Biomedical subjects

Subrata Sinha

Publications and source records attributed to Subrata Sinha.

At least 19 recordsLinked to original sources

PubMed2023

Phylogeny and evolution of SARS-CoV-2 during Delta and Omicron variant waves in India.

SARS-CoV-2 evolution has continued to generate variants, responsible for new pandemic waves locally and globally. Varying disease presentation and severity has been ascribed to inherent variant characteristics and vaccine immunity. This study analyzed genomic data from 305 whole genome sequences from SARS-CoV-2 patients before and through the third wave in India. Delta variant was reported in patients without comorbidity (97%), while Omicron BA.2 was reported in patients with comorbidity (77%). Tissue adaptation studies brought forth higher propensity of Omicron variants to bronchial tissue than lung, contrary to observation in Delta variants from Delhi. Study of codon usage pattern distinguished the prevalent variants, clustering them separately, Omicron BA.2 isolated in February grouped away from December strains, and all BA.2 after December acquired a new mutation S959P in ORF1b (44.3% of BA.2 in the study) indicating ongoing evolution. Loss of critical spike mutations in Omicron BA.2 and gain of immune evasion mutations including G142D, reported in Delta but absent in BA.1, and S371F instead of S371L in BA.1 could explain very brief period of BA.1 in December 2021, followed by complete replacement by BA.2. Higher propensity of Omicron variants to bronchial tissue, probably ensured increased transmission while Omicron BA.2 became the prevalent variant possibly due to evolutionary trade-off. Virus evolution continues to shape the epidemic and its culmination.Communicated by Ramaswamy H. Sarma.

SARS-CoV-2↗

PubMed2006

Identification and functional characterization of a novel unspliced transcript variant of HIC-1 in human cancer cells exposed to adverse growth conditions.

The wild-type p53 gene has been widely implicated in the regulation of hypermethylated in cancer-1 (HIC-1) transcription, a master growth regulatory gene with multiple promoters and, consequently, multiple alternatively spliced transcripts. We investigated the role of p53 (wild type and mutant, both endogenous and exogenous) in modulating the various HIC-1 transcripts. We discovered a novel unspliced HIC-1 transcript, identified as "f" in leukocytes and in the human cell lines U87MG (wild-type p53), U373MG (mutant p53), MCF-7 (wild-type p53), HeLa (p53 degraded by HPV18-E6 oncoprotein), and Saos-2 (p53 null). This transcript is initiated from a new transcription start site and has an intervening stop codon that would result in a possibly truncated 22-amino-acid polypeptide. When U87MG (wild-type p53) and MCF-7 cells (wild-type p53) were exposed to adverse growth conditions of serum starvation or treated with the chemotherapeutic agent cisplatin, cells underwent apoptosis and cell cycle arrest accompanied by increase in p53 and HIC-1 transcript levels. Although the increase of the HIC-1-spliced transcripts followed the increase of p53, increase in f transcript coincided with declining p53 and HIC-1 transcript and protein levels. Moreover, the levels of HIC-1 f transcript were not induced by exogenously transfected wild-type p53 in p53-mutated U373MG and p53-null Saos-2 cells, unlike the spliced transcripts that code for full-length HIC-1 protein. These findings suggest a working model wherein the status of f transcript, which is not under direct transcriptional control of wild-type p53, may influence the level of HIC-1 protein in cancer cells.

Amino Acid Sequence↗

PubMed2006

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 ( approximately 40%), locus specific microsatellite instability (LSI) was higher ( approximately 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.

Adaptor Proteins, Signal Transducing↗

PubMed2006

A phage antibody to the active site of human placental alkaline phosphatase with higher affinity to the enzyme-substrate complex.

