Shriram Venkatesan

Inactivation of chk2 and mus81 leads to impaired lymphocytes development, reduced genomic instability, and suppression of cancer.

Chk2 is an effector kinase important for the activation of cell cycle checkpoints, p53, and apoptosis in response to DNA damage. Mus81 is required for the restart of stalled replication forks and for genomic integrity. Mus81Δex3-4/Δex3-4 mice have increased cancer susceptibility that is exacerbated by p53 inactivation. In this study, we demonstrate that Chk2 inactivation impairs the development of Mus81Δex3-4/Δex3-4 lymphoid cells in a cell-autonomous manner. Importantly, in contrast to its predicted tumor suppressor function, loss of Chk2 promotes mitotic catastrophe and cell death, and it results in suppressed oncogenic transformation and tumor development in Mus81Δex3-4/Δex3-4 background. Thus, our data indicate that an important role for Chk2 is maintaining lymphocyte development and that dual inactivation of Chk2 and Mus81 remarkably inhibits cancer.

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.

Plumbagin Alters Telomere Dynamics, Induces DNA damage and cell death in human Brain Tumour Cells

Natural plant products may possess much potential in palliative therapy and supportive strategies of current cancer treatments with lesser cytotoxicity to normal cells compared to conventional chemotherapy. In the current study, anti-cancer properties of plumbagin, a plant-derived naphthoquinone, on brain cancer cells were determined. Plumbagin treatment resulted in the induction of DNA damage, cell cycle arrest and apoptosis, followed by suppression of the colony forming ability of the brain tumour cells. These effects were substantiated by upregulation of PTEN, TNFRSF1A and downregulation of E2F1 genes, along with a drop in MDM2, cyclin B1, survivin and BCL2 protein expression. Plumbagin induced elevated levels of caspase-3/7 activity as well. For the first time, we show here that plumbagin inhibits telomerase in brain tumour cells and results in telomere shortening following chronic long-term treatment. This observation implies considerable cytotoxicity of plumbagin towards cancer cells with higher telomerase activity. Collectively, our findings suggest plumbagin as a potential chemotherapeutic phytochemical in brain tumour treatment modalities.

Chromosomal instability--mechanisms and consequences.

Chromosomal instability is defined as a state of numerical and/or structural chromosomal anomalies in cells. Numerous studies have documented the incidence of chromosomal instability, which acutely or chronically may lead to accelerated ageing (tissue-wide or even organismal), cancer or other genetic disorders. Potential mechanisms leading to the generation of chromosome-genome instability include erroneous/inefficient DNA repair, chromosome segregation defects, spindle assembly defects, DNA replication stress, telomere shortening/dysfunction - to name a few. Understanding the cellular and molecular mechanisms for chromosomal instability in various human cells and tissues will be useful in elucidating the cause for many age associated diseases including cancer. This approach holds a great promise for the cytogenetic assays not only for prognosis but also for diagnostic purposes in clinical settings. In this review, a multi-dimensional approach has been attempted to portray the complexity behind the incidence of chromosome-genome instability including evolutionary implications at the species level for some of the mechanisms of chromosomal instability.

Targeting DNA-PKcs and telomerase in brain tumour cells.

