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Biomedical subjects

N Dhanasekaran

Publications and source records attributed to N Dhanasekaran.

At least 19 recordsLinked to original sources
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↗
PubMed1998

Regulation of cell proliferation by G proteins.

G Proteins provide signal transduction mechanisms to seven transmembrane receptors. Recent studies have indicated that the alpha-subunits as well as the betagamma-subunits of these proteins regulate several critical signaling pathways involved in cell proliferation, differentiation and apoptosis. Of the 17 alpha-subunits that have been cloned, at least ten of them have been shown to couple mitogenic signaling in fibroblast cells. Activating mutations in G alpha(s), G alpha(i)2, and G alpha12 have been correlated with different types of tumors. In addition, the ability of the betagamma-subunits to activate mitogenic pathways in different cell-types has been defined. The present review briefly summarizes the diverse and novel signaling pathways regulated by the alpha- as well as the betagamma-subunits of G proteins in regulating cell proliferation.

Animals↗
PubMed1998

Signaling by dual specificity kinases.

Dual specificity kinases that phosphorylate the Thr- and Tyr-residues within the TXY motif of MAP-kinases of play a central role in the regulation of various processes of cell growth. These dual specificity kinases also known as MAP kinase kinases are constituents of the sequential kinase signaling modules. Seven distinct mammalian MAP kinases kinases have been identified. Some of the unique signaling properties of these kinases are discussed here.

Animals↗
PubMed1998

G alpha q cDNA sequence from human platelets.

G-proteins play a major role in cell signaling and specific abnormalities in these proteins have been implicated in some diseases. Although, two human G alpha q coding sequences are published, they differ at functionally important regions. We have sequenced the human G alpha q cDNA synthesized from normal platelet mRNA by reverse transcription. Our results showed that the penultimate amino acid leucine is conserved in the human G alpha q sequence which has 94.4% identity in nucleotide sequence and 99.2% homology in amino acid sequence with murine sequence.

Amino Acid Sequence↗
PubMed1997

Ras-dependent signaling by the GTPase-deficient mutant of Galpha12.

Galpha12 and Galpha13 regulate diverse responses through the small GTPases Ras, CDC42, Rac, and Rho. Whereas they activate similar responses in many different cell types, they also activate more specific and critical signaling pathways in other cell types. In COS cells, in which both Galpha12 and Galpha13 stimulate Na+/H+ exchange, they do so by activating different signaling pathways. Here we report that the differential recruitment of specific small GTPases by Galpha12 and Galpha13 defines the molecular basis for their functional differences. We have observed that the stimulation of Na+/H+ exchange by the GTPase-deficient mutant of Galpha12 (Galpha12QL) requires a functional Ras and is independent of Rac/CDC42 and Jun kinase signaling module. By contrast, the stimulation of Na+/H+ exchange by Galpha13QL requires a functional Rac/CDC42 and the Jun kinase signaling module. Our results also indicate that Galpha12QL-Ras stimulation of Na+/H+ exchange involves a D609-sensitive phospholipase and protein kinase C. These studies, for the first time, describe a novel Galpha12-specific signaling pathway involving Ras, phosphatidylcholine hydrolysis, and protein kinase C in the regulation of Na+/H+ exchange.

Animals↗
PubMed1997

Platelet signal transduction defect with Galpha subunit dysfunction and diminished Galphaq in a patient with abnormal platelet responses.

G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca2+ mobilization. Present studies demonstrated that platelet phospholipase A2 (cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca2+ release induced by guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS]) was diminished in the patient's platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of alpha-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A2 analog U46619. Binding of 35S-labeled GTP[gammaS] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I2 analog) -induced rise in cAMP (mediated by Galphas) and its inhibition (mediated by Galphai) by thrombin in the patient's platelet membranes were normal. Immunoblot analysis of Galpha subunits in the patient's platelet membranes showed a decrease in Galphaq (<50%) but not Galphai, Galphaz, Galpha12, and Galpha13. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein alpha-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of Galphaq in thrombin-induced responses.

Blood Platelets↗
PubMed1997

IL-1 beta alters beta-adrenergic receptor adenylyl cyclase system function in human airway epithelial cells.