Selection of specific antibodies from large repertoires is of importance in generating antibodies to specific structural determinants and in studying structure-function relationships. Alkaline phosphatase (AP) has several isozymes with various degrees of homology and a range of common synthetic substrates. We have previously reported the generation of isozyme specific anti-enzyme antibodies to an oncofetal antigen, placental alkaline phosphatase (PLAP) by using a specific uncompetitive inhibitor, L-Phe-Gly-Gly along with the substrate para-nitrophenyl phosphate (pNPP), to elute scFvs from a phage-displayed immunoglobulin library. These antibodies were directed to the active site and inhibited enzyme activity. An uncompetitive inhibitor acts by stabilizing the enzyme-substrate (ES) complex. In the present work, we report the characteristics of a clone VE5, selected by the same method. This clone has a higher binding affinity for ES complex than for enzyme alone. This is true for all the three isozymes (placental, bone and intestinal) tested. However, the other synthetic small molecular substrate, disodium phenyl phosphate inhibits phage binding. The clone possibly binds to the conserved structures of the active site of the AP isozymes and the higher affinity binding to AP-pNPP complex reflects the method of selection. Such anti-enzyme antibodies have a possible potential role in dissecting structure-function relationship of enzymatic antigens.

Alkaline Phosphatase↗

PubMed2006

Generation and characterization of a high-affinity chimaeric antibody against hepatitis B surface antigen.

Antibody against HBsAg (hepatitis B surface antigen) is advocated for the passive immunotherapy in certain cases of hepatitis B infections. A recombinant monoclonal antibody against HBsAg would offer several advantages over the currently used polyclonal human hepatitis B immunoglobulin. 5S is a mouse monoclonal antibody that binds to HBsAg with very high affinity. However, this mouse antibody cannot be used for therapeutic purposes, as it may elicit antimouse immune responses. Chimaerization, by replacing mouse constant domains with human counterparts, can reduce the immunogenicity of this molecule. We have cloned the V(H) (heavy-chain variable region) and V(L) (light-chain variable region) genes of this mouse antibody, and fused them with C(H)1 (heavy-chain constant domain 1) of human IgG1 and C(L) (light-chain constant domain) of human kappa chain respectively. These chimaeric genes were cloned into a mammalian expression vector (pFab-CMV), which has a modular cassette coding for part of the hinge, C(H)2 and C(H)3 of human IgG1. The recombinant construct was transfected in CHO (Chinese-hamster ovary) cells to generate a stable transfectoma. The resulting transfectoma was maintained in a serum-free medium and the full-length chimaeric anti-HBsAg antibody was purified from the culture supernatant. The yield of the purified chimaeric antibody was moderate ( approximately 5.5 mg/l). We further characterized the chimaeric antibody using several in vitro techniques. It was observed that the chimaeric molecule was glycosylated and expressed in the expected heterodimeric form. This chimaeric antibody has very high affinity and specificity, similar to that of the original mouse monoclonal antibody.

Animals↗

PubMed2006

Prediction and molecular modeling of T-cell epitopes derived from placental alkaline phosphatase for use in cancer immunotherapy.

In our ongoing efforts to combat cancer, peptide-based tumor vaccines are promising as one of the several alternatives used for cancer immunotherapy and immunoprevention. We have attempted to identify T-cell epitopes suitable for the development of a peptide-based cancer vaccine directed towards placental isozyme of alkaline phosphatase (PLAP), an oncofetal antigen. After identifying amino acid residues specific to PLAP and distinct from other close PLAP homologs, we have used sequence-based immunoinformatics tools (BIMAS and SYFPEITHI) and conducted molecular modeling studies using InsightII to investigate the binding affinity of the epitopes containing the unique residues with respective MHC class I molecules. Promiscuous epitopes binding to different alleles of different class I HLA loci were analyzed to get a population coverage that is widespread. Binding affinity deduced from the modeling studies corroborated the status of most of the epitopes scoring high in BIMAS and SYFPEITHI. We have thus identified specific epitopes from PLAP that have a potential for binding to their respective MHC class I alleles with high affinity. These peptides would be analysed in experiments to demonstrate their involvement in the induction of primary cytotoxic T-cell responses in vitro, using respective HLA-restricted T-cells in our way towards the development of an effective anti-cancer vaccine in a background of diverse MHC haplotypes.

