Glial tumorigenesis: molecular alterations and identification of targets

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Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals

Neutralizing monoclonal antibodies are being found to be increasingly useful in viral infections. In hepatitis B infection, antibodies are proven to be useful for passive prophylaxis. The preS1 region (21–47a.a.) of HBV contains the viral hepatocyte-binding domain crucial for its attachment and infection of hepatocytes. Antibodies against this region are neutralizing and are best suited for immune-based neutralization of HBV, especially in view of their not recognizing decoy particles. Anti-preS1 (21–47a.a.) antibodies are present in serum of spontaneously recovered individuals. We generated a phage-displayed scFv library using circulating lymphocytes from these individuals and selected four preS1-peptide specific scFvs with markedly distinct sequences from this library. All the antibodies recognized the blood-derived and recombinant preS1 containing antigens. Each scFv showed a discrete binding signature, interacting with different amino acids within the preS1-peptide region. Ability to prevent binding of the preS1 protein (N-terminus 60a.a.) to HepG2 cells stably expressing hNTCP (HepG2-hNTCP-C4 cells), the HBV receptor on human hepatocytes was taken as a surrogate marker for neutralizing capacity. These antibodies inhibited preS1-hepatocyte interaction individually and even better in combination. Such a combination of potentially neutralizing recombinant antibodies with defined specificities could be used for preventing/managing HBV infections, including those by possible escape mutants

Clonal mutations in primary human glial tumors: evidence in support of the mutator hypothesis

<p>Abstract</p> <p>Background</p> <p>A verifiable consequence of the mutator hypothesis is that even low grade neoplasms would accumulate a large number of mutations that do not influence the tumor phenotype (clonal mutations). In this study, we have attempted to quantify the number of clonal mutations in primary human gliomas of astrocytic cell origin. These alterations were identified in tumor tissue, microscopically confirmed to have over 70% neoplastic cells.</p> <p>Methods</p> <p>Random Amplified Polymorphic DNA (RAPD) analysis was performed using a set of fifteen 10-mer primers of arbitrary but definite sequences in 17 WHO grade II astrocytomas (low grade diffuse astrocytoma or DA) and 16 WHO grade IV astrocytomas (Glioblastoma Multiforme or GBM). The RAPD profile of the tumor tissue was compared with that of the leucocyte DNA of the same patient and alteration(s) scored. A quantitative estimate of the overall genomic changes in these tumors was obtained by 2 different modes of calculation.</p> <p>Results</p> <p>The overall change in the tumors was estimated to be 4.24% in DA and 2.29% in GBM by one method and 11.96% and 6.03% in DA and GBM respectively by the other. The difference between high and lower grade tumors was statistically significant by both methods.</p> <p>Conclusion</p> <p>This study demonstrates the presence of extensive clonal mutations in gliomas, more in lower grade. This is consistent with our earlier work demonstrating that technique like RAPD analysis, unbiased for locus, is able to demonstrate more intra-tumor genetic heterogeneity in lower grade gliomas compared to higher grade. The results support the mutator hypothesis proposed by Loeb.</p

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

Molecular pathways of glial tumorigenesis

Gliomas are the most frequently occurring central nervous system tumours that include astrocytomas, anaplastic astrocytomas and glioblastoma multiforme. In spite of modern therapeutic procedures, the outcome of patients with glial tumours remains unchanged. The development of gliomas, like many other tumours, is a multistep process involving the accumulation of several genetic events which include activation of oncogenes, growth factors and their receptors and the loss of tumour suppressor genes leading to the perturbation of pathways regulating cell growth and differentiation. Genomic instability also develops during the course of glioma formation. There has been an increase in our understanding of the genesis and progression of human astrocytic tumours. This review focuses on various genetic events known to occur in the development and progression of human astrocytic tumours

Gene discovery in glioma in the context of molecular reclassification of tumors

Conventional classification of tumors, especially in terms of staging and grading is of immense importance for both prognostication as well as management strategies. However it is not a perfect system and there are many instances where tumor behaviour does not correspond to what is expected. In addition, with the onset of targeted therapy, the identification of the distinct molecular target in a subset of tumors becomes a marker of tumor behaviour as well as a target of therapy. This leads to the concept of molecular subclassification of tumors where molecular markers further refine and in some cases, alter conventional classification. We would be presenting this concept in relation to glial tumors, especially in the context of molecular markers discovered in our laboratory.</p

Alteration of a sequence with homology to human endogenous retrovirus (HERV-K) in primary human glioma: implications for viral repeat mediated rearrangement

We had earlier demonstrated that a comparison of DNA fingerprinting profiles of tumor and corresponding normal DNA from the same patient by random amplified polymorphic DNA (RAPD) analysis can readily demonstrate alterations in tumor DNA [Gene 206 (1998) 45 and J. Neuro Oncol. 48 (2000) 1]. These alterations could be used to identify changes in tumor DNA where the prior identity of the locus was not known. In this study, we report the identification, cloning and characterization of a RAPD amplified fragment which was lost in a glioma, a grade IV glioblastoma multiforme (GBM). Comparison of the RAPD profile of tumor and corresponding leucocyte DNA revealed several differences between the two. These included a band of 443 bases, which was demonstrated in the normal, but not in tumor DNA. On sequencing, this band was found to be homologous with a group of SINE sequences, which are probably derived from the human endogenous retrovirus-K (HERV-K). Homology search also reveals that HERV-K-derived sequences are interspersed, amongst others, in the tumor suppressor gene BRCA2 and the DNA repair gene XRCC1. Of particular interest is the inverted repeat pattern of HERV-derived sequences in the genes. While not demonstrating a cause effect relationship, this highlights the possible role of such virus-derived sequences in gene inactivation by recombination during tumorigenesis

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

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