Tapasya Srivastava

p53 dependent apoptosis in glioma cell lines in response to hydrogen peroxide induced oxidative stress

Generation of reactive oxygen species (ROS) is an important mode of action of many chemotherapeutic agents. Hydrogen peroxide (H(2)O(2)) is a model oxidant that has been used to study the response of cells to oxidative stress. The role of p53 in ROS induced cell death has not been consistent and has been shown to be cell type dependent. Study of cellular and molecular parameters and mechanisms involved in H(2)O(2) induced cell death in glioma cells will contribute to the understanding of response of these cells to oxidative stress. We investigated induction of cell death by H(2)O(2), and its relation to p53 in two human glial tumor derived cell lines U87MG (wild type p53) and U373MG (mutated p53). We observed that H(2)O(2) was able to induce apoptosis (as shown by morphology, flow cytometry and DNA fragmentation studies) in U87MG in a dose dependent manner. Dimethyl sulfoxide (DMSO), a known ROS scavenger, was protective to the cells. H(2)O(2) induced cell death was significantly reduced by antisense p53 oligonucleotide. Pretreatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of the redox sensitive transcription factor NF-kappa B, abrogated the increased expression of p53 protein in response to H(2)O(2), and enhanced cell survival. The U373MG cell line, having mutated p53, was comparatively resistant to H(2)O(2) induced cell death. We conclude from the study that p53, activated by NF-kappa B, is essential for H(2)O(2) induced apoptosis in glioma cells.

Frequent loss of heterozygosity and altered expression of the candidate tumor suppressor gene 'FAT' in human astrocytic tumors

BackgroundWe had earlier used the comparison of RAPD (Random Amplification of Polymorphic DNA) DNA fingerprinting profiles of tumor and corresponding normal DNA to identify genetic alterations in primary human glial tumors. This has the advantage that DNA fingerprinting identifies the genetic alterations in a manner not biased for locus.MethodsIn this study we used RAPD-PCR to identify novel genomic alterations in the astrocytic tumors of WHO grade II (Low Grade Diffuse Astrocytoma) and WHO Grade IV (Glioblastoma Multiforme). Loss of heterozygosity (LOH) of the altered region was studied by microsatellite and Single Nucleotide Polymorphism (SNP) markers. Expression study of the gene identified at the altered locus was done by semi-quantitative reverse-transcriptase-PCR (RT-PCR).ResultsBands consistently altered in the RAPD profile of tumor DNA in a significant proportion of tumors were identified. One such 500 bp band, that was absent in the RAPD profile of 33% (4/12) of the grade II astrocytic tumors, was selected for further study. Its sequence corresponded with a region of FAT, a putative tumor suppressor gene initially identified in Drosophila. Fifty percent of a set of 40 tumors, both grade II and IV, were shown to have Loss of Heterozygosity (LOH) at this locus by microsatellite (intragenic) and by SNP markers. Semi-quantitative RT-PCR showed low FAT mRNA levels in a major subset of tumors.ConclusionThese results point to a role of the FAT in astrocytic tumorigenesis and demonstrate the use of RAPD analysis in identifying specific alterations in astrocytic tumors.

Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia

Hypoxia is an integral part of tumorigenesis and contributes extensively to the neoplastic phenotype including drug resistance and genomic instability. It has also been reported that hypoxia results in global demethylation. Because a majority of the cytosine‐phosphate‐guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability. U87MG glioblastoma cells were cultured in 0.1% O2 for 6 weeks and compared with cells cultured in 21% O2 for the same duration. Real‐time PCR analysis showed a significant increase in SINE and reverse transcriptase coding long interspersed nuclear element (LINE) transcripts during hypoxia. Sequencing of bisulphite treated DNA as well as the Combined Bisulfite Restriction Analysis (COBRA) assay showed that the SINE loci studied underwent significant hypomethylation though there was patchy hypermethylation at a few sites. The inter‐alu PCR profile of DNA from cells cultured under 6‐week hypoxia, its 4‐week revert back to normoxia and 6‐week normoxia showed several changes in the band pattern indicating increased alu mediated genomic alteration. Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia. This might be a cause of genetic heterogeneity in tumours especially in variegated hypoxic environment and lead to a development of foci of more aggressive tumour cells.