Alkaline Phosphatase↗

PubMed2006

Generation and characterization of a single-gene mouse-human chimeric antibody against hepatitis B surface antigen.

BACKGROUND: Antibody against hepatitis B surface antigen (HBsAg) is used for passive immunotherapy in certain cases of hepatitis B infection. The authors have earlier reported a high-affinity mouse monoclonal (5S) against HBsAg. However, this mouse antibody cannot be used for therapeutic purposes because it may elicit antimouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. METHODS: A single-chain variable fragment (scFv), derived from the mouse monoclonal 5S, was fused with the fragment crystallisable (Fc) fragment of human IgG1. The scFv region is expected to bind to the antigen, whereas the Fc fragment can provide the effector functions required for virus neutralization. This chimeric molecule was expressed in Chinese hamster ovary (CHO) cells in serum-free medium. It was purified by affinity chromatography and characterized by in vitro binding studies. RESULTS: Purification and characterization indicated that this chimeric scFv-Fc fusion protein is secreted as a disulfide-linked, glycosylated, homodimeric molecule. The yield of the purified chimeric antibody was approximately 4.6 mg/L. In vitro analyses confirmed that this chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. CONCLUSION: Because it is a single-gene product, this chimeric scFv-Fc has the advantage of stable expression. Being chimeric and bivalent, it is expected to be less immunogenic and therefore suitable for further in vivo studies on virus neutralization.

Animals↗

PubMed2005

High affinity mouse-human chimeric Fab against hepatitis B surface antigen.

AIM: Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. METHODS: We cloned the V(H) and V(L) genes of this mouse antibody, and fused them with CH1 domain of human IgG1 and C(L) domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. coli. RESULTS: The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. This chimeric antibody fragment was further expressed in different strains of E. coli to increase the yield. CONCLUSION: We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a full-length chimeric antibody for therapeutic uses.

Animals↗

PubMed2005

Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor.

BACKGROUND: The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP) is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP). The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity. RESULTS: Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP) and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP. CONCLUSION: The results demonstrate the biochemical modulation of scFv binding. Also, the scFvs bound to the active site and denied the access to the substrate. The selection strategy could generate specific anti-enzyme antibodies to PLAP that can potentially be used for targeting, for modulating enzyme activity in in vitro and in vivo and as probes for the active site. This strategy also has a general application in selecting antibodies from combinatorial libraries to closely related molecules and conformations.

Alkaline Phosphatase↗

PubMed2005

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 x 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.

Alu Elements↗

PubMed2005

Excited-state processes in the carotenoid zeaxanthin after excess energy excitation.

Aiming for better understanding of the large complexity of excited-state processes in carotenoids, we have studied the excitation wavelength dependence of the relaxation dynamics in the carotenoid zeaxanthin. Excitation into the lowest vibrational band of the S2 state at 485 nm, into the 0-3 vibrational band of the S2 state at 400 nm, and into the 2B(u)+ state at 266 nm resulted in different relaxation patterns. While excitation at 485 nm produces the known four-state scheme (S2 --> hot S1 --> S1 --> S0), excess energy excitation led to additional dynamics occurring with a time constant of 2.8 ps (400 nm excitation) and 4.9 ps (266 nm excitation), respectively. This process is ascribed to a conformational relaxation of conformers generated by the excess energy excitation. The zeaxanthin S state was observed regardless of the excitation wavelength, but its population increased after 400 and 266 nm excitation, suggesting that conformers generated by the excess energy excitation are important for directing the population toward the S state. The S2-S1 internal conversion time was shortened from 135 to 70 fs when going from 485 to 400 nm excitation, as a result of competition between the S2-S1 internal conversion from the vibrationally hot S2 state and S2 vibrational relaxation. The S1 lifetime of zeaxanthin was within experimental error the same for all excitation wavelengths, yielding approximately 9 ps. No long-lived species have been observed after excitation by femtosecond pulses regardless of the excitation wavelength, but excitation by nanosecond pulses at 266 nm generated both zeaxanthin triplet state and cation radical.

