Biomedical subjects

R Datta

Publications and source records attributed to R Datta.

At least 55 records · Page 3Linked to original sources

PubMed1997

Activation of a CrmA-insensitive, p35-sensitive pathway in ionizing radiation-induced apoptosis.

The response of eukaryotic cells to ionizing radiation (IR) includes induction of apoptosis. However, the signals that regulate this response are unknown. The present studies demonstrate that IR treatment of U-937 cells is associated with: (i) internucleosomal DNA fragmentation; (ii) cleavage of poly(ADP-ribose) polymerase; (iii) cleavage of protein kinase C delta; and (iv) induction of an Ac-DEVD-p-nitroanilide cleaving activity. Overexpression of the cowpox protein CrmA blocked tumor necrosis factor (TNF)-induced apoptosis but had no effect on IR-induced DNA fragmentation or cleavage of poly(ADP-ribose) polymerase and protein kinase C delta. By contrast, overexpression of the baculovirus p35 protein blocked both IR- and TNF-induced apoptosis. The results further demonstrate that the IR-induced proteolytic activity is directly inhibited by the addition of purified recombinant p35, but not by CrmA. We show that the CPP32 protease is sensitive to p35 and not CrmA. We also show that IR induces activation of CPP32 and that this event, like induction of apoptosis, is sensitive to overexpression of p35 and not CrmA. These findings indicate that IR-induced apoptosis involves activation of CPP32 and that this CrmA-insensitive apoptotic pathway is distinct from those induced by TNF and certain other stimuli.

Apoptosis↗

PubMed1996

Proteolytic activation of protein kinase C delta by an ICE/CED 3-like protease induces characteristics of apoptosis.

Recent studies have shown that protein kinase C (PKC) delta is proteolytically activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. However, the relationship of PKC delta cleavage to induction of apoptosis is unknown. The present studies demonstrate that full-length PKC delta is cleaved at DMQD330N to a catalytically active fragment by the cysteine protease CPP32. The results also demonstrate that overexpression of the catalytic kinase fragment in cells is associated with chromatin condensation, nuclear fragmentation, induction of sub-G1 phase DNA and lethality. By contrast, overexpression of full-length PKC delta or a kinase inactive PKC delta fragment had no detectable effect. The findings suggest that proteolytic activation of PKC delta by a CPP32-like protease contributes to phenotypic changes associated with apoptosis.

Amino Acid Sequence↗

PubMed1996

Activation of the CPP32 protease in apoptosis induced by 1-beta-D-arabinofuranosylcytosine and other DNA-damaging agents.

The response of human myeloid leukemia cells to treatment with 1-beta-arabinofuranosylcytosine (ara-C) includes the induction of apoptosis. Ara-C induced apoptosis is associated with proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and protein kinase C (PKC) delta. However, the signals involved in this response are unknown. The present studies show that ara-C treatment of U-937 cells is associated with induction of a protease activity that cleaves the tetrapeptides Ac-DEVD-pNA and Ac-DMOD-pNA found at the cleavage sites of PARP and PKC delta, respectively. The ara-C-induced protease activity was sensitive to overexpression of the anti-apoptotic protein Bcl-xL and the baculovirus protein p35. By contrast, overexpression of the cowpox virus protein CrmA blocked apoptosis induced by engagement of the Fas receptor but not that induced by ara-C. CrmA overexpression also had no detectable effect on ara-C-induced cleavage of PKC delta. The results further show that ara-C induces activation of the CPP32 protease by a CrmA-insensitive and p35-sensitive mechanism. Similar results were obtained with cisplatinum, etoposide, and camptothecin. These findings indicate that ara-C and other DNA-damaging agents activate a CrmA-insensitive apoptotic pathway involving CPP32 and that these signals differ from those associated with apoptosis induced by the Fas receptor.

Antimetabolites, Antineoplastic↗

PubMed1996

Involvement of a CrmA-insensitive ICE/Ced-3-like protease in ceramide-induced apoptosis.

Ceramide has emerged as a novel lipid mediator of tumor necrosis factor (TNF)-induced apoptosis. However, the signals involved in this response are unknown. The present study demonstrates that ceramide-induced internucleosomal DNA cleavage is temporally associated with proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and protein kinase C (PKC) delta. Overexpression of baculovirus protein p35 blocked ceramide-induced DNA fragmentation and proteolytic activity, whereas overexpression of cowpox virus protein CrmA had no effect on these events. By contrast, TNF-induced DNA cleavage and proteolytic activity was inhibited by CrmA as well as p35. These results indicate that ceramide-induced apoptosis involves the activation of a CrmA-insensitive protease that is distinct from that induced by TNF.

