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Biomedical subjects

N Dhanasekaran

Publications and source records attributed to N Dhanasekaran.

At least 19 recordsLinked to original sources
PubMed2006

Neoplastic transformation by the gep oncogene, Galpha12, involves signaling by STAT3.

Galpha(12), the alpha-subunit of G12, which has been referred to as the gep oncogene, stimulates mitogenic pathways in different cell types and readily induces neoplastic transformation of fibroblast cell lines. Recently, we have shown that the oncogenic pathway activated by Galpha(12) involves the receptor tyrosine kinase platelet derived growth factor receptor-alpha (PDGFRalpha) and JAK3. In the present study, we demonstrate that the GTPase-deficient activated mutant of Galpha(12) activates signal transducer and activator of transcription 3 (STAT3) via PDGFRalpha as well as JAK3. Here we show that Galpha(12) stimulates the phosphorylation of STAT3 at both Tyrosine-705 and Serine-727 residues. Studies to delineate the mechanism by which Galpha(12) stimulates STAT3 have indicated that the Tyrosine-705-phosphorylation of STAT3 involves the tyrosine kinases, Janus Kinase-3 as well as Src kinase, whereas the Serine-727 phosphorylation of STAT3 occurs via the receptor tyrosine kinase, PDGFRalpha and phosphatidylinositol 3-OH kinase pathway. Our results also indicate that the coexpression of the dominant negative, DNA binding mutant of STAT3 (STAT3DB) inhibits the foci formation as well as anchorage-independent growth of Galpha(12)QL-transfectants, thereby establishing the critical role of STAT3 in Galpha(12)QL-mediated neoplastic cell growth. The results presented here demonstrate, for the first time, the ability of Galpha(12) to recruit multiple receptor-, nonreceptor-, and Ser/Thr kinases to stimulate STAT3-signaling to promote neoplastic transformation.

3T3 Cells↗
PubMed2005

Mitogenic signaling by lysophosphatidic acid (LPA) involves Galpha12.

Lysophosphatidic acid (LPA), a major G protein coupled receptor (GPCR)-activating ligand present in serum, elicits growth factor like responses by stimulating specific GPCRs coupled to heterotrimeric G proteins such as G(i), G(q), and G12/13. Previous studies have shown that the overexpression of wild-type Galpha12 (Galpha12WT) results in the oncogenic transformation of NIH3T3 cells (Galpha12WT-NIH3T3) in a serum-dependent manner. Based on the potent growth-stimulating activity of LPA and the presence of LPA and LPA-like molecules in the serum, we hypothesized that the serum-dependent neoplastic transformation of Galpha12WT-NIH3T3 cells was mediated by the stimulation of LPA-receptors (LPARs) by LPA in the serum. In the present study, using guanine nucleotide exchange assay and GST-TPR binding assay, we show that the treatment of Galpha12WT-NIH3T3 with 2 muM LPA leads to the activation of Galpha12. Stimulation of these cells with LPA promotes JNK-activation, a critical component of Galpha12-response and cell proliferation. We also show that LPA can substitute for serum in stimulating JNK-activity, DNA synthesis, and proliferation of Galpha12WT-NIH3T3 cells. LPA-mediated proliferative response in NIH3T3 cells involves Galpha12, but not the closely related Galpha13. Pretreatment of Galpha12WT-NIH3T3 cells with suramin (100 microM), a receptor-uncoupling agent, inhibited LPA-stimulated proliferation of these cells by 55% demonstrating the signal coupling between cell surface LPAR and Galpha12 in the neoplastic proliferation of NIH3T3 cells. As LPA and LPAR mediated mitogenic pathways have been shown to play a major role in tumor genesis and progression, a mechanistic understanding of the signal coupling between LPAR, Galpha12, and the downstream effectors is likely to unravel additional targets for novel cancer chemotherapies.

3T3 Cells↗
PubMed2005

Engineered Saccharomyces cerevisiae strain BioS-1, for the detection of water-borne toxic metal contaminants.

