNA repair depending on the type of DNA damage. Mouse embryonic fibroblasts from the double‐mutant mice showed a higher level of p53 with spontaneous DNA damage under unperturbed conditions, but failed to phosphorylate p53 at S23 and further induce p53 expression upon additional DNA damage. Neither Chk1 nor Chk2 is apparently essential for p53‐ or Rb‐dependent oncogene‐induced senescence. Our results suggest that the double Chk mutation leads to a high level of spontaneous DNA damage, but fails to eliminate cells with damaged DNA, which may ultimately increase cancer susceptibility independently of senescence.

Short dysfunctional telomeres impair the repair of arsenite-induced oxidative damage in mouse cells†

Telomeres and telomerase appear to participate in the repair of broken DNA ends produced by oxidative damage. Arsenite is an environmental contaminant and a potent human carcinogen, which induces oxidative stress on cells via the generation of reactive oxygen species affecting cell viability and chromosome stability. It promotes telomere attrition and reduces cell survival by apoptosis. In this study, we used mouse embryonic fibroblasts (MEFs) from mice lacking telomerase RNA component (mTERC−/− mice) with long (early passage or EP) and short (late passage or LP) telomeres to investigate the extent of oxidative damage by comparing the differences in DNA damage, chromosome instability, and cell survival at 24 and 48 h of exposure to sodium arsenite (As3+; NaAsO2). There was significantly high level of DNA damage in mTERC−/− cells with short telomeres as determined by alkaline comet assay. Consistent with elevated DNA damage, increased micronuclei (MN) induction reflecting gross genomic instability was also observed. Fluorescence in situ hybridization (FISH) analysis revealed that increasing doses of arsenite augmented the chromosome aberrations, which contributes to genomic instability leading to possibly apoptotic cell death and cell cycle arrest. Microarray analysis has revealed that As3+ treatment altered the expression of 456 genes of which 20% of them have known functions in cell cycle and DNA damage signaling and response, cell growth, and/or maintenance. Results from our studies imply that short dysfunctional telomeres impair the repair of oxidative damage caused by arsenite. The results will have implications in risk estimation as well as cancer chemotherapy. J. Cell. Physiol. 214: 796–809, 2008.

Telomere-Mediated Genomic Instability and the Clinico-Pathological Parameters in Breast Cancer

A study was undertaken to correlate telomere dysfunction and genomic instability with the histopathological grades and the estrogen and progesterone receptor status in breast cancer. Sixty‐one archived breast tissues (38 cancer tissues and 23 paired normal tissues) were used in the study. The breast tumor tissues showed significantly shorter telomeres (7.7 kb) compared with the paired adjacent tissues (9.0 kb) by Southern blot analysis. Moreover, telomere shortening was more significant in Grade III tumors than in the Grade II tumors (P = 0.05). Quantitative fluorescence in situ hybridization on paraffin tissue sections revealed a similar trend in telomere shortening. Telomere attrition was associated with telomere dysfunction as revealed by the presence of significantly higher anaphase bridges in tumor cells which was tumor grade dependent. Furthermore, estrogen receptive negative tumors displayed higher anaphase and internuclear bridges. Selected samples from each grade showed greater genomic imbalances in the higher grades than the lower grade tumors as detected by array‐comparative genomic hybridization. Telomerase activity was found to be higher in the higher grades (Grade II and III) compared with the lower grade (Grade I). The average mRNA expression of TRF1 and POT1 was lower in the tumor tissues than in the normal tissues. Tankyrase 1 mRNA expression showed a grade‐dependent increase in tumor tissues and its expression was also high in estrogen and progesterone negative tumors. The data support the notion that telomere dysfunction might be of value as a marker of aggressiveness of the tumors in breast cancer patients.

Human embryonic stem cells may display higher resistance to genotoxic stress as compared to primary explanted somatic cells.