Apoptosis↗

PubMed1995

Effects of some heavy metals on growth, pigment and antibiotic production by Streptomyces galbus.

A chromogenic strain of Streptomyces galbus 5ME-13 producing an antifungal antibiotic was used to study the effects of some heavy metals on growth, melanoid pigment and antibiotic production in culture. Mercury and nickel were highly toxic followed by cadmium while cobalt and chromium appeared to be less toxic. All the metals inhibited growth, pigment and antibiotic production with widely variable IC50 values. Cadmium and chromium at lower concentrations unusually stimulated growth and enhanced pigment production in the strain.

Antifungal Agents↗

PubMed1995

Overexpression of Bcl-XL by cytotoxic drug exposure confers resistance to ionizing radiation-induced internucleosomal DNA fragmentation.

Acquired resistance to diverse chemotherapeutic agents has been associated with overexpression of the P-glycoprotein. We have selected human U-937 cells for clones resistant to the cytotoxic agents doxorubicin (U-A20) and vincristine (U-V20). The results demonstrate that P-glycoprotein-positive U-A20 and U-V20 cells exhibit resistance to inducers of internucleosomal DNA fragmentation. Although parental U-937 cells responded to ionizing radiation with the DNA laddering characteristic of physiological cell death, the drug-resistant lines were insensitive to this effect. The U-A20 and U-V20 clones were also resistant to endonucleolytic DNA cleavage associated with exposure to tumor necrosis factor or ceramide. Previous work has demonstrated that physiological cell death is inhibited by overexpression of the Bcl-2 protein. However, analysis of Bcl-2 revealed similar levels in the parental and drug-resistant cells. In contrast, we show that U-A20 and U-V20 cells overexpress the Bcl-2-related protein, Bcl-xL. Moreover, studies with a U-937 cell line transfected with a Bcl-XL expression vector confirm resistance to ionizing radiation-induced DNA fragmentation and cell killing. These findings suggest that, unlike Bcl-2, Bcl-XL may be constitutively overexpressed as a result of selection for cytotoxic drug resistance and that Bcl-XL participates in an acquired form of multimodality resistance to chemotherapeutic agents and radiation.

Base Sequence↗

PubMed1995

Involvement of stress-activated protein kinase in the cellular response to 1-beta-D-arabinofuranosylcytosine and other DNA-damaging agents.

The cellular response to 1-beta-D-arabinofuranosylcytosine (ara-C) includes activation of Jun/AP-1, induction of c-jun transcription, and programmed cell death. The stress-activated protein (SAP) kinases stimulate the transactivation function of c-jun by amino terminal phosphorylation. The present work demonstrates that ara-C activates p54 SAP kinase. The finding that SAP kinase is also activated by alkylating agents (mitomycin C and cisplatinum) and the topoisomerase I inhibitor 9-amino-camptothecin supports DNA damage as an initial signal in this cascade. The results demonstrate that ara-C also induces binding of SAP kinase to the SH2/SH3-containing adapter protein Grb2. SAP kinase binds to the SH3 domains of Grb2, while interaction of the p85 alpha-subunit of phosphatidylinositol 3-kinase complex. The results also demonstrate that ara-C treatment is associated with inhibition of lipid and serine kinase activities of PI 3-kinase. The potential significance of the ara-C-induced interaction between SAP kinase and PI 3-kinase is further supported by the demonstration that Wortmannin, an inhibitor of PI 3-kinase, stimulates SAP kinase activity. The finding that Wortmannin treatment is also associated with internucleosomal DNA fragmentation may support a potential link between PI 3-kinase and regulation of both SAP kinase and programmed cell death.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Ionizing radiation induces rapid tyrosine phosphorylation of p34cdc2.

Eukaryotic cells respond to ionizing radiation exposure with cell cycle arrest. However, little is known about the signaling mechanisms responsible for this effect. The present work has asked whether ionizing radiation exposure is associated with changes in phosphorylation of proteins in HL-60 myeloid leukemia cells. The results demonstrate increased tyrosine phosphorylation of a M(r) 34,000 substrate. This effect was detectable at 1 to 10 min after irradiation and was induced by doses of 50 to 500 cGy. Immunoprecipitation studies further suggest that this substrate is the serine/threonine p34cdc2 protein kinase. Since p34cdc2 is required for entry into mitosis, these findings support the posttranslational modification of a cell cycle regulatory protein in the response to ionizing radiation.