Saccharomyces cerevisiae responds to extracellular toxic stimuli by increasing intracellular cyclic AMP levels, leading to activation of a cAMP-dependent protein kinase, protein kinase A (PKA). Activated PKA phosphorylates downstream substrates, including specific DNA-binding proteins, to turn off the expression of most or all of the yeast genes. Such cAMP-PKA-mediated inhibition of gene expression in response to toxic stimuli appears to be unique to S. cerevisiae. For instance, in mammalian cells, the cAMP-PKA signaling pathway is rather responsive to growth factors and hormones in addition to being primarily involved in the activation of gene expression. Activation of gene expression by the cAMP-PKA pathway in mammalian cells is due mainly to the presence of cAMP-response elements (CREs) located in the promoters of many mammalian genes, and the expression of PKA-responsive stimulatory transcription factor CRE-binding protein, commonly referred as CREBP, which binds to the CREs. Thus, activation of the cAMP-PKA signaling pathway results in the phosphorylation of CREBP by PKA, and phosphorylated CREBP transactivates specific gene expression by interacting with the cognate CRE. Based on these findings, we sought to engineer a yeast-based biosensor, in which the stress-sensing cAMP-PKA pathway of yeast is coupled to the mammalian CREBP-CRE-stimulated gene expression pathway, which drives the expression of a reporter protein, such as green fluorescent protein (GFP). As a primary step toward the development of this biosensor, we engineered a yeast strain, BioS-1, by genetically altering YPH 501, a wild-type strain of S. cerevisiae, to express human CREBP and human CRE promoter-driven GFP. Exposure of BioS-1 to varying concentrations of As3+, Fe2+, Pb2+, and Cd2+ elicits concentration-dependent expression of the GFP reporter that can be easily monitored by the fluorescence emitted by GFP. The results also indicate that the engineered BioS-1 yeast cells can detect 2.5 ppm of these toxic metals and report it through the expression of GFP within 3 h. The results presented herein demonstrate that this engineered yeast strain can detect metal toxicants and can validate the use of this prototypic yeast strain to develop a biosensor that can be used to detect and monitor cytotoxic water-borne toxic heavy metals.

Biological Assay↗
PubMed2004

Galpha13 stimulates cell migration through cortactin-interacting protein Hax-1.

Galpha13, the alpha-subunit of the heterotrimeric G protein G13, has been shown to stimulate cell migration in addition to inducing oncogenic transformation. Cta, a Drosophila ortholog of G13, has been shown to be critical for cell migration leading to the ventral furrow formation in Drosophila embryos. Loss of Galpha13 has been shown to disrupt cell migration associated with angiogenesis in developing mouse embryos. Whereas these observations point to the vital role of G13-orthologs in regulating cell migration, widely across the species barrier, the mechanism by which Galpha13 couples to cytoskeleton and cell migration is largely unknown. Here we show that Galpha13 physically interacts with Hax-1, a cytoskeleton-associated, cortactin-interacting intracellular protein, and this interaction is required for Galpha13-stimulated cell migration. Hax-1 interaction is specific to Galpha13, and this interaction is more pronounced with the mutationally or functionally activated form of Galpha13 as compared with the wild-type Galpha13. Expression of Hax-1 reduces the formation of actin stress fibers and focal adhesion complexes in Galpha13-expressing NIH3T3 cells. Coexpression of Hax-1 also attenuates Galpha(13)-stimulated activity of Rho while potentiating Galpha13-stimulated activity of Rac. The presence of a quadnary complex consisting of Galpha13, Hax-1, Rac, and cortactin indicates the role of Hax-1 in tethering Galpha13 to the cytoskeletal component(s) involved in cell movement. Whereas the expression of Hax-1 potentiates Galpha13-mediated cell movement, silencing of endogenous Hax-1 with Hax-1-specific small interfering RNAs drastically reduces Galpha13-mediated cell migration. These findings, along with the observation that Hax-1 is overexpressed in metastatic tumors and tumor cell lines, suggest a novel role for the association of oncogenic Galpha13 and Hax-1 in tumor metastasis.

Actins↗
PubMed2004

Differential regulation of Jun N-terminal kinase and p38MAP kinase by Galpha12.

Based on the findings that the overexpression of the wild-type Galpha(12) (Galpha(12)WT) result in the oncogenic transformation of NIH3T3 cells in a serum-dependent manner, a model system has been established in which the mitogenic and subsequent cell transformation pathways activated by Galpha(12) can be turned on or off by the addition or removal of serum. Using this model system, our previous studies have shown that the stimulation of Galpha(12)WT or the expression of an activated mutant of Galpha(12) (Galpha(12)QL) leads to increased cell proliferation and subsequent oncogenic transformation of NIH3T3 cells, as well as persistent activation of Jun N-terminal kinases (JNKs). In the present studies, we show that the stimulation of Galpha(12)WT or the expression of Galpha(12)QL results in a potent inhibition of p38MAPK, and that the mechanism by which Galpha(12) inhibits p38MAPK activity involves the dual specificity kinases upstream of p38MAPK. The results indicate that Galpha(12) attenuates the activation of MKK3 and MKK4, which are known to stimulate only p38MAPK or p38MAPK and JNK, respectively. The results also suggest that Galpha(12) activates JNKs specifically through the stimulation of the JNK-specific upstream kinase MKK7. These findings demonstrate for the first time that Galpha(12) differentially regulates JNK and p38MAPK by specifically activating MKK7, while inhibiting MKK3 and MKK4 in NIH3T3 cells. Since the stimulation of p38MAPK is often associated with apoptotic responses, our findings suggest that Galpha(12) stimulates cell proliferation and neoplastic transformation of NIH3T3 cells by attenuating p38MAPK-associated apoptotic responses, while activating the mitogenic responses through the stimulation of ERK- and JNK-mediated signaling pathways.