The use of human embryonic stem (hES) cells in genotoxicity screening can potentially overcome the deficiencies associated with using immortalized cell lines, primary explanted somatic cells, and live animal models. Hence this study sought to compare the responses of hES cells and primary explanted somatic cells (IMR-90 cells, human fetal lung fibroblasts) to genotoxic stress, to evaluate whether hES cells can accurately reflect the normal physiology of human somatic cells. The effects of mitomycin C (MMC) on the chromosomal stability of hESC and IMR-90 was assayed and compared by fluorescence in situ hybridization (FISH) with telomere-specific peptide nucleic acid and multicolor (m) FISH techniques. The results showed that, the percentage of aberrant cells increased from 6% in the untreated control to 57.5% at the higher dose of 0.06 microg/ml MMC (9.6-fold increase) group in the case of IMR-90 cells, whereas hES cells displayed a corresponding increase from 6% to 28% (4.6-fold increase). Telomere FISH ascertained that the main types of damage induced by MMC are chromosomal breaks and the loss of telomeric signals. No fusions were observed in all samples analyzed. This was further confirmed by mFISH, which showed that fusions and translocations were not the type of aberration induced by MMC, with no such aberrations being observed in all samples analyzed. Hence, hES cells of the H1 line are apparently more resistant to MMC-induced DNA damage, as compared to the IMR-90 cells. These results highlight possible intrinsic differences in response to damaging agents between hES cells and normal somatic cells.

Telomere-Mediated Chromosomal Instability Triggers TLR4 Induced Inflammation and Death in Mice

Background Telomeres are essential to maintain chromosomal stability. Cells derived from mice lacking telomerase RNA component (mTERC−/− mice) display elevated telomere-mediated chromosome instability. Age-dependent telomere shortening and associated chromosome instability reduce the capacity to respond to cellular stress occurring during inflammation and cancer. Inflammation is one of the important risk factors in cancer progression. Controlled innate immune responses mediated by Toll-like receptors (TLR) are required for host defense against infection. Our aim was to understand the role of chromosome/genome instability in the initiation and maintenance of inflammation. Methodology/Principal Findings We examined the function of TLR4 in telomerase deficient mTERC−/− mice harbouring chromosome instability which did not develop any overt immunological disorder in pathogen-free condition or any form of cancers at this stage. Chromosome instability was measured in metaphase spreads prepared from wildtype (mTERC+/+), mTERC+/− and mTERC−/− mouse splenocytes. Peritoneal and/or bone marrow-derived macrophages were used to examine the responses of TLR4 by their ability to produce inflammatory mediators TNFα and IL6. Our results demonstrate that TLR4 is highly up-regulated in the immune cells derived from telomerase-null (mTERC−/−) mice and lipopolysaccharide, a natural ligand for TLR4 stabilises NF-κB binding to its promoter by down-regulating ATF-3 in mTERC−/− macrophages. Conclusions/Significance Our findings implied that background chromosome instability in the cellular level stabilises the action of TLR4-induced NF-κB action and sensitises cells to produce excess pro-inflammatory mediators. Chromosome/genomic instability data raises optimism for controlling inflammation by non-toxic TLR antagonists among high-risk groups.

Clinico-Pathological Correlation of β-Catenin and Telomere Dysfunction in Head and Neck Squamous Cell Carcinoma Patients.

Background: Tumorigenesis is a complex process of accumulated alteration in function of multiple genes and pathways. Wnt signalling pathway is involved in various differentiation events during embryonic development and is conserved in various species. Objective: A multicentre collaborative initiative is undertaken to study the occurrence, prognosis and molecular mechanism of HNSCC (Head and Neck Squamous Cell Carcinoma) which is highly prevalent in eastern parts of India. From a large cohort of HNSCC tissue repository, 67 cases were selected for multi-parametric investigation. Results: 67 cases showed stable β-catenin expression. We have seen correlation, if any, of the transcription factor - β-catenin, telomere maintenance and shelterin complex proteins - TRF2, Rap1 and hTert with respect to tumor differentiation and telomere dysfunction. Immunohistochemistry of β-catenin protein showed stable and high expression in tumor when compared to stroma. MDSCC (Moderately Differentiated Squamous cell carcinoma) cases expressed nuclear expression of β-catenin in invasive fronts and showed increased genomic instability. Higher frequency of Anaphase bridges was observed ranging from <3% in normal cut margin to 13% in WDSCC (Well differentiated squamous cell carcinoma) and 18% in MDSCC (Moderately differentiated Squamous cell carcinoma). There was significant decrease in telomere length in MDSCC (<4) when compared to the normal cut margin samples (<7). Quantitative Real Time-PCR confirmed a significant correlationship between stable β-catenin expression and poor clinical and pathological outcome. Conclusion: The Stabilisation and accumulation of β-catenin was significant and correlated well with de-differentiation process as well as prognosis and therapy outcome of the patients in the cohort. Expression status of molecular markers such as β-catenin, hTert, TRF2 and RAP1 correlate significantly with the process of tumorigenesis and prognosis and may play a role in therapeutic management of Head and neck patients.