CDC2 Protein Kinase↗

PubMed1994

All-trans retinoic acid reverses phorbol ester resistance in a human myeloid leukemia cell line.

Treatment of human HL-60 leukemic cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with activation of protein kinase C (PKC) and induction of monocytic differentiation. An HL-60 variant cell line, termed HL-525, derived from long-term exposure to TPA (Homma et al, Proc Natl Acad Sci USA 83: 7316, 1986) is resistant to TPA-induced differentiation and displays decreased PKC beta expression compared with the HL-60 parent line. However, this variant exhibits features of granulocytic differentiation, including nitroblue tetrazolium reduction, when exposed to all-trans retinoic acid (ATRA). Whereas treatment of HL-525 cells with ATRA or TPA alone had no effect on features of monocytic differentiation, these agents in combination resulted in cellular adhesion, nonspecific esterase staining, and induction of the c-fms (monocyte growth factor receptor) gene. In order to measure PKC expression associated with the reversal of TPA resistance by ATRA, we exposed HL-525 cells to ATRA and analyzed PKC-mRNA and protein levels. Exposure of HL-525 cells to ATRA for 3 days resulted in induction of PKC beta transcripts, whereas there was little change in PKC alpha mRNA levels. ATRA treatment was also associated with an increase in PKC activity and an induction of cytosolic PKC beta protein levels. These findings are consistent with the hypothesis that ATRA reverses TPA resistance in HL-525 cells by enhancing the expression of PKC.

Cell Differentiation↗

PubMed1993

Coinduction of c-jun gene expression and internucleosomal DNA fragmentation by ionizing radiation.

Previous work has demonstrated that the cellular response to ionizing radiation includes transcriptional activation of the c-jun early response gene. The present studies demonstrate that this induction of c-jun expression is temporally related to the appearance of internucleosomal DNA fragmentation. These events were maximal at 6 h and transient after exposure to lethal doses (20 Gy) of ionizing radiation. We also demonstrate that N-acetyl-L-cysteine (NAC), an antioxidant, inhibits X-ray-induced c-jun expression and endonucleolytic DNA cleavage. These findings suggested that both events are mediated at least in part through the formation of reactive oxygen intermediates (ROIs). Since ROIs damage DNA and X-ray-induced DNA damage is associated with activation of poly(ADP-ribose) polymerase (ADPRP), we studied the effects of the ADPRP inhibitors 3-aminobenzamide (3-AB), nicotinamide, and theophylline. 3-AB blocked both X-ray-induced c-jun expression and internucleosomal DNA fragmentation. Similar findings were obtained with nicotinamide and theophylline. In contrast, 3-AB had little if any effect on induction of c-jun transcripts or DNA fragmentation induced by the alkylating agent mitomycin C. While c-jun expression is restricted to cells in G1 and G1/S phases, we have found that X-ray-induced c-jun transcripts are detectable throughout all phases of the cell cycle. The induction of internucleosomal DNA fragmentation by X-rays was also detectable throughout the cell cycle. Taken together, these results support the coinduction of c-jun transcription and internucleosomal DNA fragmentation by ionizing radiation. Similar studies were performed with H2O2 since this agent also results in the production of ROIs.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗

PubMed1993

Early response gene induction following DNA damage in astrocytoma cell lines.

Early response genes (ERGs) are a group of genes with low or absent expression in quiescent cells that can be induced rapidly by a variety of proliferation and differentiation stimuli. c-jun and c-fos are prototypes for this group of genes. Recent evidence suggests that DNA damaging agents such as cytotoxic drugs and ionizing radiation can elicit strong ERG induction, suggesting that these genes may be involved in DNA damage repair and/or cell death. Paralleling this interest in drug-induced ERG expression is a growing body of evidence implicating ERG expression as important to the intrinsic function of the central nervous system. We therefore set out to explore the pattern of chemotherapeutic drug- and radiation-induced c-jun and c-fos expression in neuroectodermally (astrocytic) derived cell lines. We have demonstrated that various chemotherapeutic agents and ionizing radiation can induce c-jun in a time- and concentration-dependent manner. Furthermore, this induction can be prevented by pretreatment of the cells with agents that protect against DNA damage. Finally, we have demonstrated that c-fos and c-jun expression and induction are discoordinately regulated, reflecting a difference in astrocytic cell lines compared to hematopoietic cell lines. The possible relevance of these observations to the clinical resistance of astrocytic tumors to standard chemotherapy and radiation is discussed.

