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Biomedical subjects

S V Sharma

Publications and source records attributed to S V Sharma.

At least 19 recordsLinked to original sources
PubMed2005

Molecular targeted therapy of lung cancer: EGFR mutations and response to EGFR inhibitors.

Somatic mutations within the kinase domain of the epidermal growth factor receptor (EGFR) are present in approximately 10% of non-small-cell lung cancer (NSCLC), with an increased frequency in adenocarcinomas arising in nonsmokers, women, and individuals of Asian ethnicity. These mutations lead to altered downstream signaling by the receptor and appear to define a subset of NSCLC characterized by "oncogene addiction" to the EGFR pathway, which displays dramatic responses to the reversible tyrosine kinase inhibitors gefitinib and erlotinib. The rapid acquisition of drug resistance in most cases, either through mutation of the "gateway" residue in the EGFR kinase domain or by alternative mechanisms, appears to limit the impact on patient survival. Irreversible inhibitors of EGFR display continued effectiveness in vitro against cells with acquired resistance and are now undergoing genotype-directed clinical trials. The molecular and clinical insights derived from targeting EGFR in NSCLC offer important lessons for the broader application of targeted therapeutic agents in solid tumors.

Antineoplastic Agents↗
PubMed2001

UCS15A, a non-kinase inhibitor of Src signal transduction.

Src tyrosine kinase plays key roles in signal transduction following growth factor stimulation and integrin-mediated cell-substrate adhesion. Since src-signal transduction defects are implicated in a multitude of human diseases, we have sought to develop new ways to identify small molecule inhibitors using a yeast-based, activated-src over-expression system. In the present study, we describe the identification of a unique src-signal transduction inhibitor, UCS15A. UCS15A was found to inhibit the src specific tyrosine phosphorylation of numerous proteins in v-src-transformed cells. Two of these phosphoproteins were identified as bona-fide src substrates, cortactin and Sam68. UCS15A differed from conventional src-inhibitors in that it did not inhibit the tyrosine kinase activity of src. In addition, UCS15A appeared to differ from src-destabilizing agents such as herbimycin and radicicol that destabilize src by interfering with Hsp90. Our studies suggest that UCS15A exerted its src-inhibitory effects by a novel mechanism that involved disruption of protein-protein interactions mediated by src. One of the biological consequences of src-inhibition by UCS15A was its ability to inhibit the bone resorption activity of osteoclasts in vitro. These data suggest that UCS15A may inhibit the bone resorption activity of osteoclasts, not by inhibiting src tyrosine kinase activity, but by disrupting the interaction of proteins associated with src, thereby modulating downstream events in the src signal transduction pathway.

3T3 Cells↗
PubMed2001

Stereospecific antitumor activity of radicicol oxime derivatives.

PURPOSE: Radicicol is a novel hsp90 antagonist, distinct from the chemically unrelated benzoquinone ansamycin compounds, geldanamycin and herbimycin. Both geldanamycin and radicicol bind in the aminoterminal nucleotide-binding pocket of hsp90, destabilizing the hsp90 client proteins, many of which are essential for tumor cell growth. We describe here antitumor activity of a novel oxime derivative of radicicol, KF58333. We also investigated the mechanism of antitumor activity of KF58333 in comparison with its oxime isomer KF58332. METHODS: Antiproliferative activities were determined in a panel of breast cancer cell lines in vitro. We also examined inhibition of hsp90 function and apoptosis induction in erbB2-overexpressing human breast carcinoma KPL-4 cells in vitro. Direct binding activity to hsp90 was assessed by hsp90-binding assays using geldanamycin or radicicol beads. In animal studies, we investigated plasma concentrations of these compounds after i.v. injection in BALB/c mice and antitumor activity against KPL-4 cells transplanted into nude mice. Inhibition of hsp90 function and induction of apoptosis in vivo were investigated using tumor specimens from drug-treated animals. RESULTS: KF58333 showed potent antiproliferative activity against all breast cancer cell lines tested in vitro, and was more potent than its stereoisomer KF58332. These results are consistent with the ability of KF58333 to deplete hsp90 client proteins and the induction of apoptosis in KPL-4 cells in vitro. Interestingly, KF58333, but not KF58332, showed significant in vivo antitumor activity accompanied by induction of apoptosis in KPL-4 human breast cancer xenografts. Although the plasma concentrations of these compounds were equivalent, KF58333, but not KF58332, depleted hsp90 client proteins such as erbB2, raf-1 and Akt in the tumor specimen recovered from nude mice. CONCLUSIONS: These results suggest that inhibition of hsp90 function, which causes depletion of hsp90 client proteins in tumor, contributes to the antitumor activity of KF58333, and that the stereochemistry of the oxime moiety is important for the biological activity of radicicol oxime derivatives.

