Glial tumorigenesis: molecular alterations and identification of targets

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

Background: We 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. Methods: In 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). Results: Bands 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. Conclusion: These 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

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: MIT 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 12 x 10(4) M-1 for DNA and 1.06 x 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 dosedependent manner. Biophysical outcomes are suggestive of base binding and helix contraction of nucleic acid structure upon binding with allicin. Genefal 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

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

Frequent loss of heterozygosity encompassing the hMLH1 locus in low grade astrocytic tumors

The mismatch repair genes, hMLH1 (3p22) and hMSH2 (2p21), are commonly associated with accumulation of mutations and microsatellite instability. However, the status of their gene loci itself is often not addressed. In astrocytic tumors, the heterozygosity status of these genes with reference to tumor grade has not yet been determined. We have analyzed the heterozygosity status and locus specific instability in 43 glial tumors comprising 22 low grades diffuses astrocytoma (WHO Grade II, DA) and 21 glioblastoma multiforme (Grade IV GBM) using 10 microsatellite markers at 2p and 3p to elucidate the involvement of these loci in astrocytic tumorigenesis. We observed a significantly higher loss of heterozygosity (LOH) in 3p markers encompassing the hMLH1 gene locus in DA when compared to GBM (P=0.008). In DA, while the frequency of LOH was observed to be higher in markers close to the hMLH1 gene (∼40%), locus specific microsatellite instability (LSI) was higher (∼30%) in markers localizing further to the gene. The frequency of LOH at markers on 2p, near the hMSH2 gene was, however, similar in DA and GBM (P=0.451). Our results suggest that in the astrocytic tumorigenesis, LOH at the hMLH1 gene locus is an early event in tumorigenesis. However, the mismatch repair protein expression may be regulated by other cellular factors

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

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 (H2O2) 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 H2O2 in glioma cells. HA sensitized both p53 wild type and mutated glioma cells to 0.25 mM H2O2. Potentiating effect of HA was independent of p53. Higher levels of reactive oxygen species (ROS) generation were observed in cells treated with HA+H2O2 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 4 h. The results suggest that higher levels of intracellular ROS, generated by HA+H2O2 act as a molecular switch in activating a rapidly acting p53-independent mitochondrial apoptotic pathway

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