Astrocytoma↗

PubMed1993

Reactive oxygen intermediates target CC(A/T)6GG sequences to mediate activation of the early growth response 1 transcription factor gene by ionizing radiation.

The cellular response to ionizing radiation includes induction of the early growth response 1 gene (EGR1). The present work has examined the involvement of reactive oxygen intermediates (ROIs) in this response. Exposure of human HL-525 cells, an HL-60 subclone deficient in protein kinase C-mediated signaling, to both ionizing radiation and H2O2 was associated with increases in EGR-1 transcripts. These increases in EGR-1 expression were inhibited by the antioxidant N-acetyl-L-cysteine (NAC). Nuclear run-on assays demonstrate that NAC inhibits the activation of EGR1 transcription by these agents. Previous studies have shown that induction of EGR1 by x-rays is conferred by serum response or CC(A/T)6GG (CArG) elements. The present studies demonstrate similar findings with H2O2 and the finding that activation of the EGR1 promoter region containing CArG elements is abrogated by NAC. Moreover, we show that NAC inhibits the ability of a single CArG box to confer x-ray and H2O2 inducibility when linked to a heterologous promoter. Taken together, these findings indicate that ROIs induce EGR1 transcription by activation of CArG elements.

Actins↗

PubMed1993

Transcriptional regulation of the early growth response 1 gene in human myeloid leukemia cells by okadaic acid.

The early growth response 1 (EGR-1) gene is induced after mitogenic stimulation of diverse cell types. The present work has examined the effects of okadaic acid, an inhibitor of protein phosphatases 1 and 2A, on EGR-1 expression during monocytic differentiation of U-937 myeloid leukemia cells. Treatment of U-937 cells with okadaic acid was associated with transient increases in EGR-1 mRNA levels. These increases were maximal at 6 h and occurred in the absence of de novo protein synthesis. Nuclear run-on assays demonstrated that although EGR-1 transcription is detectable in untreated U-937 cells, this rate is increased 6-fold by okadaic acid. Sequences responsive to okadaic acid-induced signals were determined by deletion analysis of the EGR-1 promoter. The results demonstrate that okadaic acid-induced EGR-1 transcription is dependent on the presence of CC (A/T)6 GG (CArG) motifs. The EGR-1 promoter contains six CArG boxes. However, only the 5'-most distal (first) CArG sequence conferred okadaic acid inducibility. A 40-base pair oligomer corresponding to the first CArG element also conferred okadaic acid inducibility of the minimal thymidine kinase gene promoter. In contrast, there was no inducibility using a similar oligomer containing a mutated CArG box. Finally, binding of nuclear proteins to the first CArG sequence was similar for control and okadaic acid-treated cells. Taken together, these results suggest that okadaic acid activates EGR-1 transcription and that this event is mediated at least in part by a single CArG element.

Ethers, Cyclic↗

PubMed1993

Ionizing radiation down-regulates histone H1 gene expression by transcriptional and post-transcriptional mechanisms.

The cellular response to ionizing radiation includes growth arrest and DNA repair. However, little is known about the regulation of gene expression by this agent. The present studies demonstrate that exposure to ionizing radiation is associated with a dose-dependent decrease in histone H1 gene expression. Following treatment with 20 Gy, this effect was transient, detectable at 15-30 min, and maximal at 6 h. Nuclear run-on assays demonstrate that this down-regulation is controlled at least in part by transcriptional mechanisms. We also demonstrate that inhibition of protein synthesis with cycloheximide abrogates the down-regulation of both histone H1 gene transcription and mRNA levels in irradiated cells. The results demonstrate that treatment with ionizing radiation is associated with a decrease in the stability of the histone H1 transcript, and that this effect is reversed by inhibition of protein synthesis. These findings demonstrate that ionizing radiation activates at least two distinct signaling pathways that control histone H1 expression at the transcriptional and post-transcriptional levels.

Dose-Response Relationship, Radiation↗

PubMed1993

All-trans retinoic acid induces monocyte growth factor receptor (c-fms) gene expression in HL-60 leukemia cells.