Animals↗
PubMed1999

Interaction of radicicol with members of the heat shock protein 90 family of molecular chaperones.

The Hsp90 family of proteins in mammalian cells consists of Hsp90 alpha and beta, Grp94, and Trap-1 (Hsp75). Radicicol, an antifungal antibiotic that inhibits various signal transduction proteins such as v-src, ras, Raf-1, and mos, was found to bind to Hsp90, thus making it the prototype of a second class of Hsp90 inhibitors, distinct from the chemically unrelated benzoquinone ansamycins. We have used two novel methods to immobilize radicicol, allowing for detailed analyses of drug-protein interactions. Using these two approaches, we have studied binding of the drug to N-terminal Hsp90 point mutants expressed by in vitro translation. The results point to important drug contacts with amino acids inside the N-terminal ATP/ADP-binding pocket region and show subtle differences when compared with geldanamycin binding. Radicicol binds more strongly to Hsp90 than to Grp94, the Hsp90 homolog that resides in the endoplasmic reticulum. In contrast to Hsp90, binding of radicicol to Grp94 requires both the N-terminal ATP/ADP-binding domain as well as the adjacent negatively charged region. Radicicol also specifically binds to yeast Hsp90, Escherichia coli HtpG, and a newly described tumor necrosis factor receptor-interacting protein, Trap-1, with greater homology to bacterial HtpG than to Hsp90. Thus, the radicicol-binding site appears to be specific to and is conserved in all members of the Hsp90 family of molecular chaperones from bacteria to mammals, but is not present in other molecular chaperones with nucleotide-binding domains.

3T3 Cells↗
PubMed1998

Rapid recruitment of p120RasGAP and its associated protein, p190RhoGAP, to the cytoskeleton during integrin mediated cell-substrate interaction.

The interaction of cells with their substrate triggers cascades of signal transduction that result in profound changes in cell morphology. The nature of these signals and how they are integrated to orchestrate changes in cell shape are beginning to be elucidated. In particular, adhesive interactions between cells and their substrate, mediated by cell-surface integrins and extracellular matrix (ECM) proteins, appear to result in massive rearrangement of the cell cytoskeleton via the small G-protein, Rho. Here we show that in mouse fibroblasts, the interaction between cells and their substrate results in the rapid recruitment to the cytoskeleton of RasGAP (p120RasGAP), its associated protein of 190 kilodaltons, the GTPase activating protein for RhoA (p190RhoGAP) and the focal adhesion kinase (p125FAK). Similar results were obtained when cells were plated on ECM proteins, such as fibronectin, suggesting that the phenomenon is integrin mediated. These studies suggest that in fibroblasts, cell-substrate interaction triggered by integrin engagement result in the recruitment to the cytoskeleton of signaling molecules such as p120RasGAP, p190RhoGAP and p125FAK and may be involved in the formation of membrane cytoskeleton-associated signaling complexes that are important in cytoarchitectural reorganization.

3T3 Cells↗
PubMed1998

Radicicol leads to selective depletion of Raf kinase and disrupts K-Ras-activated aberrant signaling pathway.

Activation of Ras leads to the constitutive activation of a downstream phosphorylation cascade comprised of Raf-1, mitogen-activated protein kinase (MAPK) kinase, and MAPK. We have developed a yeast-based assay in which the Saccharomyces cerevisiae mating pheromone-induced MAPK pathway relied on co-expression of K-Ras and Raf-1. Radicicol, an antifungal antibiotic, was found to inhibit the K-ras signaling pathway reconstituted in yeast. In K-ras-transformed, rat epithelial, and K-ras-activated, human pancreatic carcinoma cell lines, radicicol inhibited K-Ras-induced hyperphosphorylation of Erk2. In addition, the level of Raf kinase was significantly decreased in radicicol-treated cells, whereas the levels of K-Ras and MAPK remained unchanged. These results suggest that radicicol disrupts the K-Ras-activated signaling pathway by selectively depleting Raf kinase and raises the possibility that pharmacological destabilization of Raf kinase could be a new and powerful approach for the treatment of K-ras-activated human cancers.

Animals↗
PubMed1998

Targeting of the protein chaperone, HSP90, by the transformation suppressing agent, radicicol.

Radicicol, a macrocyclic anti-fungal antibiotic, has the ability to suppress transformation by diverse oncogenes such as Src, Ras and Mos. Despite this useful property, the mechanism by which radicicol exerts its anti-transformation effects is currently unknown. To understand the transformation-suppressing effects of radicicol, a biotinylated derivative of radicicol was chemically synthesized and used as a probe in a Western-blot format to visualize cellular proteins that interact with radicicol. In transformed and untransformed mouse fibroblasts, the most prominent cellular protein that bound to radicicol had a molecular weight of approximately 90 kDa. Further analysis revealed that this protein was the mouse homologue of the 90 kDa heat shock protein (HSP90). This was confirmed by demonstrating the ability of radicicol to specifically bind purified human HSP90. Specificity of binding was demonstrated by the inhibition of binding of biotinylated radicicol by the native drug. Taken together with other studies the present observations suggest that the anti-transformation effects of radicicol may be mediated, at least in part, by the association of radicicol with HSP90 and the consequent dissociation of the Raf/HSP90 complex leading to the attenuation of the Ras/MAP kinase signal transduction pathway.