Sensitizing glioma cells to cisplatin by abrogating the p53 response with antisense oligonucleotides

Most gene therapy strategies related to p53 concentrate on the restoration of the activity of mutant p53, as several observations indicate that tumors and cell lines having the mutant gene are resistant to chemotherapy. However, as there is also some evidence to the contrary, we studied the relationship of the p53 status to the cellular response of glioma cells that were exposed to cisplatin. At a concentration of 2.5 μg/ml (which is about half the peak pharmacological blood level reached during chemotherapy), U373MG glioma cells, which had a mutant p53 gene, were more sensitive to the drug as compared to U87MG glioma cells (with normal p53). The U373MG cells responded with apoptosis while U87MG cells responded with a G2–M arrest. In U87MG cells, blocking the p53 response by antisense oligonucleotides also sensitized the cells to 2.5 μg/ml cisplatin, and shifted the cellular response from arrest to caspase 3-mediated apoptosis. A sensitive, p53-independent, mechanism for chemotherapy-induced apoptosis suggests that, in some cases, p53 abrogation by gene therapy or small molecule-based strategies could be a viable therapeutic strategy.

Increased hMSH2 Protein Expression in Glioblastoma Multiforme

AbstracthMSH2 and hMLH1 are the most commonly studied mismatch repair proteins and their absence is associated with microsatellite instability (MSI) especially in hereditary non-polyposis colorectal cancer, and also in some sporadic tumors. However, there are some tumors, namely, urothelial neoplasms and salivary gland tumors, where overexpression of the proteins has been reported, though the implications of these findings are not very clear. There is no report on the expression of these proteins in different grades of human astrocytic tumors. We have studied the expression pattern of hMSH2 and hMLH1 in high (Grade IV, glioblastoma multiforme (GBM)) and low (Grade II, astrocytoma (AS)) grade primary human gliomas by immunohistochemistry. We observed that there was a significantly higher expression of hMSH2 protein in 28 GBM (mean 703.07 ± 236.28) as compared with 27 AS (mean 307.03 ± 204.71), p = 1.47 × 10−8 by a two-tailed t-test of unpaired samples. However, for hMLH1 no such difference was observed, mean counts being 543.29 ± 320.35 for 27 GBM and 505.92 ± 342.37 for 26 AS, p = 0.67. A small proportion of tumors was observed to be immunonegative for either of the proteins in both high- and low-grade tumors. While MSI has been shown previously to be infrequent in human astrocytic tumors, the implications of the overexpression of hMSH2 in GBM are not clear.

Nucleic acid binding properties of allicin: Spectroscopic analysis and estimation of anti-tumor potential

BACKGROUND Allicin has received much attention due to its anti-proliferative activity and not-well elucidated underlying mechanism of action. This work focuses towards determining the cellular toxicity of allicin and understanding its interaction with nucleic acid at molecular level. METHODS MTT assay was used to assess the cell viability of A549 lung cancer cells against allicin. Fourier transform infrared (FTIR) and UV-visible spectroscopy were used to study the binding parameters of nucleic acid-allicin interaction. RESULTS Allicin inhibits the proliferation of cancer cells in a concentration dependent manner. FTIR spectroscopy exhibited that allicin binds preferentially to minor groove of DNA via thymine base. Analysis of tRNA allicin complex has also revealed that allicin binds primarily through nitrogenous bases. Some amount of external binding with phosphate backbone was also observed for both DNA and RNA. UV visible spectra of both DNA allicin and RNA allicin complexes showed hypochromic shift with an estimated binding constant of 1.2×10(4)M(-1) for DNA and 1.06×10(3)M(-1)for RNA binding. No major transition from the B-form of DNA and A-form of RNA is observed after their interaction with allicin. CONCLUSIONS The results demonstrated that allicin treatment inhibited the proliferation of A549 cells in a dose-dependent manner. Biophysical outcomes are suggestive of base binding and helix contraction of nucleic acid structure upon binding with allicin. GENERAL SIGNIFICANCE The results describe cytotoxic potential of allicin and its binding properties with cellular nucleic acid, which could be helpful in deciphering the complete mechanism of cell death exerted by allicin.

Recent advances in targeted therapy for glioblastoma

Glioblastomas are the most common form of brain tumor with a very dismal prognosis. While a standard treatment regimen of surgery followed by chemo/radiotherapy is currently used, this has only marginally improved the survival time of patients with little benefit on tumor recurrence. Although many molecular targets have already been identified and tested in clinical trials, very few are approved for use in clinics. Efforts are ongoing to target newer molecules that could be used for drug development. This review provides up-to-date information on the drugs and their molecular targets, which are currently in different stages of clinical trials. Since multiple signaling pathways are deregulated, it appears that the use of combination drugs along with personalized targeting approach would provide better therapy in the future.