BackgroundPatients suffering from brain tumours such as glioblastoma and medulloblastoma have poor prognosis with a median survival of less than a year. Identifying alternative molecular targets would enable us to develop different therapeutic strategies for better management of these tumours.MethodsGlioblastoma (MO59K and KNS60) and medulloblastoma cells (ONS76) were used in this study. Telomerase inhibitory effects of MST-312, a chemically modified-derivative of epigallocatechin gallate, in the cells were assessed using telomere repeat amplification protocol. Gene expression analysis following MST-312 treatment was done by microarray. Telomere length was measured by telomere restriction fragments analysis. Effects of MST-312 on DNA integrity were evaluated by single cell gel electrophoresis, immunofluorescence assay and cytogenetic analysis. Phosphorylation status of DNA-PKcs was measured with immunoblotting and effects on cell proliferation were monitored with cell titre glow and trypan blue exclusion following dual inhibition.ResultsMST-312 showed strong binding affinity to DNA and displayed reversible telomerase inhibitory effects in brain tumour cells. In addition to the disruption of telomere length maintenance, MST-312 treatment decreased brain tumour cell viability, induced cell cycle arrest and double strand breaks (DSBs). DNA-PKcs activation was observed in telomerase-inhibited cells presumably as a response to DNA damage. Impaired DNA-PKcs in MO59J cells or in MO59K cells treated with DNA-PKcs inhibitor, NU7026, caused a delay in the repair of DSBs. In contrast, MST-312 did not induce DSBs in telomerase negative osteosarcoma cells (U2OS). Combined inhibition of DNA-PKcs and telomerase resulted in an increase in telomere signal-free chromosomal ends in brain tumour cells as well. Interestingly, continual exposure of brain tumour cells to telomerase inhibitor led to population of cells, which displayed resistance to telomerase inhibition-mediated cell arrest. DNA-PKcs ablation in these cells, however, confers higher cell sensitivity to telomerase inhibition, inducing cell death.ConclusionsEfficient telomerase inhibition was achieved with acute exposure to MST-312 and this resulted in subtle but significant increase in DSBs. Activation of DNA-PKcs might indicate the requirement of NHEJ pathway in the repair telomerase inhibitor induced DNA damage. Therefore, our results suggest a potential strategy in combating brain tumour cells with dual inhibition of telomerase and NHEJ pathway.

Synergistic Interaction of Rnf8 and p53 in the Protection against Genomic Instability and Tumorigenesis

Rnf8 is an E3 ubiquitin ligase that plays a key role in the DNA damage response as well as in the maintenance of telomeres and chromatin remodeling. Rnf8−/− mice exhibit developmental defects and increased susceptibility to tumorigenesis. We observed that levels of p53, a central regulator of the cellular response to DNA damage, increased in Rnf8−/− mice in a tissue- and cell type–specific manner. To investigate the role of the p53-pathway inactivation on the phenotype observed in Rnf8−/− mice, we have generated Rnf8−/−p53−/− mice. Double-knockout mice showed similar growth retardation defects and impaired class switch recombination compared to Rnf8−/− mice. In contrast, loss of p53 fully rescued the increased apoptosis and reduced number of thymocytes and splenocytes in Rnf8−/− mice. Similarly, the senescence phenotype of Rnf8−/− mouse embryonic fibroblasts was rescued in p53 null background. Rnf8−/−p53−/− cells displayed defective cell cycle checkpoints and DNA double-strand break repair. In addition, Rnf8−/−p53−/− mice had increased levels of genomic instability and a remarkably elevated tumor incidence compared to either Rnf8−/− or p53−/− mice. Altogether, the data in this study highlight the importance of p53-pathway activation upon loss of Rnf8, suggesting that Rnf8 and p53 functionally interact to protect against genomic instability and tumorigenesis.

Biomarkers of ionizing radiation exposure: A multiparametric approach

Humans are exposed to ionizing radiation not only through background radiation but also through the ubiquitous presence of devices and sources that generate radiation. With the expanded use of radiation in day.to.day life, the chances of accidents or misuse only increase. Therefore, a thorough understanding of the dynamic effects of radiation exposure on biological entities is necessary. The biological effects of radiation exposure on human cells depend on much variability such as level of exposure, dose rate, and the physiological state of the cells. During potential scenarios of a large.scale radiological event which results in mass casualties, dose estimates are essential to assign medical attention according to individual needs. Many attempts have been made to identify biomarkers which can be used for high throughput biodosimetry screening. In this study, we compare the results of different biodosimetry methods on the same irradiated cells to assess the suitability of current biomarkers and push forward the idea of employing a multiparametric approach to achieve an accurate dose and risk estimation.