Inflammatory cells release a variety of cytokines, including interleukin (IL)-1 beta, into the airway in asthma. This study examined the effects of human IL-1 beta on the function of the beta-adrenergic receptor (beta AR)-adenylyl cyclase (AC) system in BEAS-2B cells, a human airway epithelial cell line. IL-1 beta markedly increased beta AR density (Bmax; P < 0.001) primarily by increasing the percentage of the beta 2AR subtype (from 67 to 91%; P < 0.001). Bmax increased monotonically over time in response to 200 pM IL-1 beta and was approximately 2.5-fold greater than control cells between 36 and 42 h. In contrast, the concentration response of Bmax to IL-1 beta given for 18 h was biphasic. Bmax increased with IL-1 beta concentrations from 2 to 200 pM, but, at > 200 pM, it decreased progressively toward control values. IL-1 beta-induced increases in Bmax with IL-1 beta were associated with approximately threefold increases in beta 2 AR mRNA and were blocked by the protein synthesis inhibitor cycloheximide. Despite the marked increase in Bmax, however, IL-1 beta depressed adenosine 3',5'-cyclic monophosphate (cAMP) responses to isoproterenol and forskolin, a direct activator of AC (P < 0.001 by analysis of variance for both). The inhibitory effect of IL-1 beta on cAMP production appeared to be explained by increases in the activity of an inhibitory GTP binding protein because IL-1 beta treatment increased the activity of a pertussis toxin ADP-ribosylated Gi alpha protein by approximately 2.5-fold; and pretreatment of intact cells with pertussis toxin inhibited the effect of IL-1 beta on cAMP production. These data indicate that IL-1 beta-mediated changes in the beta AR-AC system function in airway epithelial cells are complex and involve expression of receptor protein, GTP binding protein, and possibly AC itself. Increases in IL-1 beta may contribute to abnormalities in airway function in subjects with asthma.

Adenylyl Cyclases↗
PubMed1996

Signaling by the G12 class of G proteins.

The G12 class of G proteins are defined by the alpha-subunits of mammalian G12 and G13. Biochemical and mutational characterization of G alpha 12/13 have identified several novel signaling pathways regulated by these alpha-subunits. Studies with the constitutively activated mutants of G alpha 12 and G alpha 13 have indicated that they stimulate mitogenic signaling pathways leading to the oncogenic transformation of fibroblast cell lines. Recent analyses have indicated that G alpha 12 and G alpha 13 regulate cytoplasmic as well as nuclear signaling events such as activation of the Jun N-terminal kinase signaling module, Na+/H+ exchangers, focal adhesion assemblies, and transcriptional activation of specific primary response genes. The emerging view suggests that these signaling events represent an integrated response regulated by G12 and G13. This review discusses the diverse signaling responses regulated by G12 and G13, and the interrelationship of these responses.

Amino Acid Sequence↗
PubMed1995

The photoactivatable NAD+ analogue [32P]2-azido-NAD+ defines intra- and inter-molecular interactions of the C-terminal domain of the G-protein G alpha t.

Recently, we reported the synthesis and use of [32P]2-azido-NAD+ as a probe to study the structural organization of G-proteins. Pertussis toxin was used to 'tether' [32P]2-azido-ADP-ribose of [32P]2-azido-NAD+ to Cys347 of the alpha subunit of the G-protein Gt. Light activation of the azide moiety covalently cross-linked the domain containing Cys347 at the C-terminus of alpha t with neighbouring intra- and inter-molecular domains of holo-transducin. The radiolabel from [32P]2-azido-ADP-ribose was then transferred to the 'acceptor' domain by cleaving the thioglycosidic bond between Cys347 and [32P]2-azido-ADP- ribose with mercuric acetate. ADP-ribosylation followed by photocross-linking of holo-transducin indicated intramolecular interactions of the C-terminal domain with other alpha t domains and intermolecular interactions with holotransducin alpha and gamma subunits. The radiolabelled peptides, which were radiolabelled because of the transfer of the photoactive moiety, were identified by utilizing 2-(2'-nitrophenylsulphenyl)-3-methyl-3'- bromoindolenine ('BNPS-skatole') and CNBr. The results indicate that the C-terminus of alpha t interacts with both N-terminal and C-terminal domains within the alpha t molecular. Mapping the interacting sites between cross-linked alpha dimers and alpha trimers indicates that the C-terminal domain of alpha t is involved in the formation of alpha t homopolymers in solution. In addition, our studies place the beta gamma subunit in close proximity to Cys347 of alpha t, as indicated by the transfer of [32P]2-azido-ADP-ribose from Cys347 to the gamma subunit, which was further localized to the C-terminal half of gamma t. The studies presented here identify the C-terminal intra- and inter-molecular interactions of the alpha subunit of holo-transducin.