Heterozygosity status of 1p and 19q and its correlation with p53 protein expression and EGFR amplification in patients with astrocytic tumors: novel series from India

There are few reports of loss of heterozygosity (LOH) of 1p and 19q in astrocytic tumors, especially glioblastoma multiforme (GBM). We evaluated 1p and 19q (either or both) heterozygosity status in 71 astrocytomas, including 6 pediatric cases: 20 diffuse astrocytomas (DA), 9 anaplastic astrocytomas (AA), and 42 GBM. In the GBMs, p53 protein expression was assessed by immunohistochemistry and epidermal growth factor receptor (EGFR) gene amplification by fluorescence in situ hybridization; TP53 sequencing was done in 15 of the GBMs. In adults, LOH of 1p or 19q was detected in 16% of DAs and 50% of GBMs; none of the AAs showed this alteration. In GBMs, LOH of 19q was most common (26%), followed by combined 1p and 19q LOH (13%) and 1p LOH (10%). Pediatric GBMs also harbored isolated 1p and 19q LOH (50%). Notably, LOH of 1p or 19q LOH was more frequent in p53 immunopositive secondary GBMs (61%) than in primary GBMs (17%). This suggests that LOH of 1p and 19q may be acquired during progression to secondary GBMs. Thus, 1p and 19q LOH can occur in astrocytic tumors, most commonly in secondary GBMs without morphological correlation with an oligodendroglial histology. The clinical significance of recognition of this subset of GBMs is based on several recent reports of association with better prognosis, although long-term follow-up studies are required.

Inter-alu PCR detects high frequency of genetic alterations in glioma cells exposed to sub-lethal cisplatin.

Increased genomic instability contributes to higher frequency of secondary drug resistance and neoplastic progression in tumors as well as in cells exposed to sub‐lethal concentrations of chemotherapeutic agents. We have used PCR based DNA fingerprinting techniques of randomly amplified polymorphic DNA (RAPD) and inter‐alu PCR to study this phenomenon in the tumor genome. The choice of the primer, either random (for RAPD) or specific (inter‐alu PCR) can determine the nature of alterations being assessed. We have compared the inter‐alu PCR and RAPD profiles of U87MG glioblastoma cells exposed to sequentially increasing low doses of cisplatin for 24 passages to that of untreated controls. Inter‐alu PCR, with 2 primers, demonstrated a number of alterations in the treated cells, in the form of loss / gain and changes in the intensity of bands. No changes were observed by RAPD analysis with 5 primers, however, indicating a preferential increase in the alu mediated recombination frequency in the treated cells (p = 1.866 × 10−4). The number of changes observed with respect to the corresponding leucocyte DNA in the inter‐alu PCR profile of 26 primary tumors (Grade II = 13; Grade IV = 13), resected before chemotherapy, for the 2 inter‐alu primers was very small. We present a novel application of the inter‐alu PCR in detecting alterations in long term cultured cells at low dose exposure to a chemotherapeutic agent. Our results suggest that alu mediated recombination may be important in cells exposed to sub‐lethal doses of cisplatin but not in the genesis of primary glioma.

Hydroxylamine potentiates the effect of low dose hydrogen peroxide in glioma cells independent of p53

We had earlier shown that higher concentration of hydrogen peroxide (H(2)O(2)) induced p53-dependent apoptosis in glioma cell line with wild type p53 but had minimal effect on cells with mutated p53. Here we show a potentiating effect of hydroxylamine (HA), an inhibitor of catalase, on a nontoxic dose of H(2)O(2) in glioma cells. HA sensitized both p53 wild type and mutated glioma cells to 0.25 mM H(2)O(2). Potentiating effect of HA was independent of p53. Higher levels of reactive oxygen species (ROS) generation were observed in cells treated with HA+H(2)O(2) as compared to cells treated with each component alone in both the cell lines. Dimethyl sulfoxide (DMSO) protected cells. Cytosolic cytochrome c and activated caspase 3 were detected at 4h. The results suggest that higher levels of intracellular ROS, generated by HA+H(2)O(2) act as a molecular switch in activating a rapidly acting p53-independent mitochondrial apoptotic pathway.