Animals↗
PubMed2004

The microrevolution: applications and impacts of microarray technology on molecular biology and medicine (review).

Microarray is a technique that provides a global analysis of gene expression at the level of transcription. Genetic and epigenetic changes underlie neoplastic transformation, cardiovascular disease, some psychiatric illnesses, and a growing list of disease pathogeneses and therapeutic responses. The profile of genes expressed by different cells (gene up- and downregulation under different conditions) determines their phenotype, and thus provides insights into the molecular basis for health and disease. Microarray technology combines standard molecular techniques with high-throughput screening to monitor the expression of up to 38,500 genes. A single experiment can assay gene expression across the entire genome under experimental or clinical conditions. Microarray therefore extends wide bridges between basic science and clinical medicine. This review describes the principles behind transcriptional profiling, experimental designs, and data analyses in the context of basic and clinical sciences. In addition, we survey the Microrevolutionized field of clinical cancer research by reviewing the most recent and innovative microarray studies of breast carcinoma.

Breast Neoplasms↗
PubMed2003

Apoptosis of granulosa cells: a review on the role of MAPK-signalling modules.

Recent studies suggest that ovarian follicular atresia is associated with DNA fragmentation and degeneration of granulosa cells, the hallmark of programmed cell death or apoptosis. Apoptosis of granulosa cells play a major role in follicular atresia. These studies have also demonstrated the involvement of tumour suppressors, apoptotic proteins and survival factors. These factors contribute to the developmental decision as to whether the ovarian follicles mature or undergo atresia. However, the precise temporal and molecular events involved in the apoptotic pathways in this process need to be elucidated. The present report summarizes the role of Jun N-terminal kinase (JNK), p38 mitogen activated protein kinase (p38 MAPK), and extracellular-signal regulated kinase (ERK)-signalling module in the regulation of pro- and anti-apoptotic factors of the granulosa cells in regulating follicular atresia. The findings presented here suggest that the loss of tropic hormone support is translated into the attenuation of Raf-1-MAPK/ERK kinase (MEK)-ERK-signalling pathway of the granulosa cells and this results in the decreased phosphorylation of the pro-apoptotic BAD.

Animals↗
PubMed2002

JLP: A scaffolding protein that tethers JNK/p38MAPK signaling modules and transcription factors.

Extracellular signals are transduced into cells through mitogen-activated protein kinases (MAPKs), which are activated by their upstream kinases. Recently, families of scaffolding proteins have been identified to tether specific combinations of these kinases along specific signaling pathways. Here we describe a protein, JLP (c-Jun NH2-terminal kinase-associated leucine zipper protein), which acts as a scaffolding protein to bring together Max and c-Myc along with JNK (c-Jun NH2-terminal kinase) and p38MAPK, as well as their upstream kinases MKK4 (MAPK kinase 4) and MEKK3 (MAPK kinase kinase 3). Thus, JLP defines a family of scaffolding proteins that bring MAPKs and their target transcription factors together for the execution of specific signaling pathways.

3T3 Cells↗
PubMed2002

Determining cellular role of G alpha 12.

Using the expression strategies described here, we have demonstrated a model system whereby the sequential signaling events involved in cell proliferation and subsequent transformation regulated by G alpha 12 can be investigated. The model system presented here can also be used to study the temporal interrelationships between small GTPases, kinases, and other signaling proteins involved in G alpha 12-signaling pathways. Further analyses using this model system and the strategies presented here should provide valuable clues in defining the signaling network regulated by G alpha 12 in stimulating cell proliferation and oncogenic transformation.

3T3 Cells↗
PubMed2001

Transforming G proteins.