Rad54 and Mus81 cooperation promotes DNA damage repair and restrains chromosome missegregation

Rad54 and Mus81 mammalian proteins physically interact and are important for the homologous recombination DNA repair pathway; however, their functional interactions in vivo are poorly defined. Here, we show that combinatorial loss of Rad54 and Mus81 results in hypersensitivity to DNA-damaging agents, defects on both the homologous recombination and non-homologous DNA end joining repair pathways and reduced fertility. We also observed that while Mus81 deficiency diminished the cleavage of common fragile sites, very strikingly, Rad54 loss impaired this cleavage to even a greater extent. The inefficient repair of DNA double-strand breaks (DSBs) in Rad54−/−Mus81−/− cells was accompanied by elevated levels of chromosome missegregation and cell death. Perhaps as a consequence, tumor incidence in Rad54−/−Mus81−/− mice remained comparable to that in Mus81−/− mice. Our study highlights the importance of the cooperation between Rad54 and Mus81 for mediating DNA DSB repair and restraining chromosome missegregation.

Telomere Biology—Insights into an Intriguing Phenomenon

Bacteria and viruses possess circular DNA, whereas eukaryotes with typically very large DNA molecules have had to evolve into linear chromosomes to circumvent the problem of supercoiling circular DNA of that size. Consequently, such organisms possess telomeres to cap chromosome ends. Telomeres are essentially tandem repeats of any DNA sequence that are present at the ends of chromosomes. Their biology has been an enigmatic one, involving various molecules interacting dynamically in an evolutionarily well-trimmed fashion. Telomeres range from canonical hexameric repeats in most eukaryotes to unimaginably random retrotransposons, which attach to chromosome ends and reverse-transcribe to DNA in some plants and insects. Telomeres invariably associate with specialised protein complexes that envelop it, also regulating access of the ends to legitimate enzymes involved in telomere metabolism. They also transcribe into repetitive RNA which also seems to be playing significant roles in telomere maintenance. Telomeres thus form the intersection of DNA, protein, and RNA molecules acting in concert to maintain chromosome integrity. Telomere biology is emerging to appear ever more complex than previously envisaged, with the continual discovery of more molecules and interplays at the telomeres. This review also includes a section dedicated to the history of telomere biology, and intends to target the scientific audience new to the field by rendering an understanding of the phenomenon of chromosome end protection at large, with more emphasis on the biology of human telomeres. The review provides an update on the field and mentions the questions that need to be addressed.

Effects of rapamycin on the mechanistic target of rapamycin (mTOR) pathway and telomerase in breast cancer cells

The mTOR pathway and the enzyme telomerase are two key players commonly upregulated in cancers. They render survival and proliferative advantage to cancer cells, and are regarded as attractive anticancer targets. Rapamycin, a macrolide antibiotic and mTOR inhibitor, has recently also been implicated in telomerase inhibition and telomere attrition, although the mechanisms remain poorly understood. Using breast cancer cells (MCF-7 and MDA-MB-231) wherein telomerase activity and mTOR pathway are concurrently overexpressed, this study sought to unravel novel mechanisms by which rapamycin may affect these pathways. Short term treatment with an acute dose of rapamycin inhibited the mTOR pathway and telomerase activity and induced G1 arrest. This arrest was independent of cyclin D1 and p21 levels and was not mediated by DNA damage in both cell types. While long term treatment with a clinically relevant dose of rapamycin resulted in compromised population doubling capacity and mTOR pathway inhibition, there was no effect on telomere functionality and telomerase activity as evidenced by our assessments of hTERT protein levels, in vitro telomerase activity, telomere length and telomere FISH analyses. We also found that sustained rapamycin treatment leading to Akt activation may play a role in resistance in the more invasive MDA-MB-231 cells. In summary, rapamycin specifically inhibits the activation of mTOR pathway. Moreover, we show for the first time that while acute short-term treatment with rapamycin induces telomerase inhibition, it does not affect telomerase activity nor does it inflict telomere dysfunction in breast cancer cells upon chronic long-term treatment with a clinically relevant dose. These findings may be useful while designing combinatorial treatment strategies with rapamycin inhibition in the clinic.