All-trans-retinoic (ATRA) treatment of patients with acute promyelocytic leukemia results in differentiation of the malignant cells and a high complete remission rate. ATRA treatment induced granulocytic differentiation in HL-60 cells as assessed by nitroblue tetrazolium (NBT) reduction, but had no effect on non-specific esterase (NSE) straining, as expected in cells maturing along the monocytic lineage. However, our results demonstrate that ATRA (0.1-10 microM) induces expression of the c-fms (monocyte colony-stimulating factor receptor) gene in HL-60 cells. This effect was detectable after 2 days and expression was maximal at 5 days. Similar results were obtained during treatment with cis-retinoic acid (CRA), hexamethylene bisacetamide (HMBA), or dimethyl sulfoxide (DMSO). The results also demonstrate that ATRA-induced c-fms expression is potentiated by exposure to tumor necrosis factor alpha (TNF alpha) or dibutyryl cyclic adenosine monophosphate (cAMP). The induction of c-fms transcripts by ATRA is associated with induction of M-CSF-binding ability, suggesting cell surface expression of the monocyte growth factor receptor. Our results indicate that retinoic acid can induce features of both monocytic and granulocytic differentiation in HL-60 cells.

Carboxylesterase↗

PubMed1992

Ionizing radiation activates transcription of the EGR1 gene via CArG elements.

The present studies have examined the effects of ionizing radiation on control of the early growth response 1 (EGR1) gene. Exposure of human HL-525 cells to x-rays was associated with increases in EGR1 mRNA levels. Nuclear run-on assays showed that this effect is related at least in part to activation of EGR1 gene transcription. Sequences responsive to ionizing radiation-induced signals were determined by deletion analysis of the EGR1 promoter. The results demonstrate that x-ray inducibility of the EGR1 gene is conferred by a region containing six serum response or CC(A+T-rich)6GG (CArG) motifs. Further analysis confirmed that the region encompassing the three distal or upstream CArG elements is functional in the x-ray response. Moreover, this region conferred x-ray inducibility to a minimal thymidine kinase gene promoter. Taken together, these results indicate that ionizing radiation induces EGR1 transcription through CArG elements.

Base Sequence↗

PubMed1992

Involvement of reactive oxygen intermediates in the induction of c-jun gene transcription by ionizing radiation.

Previous work has demonstrated that the cellular response to ionizing radiation includes transcriptional activation of the c-jun gene. The signaling events responsible for this response, however, remain unclear. The present studies have examined the effects of ionizing radiation on c-jun expression in a variant of HL-60 cells, designated HL-525, which is deficient in protein kinase C (PKC)-mediated signal transduction. The results demonstrate that these cells express low levels of PKC alpha and PKC beta transcripts and exhibit an attenuated induction of c-jun expression following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, HL-525 cells respond to ionizing radiation with an increase in c-jun mRNA which is more pronounced than that in wild-type HL-60 cells. These cells similarly respond to ionizing radiation with increased expression of the jun-B, jun-D, c-fos, and fos-B genes. Nuclear run-on assays demonstrate that X-ray-induced c-jun expression in HL-525 cells is regulated by increases in the rate of c-jun gene transcription. Moreover, mRNA stability studies in irradiated HL-525 cells demonstrate that the half-life of c-jun transcripts is prolonged compared to that in wild-type cells. Studies with N-acetyl-L-cysteine (NAC), an antioxidant, suggest that X-ray-induced transcriptional activation of the c-jun gene is mediated at least in part through the formation of reactive oxygen intermediates (ROIs). In this context, H2O2 also induced c-jun expression in HL-525 cells, and this effect was inhibited by NAC.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Nucleus↗

PubMed1992

Transcriptional regulation of cytokine expression by diethyldithiocarbamate in human HL-60 promyelocytic leukemia cells.

Diethyldithiocarbamate (DDTC) is an investigational agent used to ameliorate chemotherapy- or radiotherapy-induced myelosuppression. We studied the effects of DDTC on the regulation of hematopoietic cytokine production in human myeloid cells. The results demonstrated that DDTC decreases proliferation of human HL-60 promyelocytic leukemia cells in a concentration-dependent manner. DDTC treatment also increased interleukin-alpha (IL-1 alpha), IL-1 beta, and tumor necrosis factor (TNF) expression in these cells. Similar findings were obtained in normal human peripheral blood monocytes. Peak induction of these cytokines occurred 6-12 hr after exposure to DDTC; levels returned to those in control cells by 24-48 hr in HL-60 cells. This effect was specific for IL-1 and TNF in that there was no detectable increase in IL-3, macrophage colony-stimulating factor or granulocyte/macrophage colony-stimulating factor RNA expression. Transcriptional run-on analysis demonstrated that exposure to DDTC increased the rate of TNF gene transcription in HL-60 cells. These data suggest that the myeloprotective effects of DDTC may be mediated, at least in part, by the induction of TNF, IL-1 alpha, and IL-1 beta.

Cytokines↗