Heterotrimeric guanine nucleotide binding proteins, commonly known as G proteins form a super-family of signal transduction proteins. They are peripherally associated with the plasma membrane and provide signal coupling to seven transmembrane surface receptors. G proteins are composed of monomers of alpha, beta, and gamma subunits. The beta- and gamma-subunits are tightly associated. The receptors activated by the appropriate "signal", interact catalytically with specific G-proteins to mediate guanine nucleotide exchange at the GDP/GTP binding site of the G-protein alpha-subunits, thus displacing the bound GDP for GTP. The GTP bound form of the g-protein alpha-subunit and in some cases the free betagamma-subunits initiate cellular response by altering the activity of specific effector molecules. Recent studies have indicated that the asyncronous activation of these proteins can lead to the oncogenic transformation of different cell types. The mechanism by which G-proteins regulate the various cell functions appear to involve a complex net-working between different signaling pathways. This review summarizes the signaling mechanisms involved in the regulation of cell proliferation by these transforming G proteins.

Cell Transformation, Neoplastic↗
PubMed2001

Activated mutant of Galpha(12) enhances the hyperosmotic stress response of NIH3T3 cells.

Heterotrimeric G protein G12 stimulates diverse physiological responses including the activities of Na+/H+ exchangers and Jun kinases. We have observed that the expression of the constitutively activated, GTPase-deficient mutant of Galpha(12) (Galpha(12)QL) accelerates the hyperosmotic response of NIH3T3 cells as monitored by the hyperosmotic stress-stimulated activity of JNK1. The accelerated response appears to be partly due to the increased basal activity of JNK since cell lines-such as NIH3T3 cells expressing JNK1-in which JNK activity is elevated, show a similar response. NIH3T3 cells expressing Galpha(12)QL also display heightened sensitivity to hyperosmotic stress. This is in contrast to JNK1-NIH3T3 cells that failed to enhance sensitivity although they do exhibit an accelerated hyperosmotic response. Reasoning that the increased sensitivity seen in Galpha(12)QL cells is due to a signaling component other than JNK, the effect of dimethyamiloride, an inhibitor of Na+/H+ exchanger in this response, was assessed. Treatment of vector control NIH3T3 cells with 50 microM dimethylamiloride potently inhibited their hyperosmotic response whereas the response was only partially inhibited in Galpha(12)QL-NIH3T3 cells. These results, for the first time, identify that NHEs are upstream of the JNK module in the hyperosmotic stress-signaling pathway and that Galpha(12) can enhance this response by modulating either or both of these components namely, JNKs and NHEs in NIH3T3 cells.

3T3 Cells↗
PubMed2000

Single amino acid of g(alpha16) (Ala(228)) is responsible for the ability of chemoattractant C5a receptor to induce G(alpha16)-mediated inositol phosphate release.

Our previous study suggested that the region encompassing residues 220-240 on G(alpha16) is important in coupling with C5a receptor (Lee et al. (1995) Mol. Pharmacol. 47, 218-223). When aligned sequences are compared in the residue 220-240 segment of G(alpha16), there is a block of eight amino acids extending from residue 227 to residue 234 (227-Ile-Ala-Leu-Ile-Tyr-Leu-Ala-Ser-234) in G(alpha16) that is replaced by a heterologous block extending from amino acid residue 224 to residue 231 (224-Thr-Ser-Ile-Met-Phe-Leu-Val-Ala-231) in G(alpha11). In order to identify the specific amino acid residue necessary for coupling to C5a receptor within the extension of eight amino acids in G(alpha16), a series of chimeric G(alpha11)/G(alpha16) cDNA constructs and mutant G(alpha16) cDNAs were expressed. Then the ability of chimeras and mutant proteins to mediate C5a-induced release of inositol phosphate in transfected Cos-7 cells was tested. The results show that single amino acid Ala(228) is responsible for conferring about 40-50% of the activity of G(alpha16) induced by C5a receptor stimulation.

Alanine↗
PubMed2000

Cisplatin-resistance involves the defective processing of MEKK1 in human ovarian adenocarcinoma 2008/C13 cells.

Cisplatin has been widely used as a chemotherapeutic agent to treat different types of tumors. However, its use is limited by the ability of the tumor cells to develop cisplatin-resistance. The molecular lesion that produces cisplatin-resistance is poorly understood. In this report, we show that cisplatin activates a robust apoptotic pathway involving the activation of JNK and p38MAPK whereas it fails to elicit such a response in cisplatin-resistant 2008/C13 cells. Analysis of the defective apoptotic pathway in 2008/C13 cells indicates that these cells are deficient in the proteolytic activation of MEKK1 by caspase-3. The blunted activity of caspase-3 appears to be closely related to the increased levels of the anti-apoptotic protein Bcl-xL seen in the resistant cells. These studies, for the first time, demonstrate that inadequate caspase-3 processing and MEKK1 activation can lead to a drug-resistant phenotype.