Lasers↗

PubMed2005

Expression and structure of interleukin 4 receptors in primary meningeal tumors.

BACKGROUND: It was reported previously that malignant human tumors, like glioma and medulloblastoma, express high-density interleukin (IL-4) receptor mRNA and protein. Because IL-4 receptors (R) are sensitive targets for targeted therapeutics, knowledge of the expression of these receptors in other central nervous system tumors is of great interest. In this study, the authors examined the expression and subunit composition of IL-4R complex in primary human meningiomas. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) analysis for IL-13Ralpha1, IL-4Ralpha and IL-2Rgammac was performed on total RNA extracted from 35 meningiomas and a normal human brain tissue sample. Results were confirmed in nine randomly selected tumors by quantitative real-time PCR and in situ immunofluorescence assay. RESULTS: Transcripts for the IL-4Ralpha and IL-13Ralpha1 chains were overexpressed in meningiomas compared with normal brain tissue. The levels of IL-4Ralpha mRNA appeared to be higher compared with the levels of IL-13Ralpha1 mRNA. The results also showed that tumors with higher disease grade tended to have increased mRNA expression for the IL-4Ralpha chain. This IL-4Ralpha mRNA overexpression appeared to be more frequent in younger patients (age < 37 years). The transcripts for IL-2Rgammac chain were not detected in any of the tumor samples or in normal brain tissue. Quantitative real-time PCR confirmed the results of the RT-PCR analysis. Meningiomas also demonstrated a bright immunofluorescent staining for the IL-4Ralpha and IL-13Ralpha1 chains but no staining for IL-2Rgammac. CONCLUSIONS: Expression of the IL-4Ralpha and IL-13Ralpha1 chains and absence of IL-2gammac expression established that meningiomas expressed type II IL-4Rs. These receptors may serve as a target for cytotoxin/immunotoxin therapy in patients with meningioma who are not amenable to surgical resection or for recurrent tumors.

Adult↗

PubMed2005

Apoptosis and proliferation: correlation with p53 in astrocytic tumours.

Apoptosis and cell proliferation occur simultaneously in tumour tissue with tumour suppressor gene, p53 being one of the key players in the complex relationship between these two key phenomena. We, as well as several other groups, have earlier demonstrated the association of p53 immunopositivity with increased degree of cell proliferation in astrocytic tumours. Here we have studied the extent of apoptosis in 62 primary human astrocytic tumours [25 Diffuse Astrocytoma (DA), 9 Anaplastic Astrocytoma (AA) and 28 Glioblastoma multiforme (GBM)] in relation to tumour grade, proliferative status and p53 protein expression. Apoptosis was measured by the TUNEL assay while, cell proliferation (MIB-1 index) and p53 protein immunoreactivity were evaluated by immunohistochemical staining using MIB-1 and DO-1 monoclonal antibodies respectively. The apoptotic index (AI) was greater in GBM than in AA or DA, and more in tumours with p53 immunopositivity than in those without. The most striking observation was the strong correlation between Apoptotic index (AI) and proliferation index (PI) in p53 negative GBM (r=0.766, P < 0.005). However this was not observed in p53 +ve GBM or in low grade DA either p53 positive or negative. Taking p53 negativity in IHC as evidence of a functional gene/protein, this extends the link between proliferation and apoptosis, hitherto observed only in cultured cells with functional p53, to a subset of solid tumours.

Apoptosis↗

PubMed2005

Problems in using statistical analysis of replacement and silent mutations in antibody genes for determining antigen-driven affinity selection.