3T3 Cells↗
PubMed1997

Specific changes in rasGAP-associated 62 kilodalton protein during integrin mediated cell-substrate interaction.

A cascade of signal transduction events is initiated when cells make contact with each other or with a substrate. The nature of these signal transduction pathways is beginning to be elucidated. In particular, adhesive interactions between cells and their substrate, mediated by cell-surface integrins and extracellular matrix proteins, appears to activate the MAP kinase pathway. Here we show that in mouse fibroblasts and rat epithelial cells, tyrosine phosphorylation of a 62 kilodalton rasGAP-associated protein (GAPa-p62) is decreased upon cell-substrate interaction. Interaction between fibroblasts and various extracellular matrices such as fibronectin, vitronectin and collagen IV, but not laminin, results in tyrosine dephosphorylation of GAPa-p62. Cell-substrate mediated tyrosine dephosphorylation of GAPa-p62 is defective in transformed cell lines, suggesting a possible role for p62 in tumorigenic transformation. These studies suggest that in fibroblasts, and perhaps even in epithelial cells, the signal transduction pathway(s) triggered by different integrin engagement events converge on the rasGAP protein and alter the tyrosine phosphorylation and/or association of GAPa-p62.

3T3 Cells↗
PubMed1996

HLA class-I and class-II antigen association in rheumatoid arthritis at Varanasi, India.

Clinical presentation of rheumatoid arthritis (RA) and its severity differs in different races. Genetic factors play a significant role in its predeliction. The present study was undertaken to find out association of HLA class I and class II antigens with rheumatoid arthritis prevalent in Asian Indians residing at Varanasi. Ninety rheumatoid arthritis patients strictly fulfilling American Rheumatism Association criteria were screened for prevalent HLA class I and class II antigen by Terasaki Microlympho-cytotoxicity test. Results were compared with 100 healthy controls and 35 Seronegative Spondyloarthritides cases (SSA). Rheumatoid arthritis patients showed increased frequency of HLA-A2 and B40 antigens compared to healthy controls (p < .001). SSA patients showed significantly increased Phenotype frequency (PF) of HLA-B27 (p < .0001) and B40 (p < .001). Significant detection of HLA-A2 exclusively in RA patients suggests a more positive association of A2 in rheumatoid arthritis at Varanasi. HLA-B40 could not be attributed absolute significance of association with SSA or RA as it showed increased frequency in both diseases.

Adult↗
PubMed1996

Radicicol inhibits tyrosine phosphorylation of the mitotic Src substrate Sam68 and retards subsequent exit from mitosis of Src-transformed cells.

Sam68 (Src-associated in mitosis 68 kDa) is a protein that associates with and is tyrosine phosphorylated by Src in a mitosis-specific manner, thereby raising the possibility of a role for Src in the regulation of the cell cycle. This study examines the effects of radicicol, a Src tyrosine kinase inhibitor, upon both the phosphorylation of Sam68 and mitotic progression in Src-transformed mouse fibroblasts. Radicicol reversibly inhibits the mitosis-specific tyrosine phosphorylation of Sam68 in vivo, as determined by antiphosphotyrosine immunoblotting. Radicicol inhibits the tyrosine phosphorylation of both free and Src-associated Sam68, suggesting the presence of two intracellular pools of tyrosine phosphorylated Sam68 in mitotic cells. In addition, radicicol treatment has no effect on the ability of cells to enter mitosis, indicating that tyrosine phosphorylation of Sam68 is probably not important for cells to enter mitosis. However, radicicol reversibly retards the exit of cells from mitosis, as determined by flow cytometric analyses. Radicicol mediated inhibition of Sam68 tyrosine phosphorylation, and its concurrent ability to block mitotic exit suggests the possibility of a significant role for Src kinase and this unique mitotic substrate, Sam68, in cell cycle regulation.

3T3 Cells↗
PubMed1995

In vivo and in vitro serine/threonine phosphorylations of epidermal growth factor receptor upon entry into the cell cycle.