Adenocarcinoma↗
PubMed1999

Apoptosis of ovarian granulosa cells: correlation with the reduced activity of ERK-signaling module.

Apoptosis of the ovarian granulosa cells plays a crucial role in the determination of the number of follicles destined to ovulate in each reproductive cycle. While the activation of specific apoptotic pathway or the inactivation of cell survival pathway can initiate apoptosis, the signaling mechanism(s) involved in initiating the onset of apoptosis in granulosa cells is not fully understood. In the present study, using granulosa cells derived from eCG-primed immature rats, we investigated the temporal signaling events involved in the onset of apoptosis in the granulosa cells. The administration of 15 IU of eCG to 21-day-old immature female rats stimulate the growth and development of ovarian follicles until 72 h, after which the granulosa cells of the ovarian follicles undergo apoptosis due to the waning levels of tropic hormonal support. An analysis of the signaling events leading to apoptosis indicates that the DNA fragmentation can be seen in these cells from 96 h. A small increase in the levels of the pro-apoptotic factor Bax can be seen from 96 h while an increase in the activity of JNK can be seen from 108 h onwards. By contrast, a reduction in ERK signaling can be seen by 48 h. Similar reduction in Raf-1 kinase activity can be discerned from 48 h onwards. A concomitant decrease in the phosphorylated form of Bad can also be detected. These findings taken together, suggest that the loss of tropic hormone support is translated into the attenuation of Raf-1-MEK-ERK signaling pathway and this reduction along with a reduction in the levels of phosphorylated form of Bad triggers the onset of apoptosis in the ovarian granulosa cells.

Animals↗
PubMed1999

Oncogenic mutant of Galpha12 stimulates cell proliferation through cycloxygenase-2 signaling pathway.

Expression of the GTPase-deficient, activated mutant alpha-subunit of the heterotrimeric G protein G12 (Galpha12QL) leads to the neoplastic transformation of fibroblast cell lines. The mitogenic pathway regulated by Galpha12QL includes an extensive signaling network involving several small GTPases and various kinases. In addition, Galpha12QL has been shown to potentiate the serum-induced phospholipase-A2 activity in NIH3T3 cells. In the present study, we demonstrate that cycloxygenase-2 (COX-2) pathway is involved in the mitogenic pathway activated by Galpha12QL. Expression of Galpha12QL and not Galpha13QL, stimulates the serum-induced release of arachidonic acid in NIH3T3 cells. Furthermore, expression of Galpha12QL or the stimulation of wild-type Galpha12 induces the expression of COX-2. Our results also indicate that the COX-2 inhibitor acutely disrupts the DNA-synthesis stimulated by Galpha12QL in NIH3T3 cells. These studies, for the first time, identify the crucial role of COX-2 in Galpha12-mediated regulation of cell proliferation and suggest a role for prostaglandin-derived autocrine loop in Galpha12-mediated signaling pathways.

3T3 Cells↗
PubMed1999

GTPase deficient mutant of G(alpha13) regulates the expression of Egr-1 through the small GTPase Rho.

The alpha-subunit of the heterotrimeric G protein G13 regulate cell growth, differentiation and apoptosis in different cell types. Expression of the constitutively activated mutant of G(alpha)13 (G(alpha13)QL) increases the expression of Egr-1, an immediate-early response gene that is identified to be involved in cell growth, differentiation, and apoptosis. Here we report that G(alpha13)QL activates the promoter of Egr-1 through specific sequence which includes the characteristic CArG boxes. We also demonstrate that the G(alpha13)QL activation of Egr-1 promoter is mediated by the Ras-like small GTPase Rho.

3T3 Cells↗
PubMed1999

Signaling pathways and diabetic embryopathy.

Diabetic embryopathy is the leading cause of neonatal death and/or congenital malformations in infants of diabetic mothers. Because the development of the embryo critically depends on the maternal and the embryonic signaling pathways, a defective signaling mechanism between the maternal and the embryonic tissues appears to be involved in the etiology of diabetic embryopathy. Analyses of the recent studies from different laboratories suggest a "multifactorial" basis for diabetic embryopathy. These studies suggest that a wide variety of signal-transducers converge towards the regulation of elcosanoid signaling pathway which appears to be the critical pathway involved in diabetic embryopathy. The characterization of the regulatory components of this pathway is likely to identify the signaling loci susceptible for the therapeutic intervention.

Arachidonic Acid↗