The analysis of molecular signatures of antigen-driven affinity selection of B cells is of immense use in studies on normal and abnormal B cell development. Most of the published literature compares the expected and observed frequencies of replacement (R) and silent (S) mutations in the complementarity-determining regions (CDRs) and the framework regions (FRs) of antibody genes to identify the signature of antigenic selection. The basic assumption of this statistical method is that antigenic selection creates a bias for R mutations in the CDRs and for S mutations in the FRs. However, it has been argued that the differences in intrinsic mutability among different regions of an antibody gene can generate a statistically significant bias even in the absence of any antigenic selection. We have modified the existing statistical method to include the effects of intrinsic mutability of different regions of an antibody gene. We used this method to analyse sequences of several B cell-derived monoclonals against T-dependent antigens, T-independent antigens, clones derived from lymphoma and amyloidogenic clones. Our sequence analysis indicates that even after correcting for the intrinsic mutability of antibody genes, statistical parameters fail to reflect the role of antigen-driven affinity selection in maturation of many clones. We suggest that, contrary to the basic assumption of such statistical methods, selection can act both for and against R mutations in the CDR as well as in the FR regions. In addition we have identified different methodological difficulties in the current uses of such statistical analysis of antibody genes.

Animals↗

PubMed2004

Supratentorial glioblastoma in adults: identification of subsets and their clinical correlation.

The concept of different genetic pathways leading to glioblastoma multiforme (GBM) has gained considerable acceptance, and two major groups are now described, primary or de novo GBM and secondary GBM. The present study was undertaken to elucidate whether additional pathways exist and to determine whether there is any correlation between these different variants and clinical parameters, such as age, duration of symptoms, and outcome. For this purpose, immunophenotyping was performed to study the simultaneous expression of p53 protein and epidermal growth factor receptor (EGFR) in 58 cases of adult supratentorial GBM. By this method, four variants of GBM could be distinguished: 34% were p53 positive only, 38% were EGFR positive only, 14% were double negative (p53 negative/EGFR negative), and 14% were double positive (p53 positive/EGFR positive). Interestingly, all nine cases of secondary GBM in which there was clinical and histological evidence of progression from a preexisting low-grade lesion were p53 positive. Differences were observed with regard to the age distribution of the four variants, in that the p53 negative/EGFR negative tumors occurred most frequently in the younger age group (21-40 years). In the elderly group (61-80 years), two-thirds of the tumors were p53 negative/EGFR positive primary GBMs, and no case of the double positive or double negative variant was encountered. The differences in duration of symptoms and symptom-free survival according to age group and genetic subset were not statistically significant. There were no differences in outcome within each age category for any GBM variant, although the longest mean symptom-free survival was noted among patients aged 41-60 years with the p53 positive/EGFR negative variant. This study therefore indicates that at least four subsets of GBM exist, but despite different genotypes, the biologic behavior remains similar. Other genetic alterations therefore need to be investigated to identify prognostic makers.

Adult↗

PubMed2004

Increased hMSH2 protein expression in glioblastoma multiforme.

hMSH2 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 hMLHI 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 x 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.

Adaptor Proteins, Signal Transducing↗

PubMed2004

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 microg/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 microg/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.

Antineoplastic Agents↗

PubMed2003

Loss of heterozygosity of a locus in the chromosomal region 17p13.3 is associated with increased cell proliferation in astrocytic tumors.

We had previously reported that loss of heterozygosity (LOH) of the D17S379 locus on 17p13.3 was significantly more frequent in high-grade gliomas (anaplastic astrocytoma, AA; glioblastoma multiforme, GBM) than in those of a low-grade diffuse astrocytoma (DA); however, this was independent of alterations at the TP53 locus, We also showed that LOH of D17S379 was associated with positive staining for p53 protein on immunohistochemistry, but LOH of the TP53 gene had no such association. In this work we show that cell proliferation as determined by MIB-1 labeling index (LI) was significantly higher in tumors with LOH of D17S379 than those with no LOH (NLOH). In accord with our previous results, p53 protein immunopositivity was also associated with increased MIB-1 LI; however, we observed no such association of LI with TP53 LOH. The results further confirm that alteration of one or more putative tumor suppressor loci at 17p13.3 is associated with increased proliferation in astrocytic tumors.

Adult↗