Protein phosphorylation and dephosphorylation is one of the main mechanisms of cell cycle regulation. This study examines the modulation of epidermal growth factor receptor phosphorylation as cells emerge from quiescence and enter the S phase of the cell cycle. The epidermal growth factor receptor is phosphorylated primarily on serine and threonine, but not on tyrosine residues, in an S phase-dependent fashion, as determined by phosphoamino acid analysis and anti-phosphotyrosine immunoblotting. These phosphorylations occur both in vitro and in vivo and are ligand independent. Some of the sites that are phosphorylated in vitro also appear to be phosphorylated in vivo, as determined by two-dimensional tryptic phosphopeptide analysis. At least one of the in vivo phosphorylation sites is phosphorylated by mitogen-activated protein kinase. Although the mechanism for this ligand-independent phosphorylation is not known, its correlation with emergence from quiescence and entry into the cell cycle suggests that the phosphorylation of epidermal growth factor receptor on serine and threonine residues may have heretofore unknown role(s) in cell cycle entry and progression.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1993

Changing profile of haematogenous osteomyelitis in a teaching hospital.

A total of 155 consecutive patients of osteomyelitis were studied clinically, radiologically and bacteriologically. The follow up ranged from 2 1/2 to 4 1/2 yr (average 3.5 yr). Age varied from 8 months to 50 yr. Onset of the disease was acute in 106 and insidious in 49 patients. Of these 116 cases were treated conservatively which include 12 treated by incision and drainage. The rest underwent surgery (saucerization, curettage, sequestrectomy etc.). There was a trend for osteomyelitis to shift from the known incidence in early age to adulthood, acute to insidious onset and infection by Gram positive to Gram negative organisms.

Adolescent↗
PubMed1993

Melittin-induced hyperactivation of phospholipase A2 activity and calcium influx in ras-transformed cells.

The activated ras oncogene is a key mediator of cellular transformation and is present in a wide variety of primary human neoplasms. The biochemical role of the ras oncogene in cellular transformation is at present unclear, and hence approaches to control its activities in transformed cells have met with limited success. Previous studies have demonstrated the ability of melittin, a 26 amino acid amphipathic peptide from bee venom, to specifically counterselect for cells in culture that express high levels of the ras oncogene product. The biochemical basis for this counterselection is currently unknown. This study demonstrates the ability of melittin to hyperactivate phospholipase A2 (PLA2) in ras-transformed cells by the mediation of enhanced influx of calcium ions (Ca2+). This hyperactivation of PLA2 and Ca2+ mobilization in ras-transformed cells by melittin is mimicked by the calcium ionophore, A23187. Both melittin- and A23187-mediated PLA2 hyperactivation require Ca2+. However, the action of melittin is strongly dependent on extracellular Ca2+, whereas that of A23187 is not. Melittin-induced Ca2+ influx and PLA2 hyperactivation is inhibited by manganese ions (Mn2+). These studies reveal a close correlation between the extent of PLA2 hyperactivation and Ca2+ mobilization, suggesting a causal relationship.

3T3 Cells↗
PubMed1992

Allogenic decalbone in the repair of benign cystic lesions of bone.

Forty-six cases of benign cystic lesions of bone were treated by curettage and compact filling using partially decalcified allogenic bone graft (Decalbone); of these, 35 were available for study. Decalbone was prepared by partial decalcification with 0.6 N hydrochloric acid (HCl) of human bones generally obtained from freshly amputated limbs. The commonest lesions of bone were giant cell tumours (14) and aneurysmal bone cysts (15), and the commonest bones involved were the femur (23) and the tibia (12). There was one failure and four recurrences. Five cases were infected but this did not interfere with healing of the primary lesion. Radiological incorporation of the graft was seen at about 3 months in unicameral bone cysts, at 4-6 months in aneurysmal bone cysts and at 6-9 months in giant cell tumours. There was no recurrence in any case of giant cell tumour, but three aneurysmal bone cysts recurred. There was no clinical immune reaction.

Adult↗
PubMed1992

Reconstruction for xanthoma of the Achilles tendon.

Two patients with bilateral xanthoma of the Achilles tendon were treated by wide resection of the tumour. Reconstruction was carried out by a distally based flap fashioned from the remaining proximal tendon and gastrocnemius aponeurosis.

Achilles Tendon↗
PubMed1992

Macrodystrophia lipomatosa: radiographic observations.

23 cases of macrodystrophia lipomatosa (MDL) are reported showing a wide spectrum of radiographic findings. Typical findings were hypertrophy of all the mesodermal tissues of the affected digits with dramatic overgrowth of fat. Phalanges were enlarged both in length and transverse diameter, but the trabecular pattern was maintained. In one patient, the phalanges and metatarsals were elongated but thinned. In another case, all the phalanges and metatarsals of the great toe were small. The little toe was also involved in two cases. Articular surfaces were slanting. There was a high incidence of palmar and plantar involvement. In a few cases the forearm and leg were also involved. Other uncommon features observed were early maturation of epiphyseal centres of ossification of phalanges and metatarsals, syndactyly, polydactyly, brachydactyly and symphalangism. Angiography was uncharacteristic.

Adolescent↗