Adenosine Diphosphate↗
PubMed1995

G alpha 12 and G alpha 13 stimulate Rho-dependent stress fiber formation and focal adhesion assembly.

Rho, a member of the Ras superfamily of GTP-binding proteins, regulates actin polymerization resulting in the formation of stress fibers and the assembly of focal adhesions. In Swiss 3T3 cells, heterotrimeric G protein-coupled receptors for lysophosphatidic acid and gastrin releasing peptide stimulate Rho-dependent stress fiber and focal adhesion formation. The specific heterotrimeric G protein subunits mediating Rho-dependent stress fiber and focal adhesion formation have not been defined previously. We have expressed GTPase-deficient, constitutively activated G protein alpha subunits and mixtures of beta and gamma subunits in Swiss 3T3 cells. Measurement of actin polymerization and focal adhesion formation indicated that GTPase-deficient alpha 12 and alpha 13, but not the activated forms of alpha 12 or alpha q stimulated stress fiber and focal adhesion assembly. Combinations of beta and gamma subunits were unable to stimulate stress fiber or focal adhesion formation. G alpha 12- and alpha 13-mediated stress fiber and focal adhesion assembly was inhibited by botulinum C3 exoenzyme, which ADP-ribosylates and inactivates Rho, indicating that alpha 12 and alpha 13, but not other G protein alpha subunits or beta gamma complexes, regulate Rho-dependent responses. The results define the integration of G12 and G13 with the regulation of the actin cytoskeleton.

3T3 Cells↗
PubMed1995

Activation of Jun kinase/stress-activated protein kinase by GTPase-deficient mutants of G alpha 12 and G alpha 13.

Signal transduction pathways regulated by G12 and G13 heterotrimeric G proteins are largely unknown. Expression of activated, GTPase-deficient mutants of alpha 12 and alpha 13 alter physiological responses such as Na+/H+ exchanger activity, but the effector pathways controlling these responses have not been defined. We have found that the expression of GTPase-deficient mutants of alpha 12 (alpha 12Q229L) or alpha 13 (alpha 13Q226L) leads to robust activation of the Jun kinase/stress-activated protein kinase (JNK/SAPK) pathway. Inducible alpha 12Q229L and alpha 13Q226L expression vectors stably transfected in NIH 3T3 cells demonstrated JNK/SAPK activation but not extracellular response/mitogen-activated protein kinase activation. Transient transfection of alpha 12Q229L and alpha 13Q226L also activated the JNK/SAPK pathway in COS-1 cells. Expression of the GTPase-deficient mutant of alpha q (alpha qQ209L) but not alpha i (alpha iQ205L) or alpha s (alpha sQ227L) was also able to activate the JNK/SAPK pathway. Functional Ras signaling was required for alpha 12Q229L and alpha 13Q226L activation of the JNK/SAPK pathway; expression of competitive inhibitory N17Ras inhibited JNK/SAPK activation in response to both alpha 12Q229L and alpha 13Q226L. The results describe for the first time a Ras-dependent signal transduction pathway involving JNK/SAPK regulated by alpha 12 and alpha 13.

3T3 Cells↗
PubMed1995

Analysis of G protein alpha subunit mRNA abundance in preimplantation mouse embryos using a rapid, quantitative RT-PCR approach.

We have developed a novel reverse transcription-polymerase chain reaction (RT-PCR)-based approach for systematically quantifying in a single experiment the abundances of many different mRNAs in preimplantation mouse embryos. With this approach, the entire mRNA population from a small number of embryos is amplified while preserving the relative abundance of each mRNA in the cDNA population. The cDNA is analyzed by quantitative hybridization to radiolabeled probes. The approach is very sensitive and provides reliable, quantitative data regarding changes in mRNA abundance. A major advantage of this method is that estimates of mRNA copy number can be obtained and compared between different mRNAs. With this approach, we analyzed the patterns of expression of nine G protein alpha subunit mRNAs (G alpha s, G alpha i, G alpha q, G alpha o, and G alpha 11-15) in oocytes, eggs, and preimplantation embryos from fertilization to the blastocyst stage. Six alpha subunit mRNAs were expressed at significant levels, all of which underwent significant temporal alterations in expression. The mRNAs encoding some alpha subunit types were expressed predominantly in the egg and 1-cell embryo, underwent sharp reductions during the 2-cell stage, and were re-expressed between the 8-cell and blastocyst stages. One alpha subunit mRNA increased in abundance at the early blastocyst stage. The possible significance of these alterations in G protein mRNA abundance to embryonic development is discussed.

Animals↗