Reactive oxygen species play an important role in the activation of heat shock factor 1 in ischemic-reperfused heart.
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BACKGROUND: The myocardial protective role of heat shock protein (HSP) has been demonstrated. Recently, we reported that ischemia/reperfusion induced a significant activation of heat shock factor (HSF) 1 and an accumulation of mRNA for HSP70 and HSP90. We examined the role of reactive oxygen species (ROSs) in the induction of stress response in the ischemic-reperfused heart. METHODS AND RESULTS: Rat hearts were isolated and perfused with Krebs-Henseleit buffer by the Langendorff method. Whole-cell extracts were prepared for gel mobility shift assay using oligonucleotides containing the heat shock element. Induction of mRNA for HSP70 and HSP90 was examined by Northern blot analysis. Repetitive ischemia/reperfusion, which causes recurrent bursts of free radical generation, resulted in burst activation of HSF1, and this burst activation was significantly reduced with either allopurinol 1 mmol/L (an inhibitor of xanthine oxidase) or catalase 2x10(5) U/L (a scavenger of H2O2). Significant activation of HSF1 was observed on perfusion with buffer containing H2O2 150 micromol/L or xanthine 1 mmol/L plus xanthine oxidase 5 U/L. The accumulation of mRNA for HSP70 or HSP90 after repetitive ischemia/reperfusion was reduced with either allopurinol or catalase. CONCLUSIONS: Our findings demonstrate that ROSs play an important role in the activation of HSF1 and the accumulation of mRNA for HSP70 and HSP90 in the ischemic-reperfused heart. (+info)
Human, rat, and mouse kidney cells express functional erythropoietin receptors.
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BACKGROUND: Erythropoietin (EPO), secreted by fibroblast-like cells in the renal interstitium, controls erythropoiesis by regulating the survival, proliferation, and differentiation of erythroid progenitor cells. We examined whether renal cells that are exposed to EPO express EPO receptors (EPO-R) through which analogous cytokine responses might be elicited. METHODS: Normal human and rat kidney tissue and defined cell lines of human, rat, and mouse kidney were screened, using reverse transcription-polymerase chain reaction, nucleotide sequencing, ligand binding, and Western blotting, for the expression of EPO-R. EPO's effects on DNA synthesis and cell proliferation were also examined. RESULTS: EPO-R transcripts were readily detected in cortex, medulla, and papilla of human and rat kidney, in mesangial (human, rat), proximal tubular (human, mouse), and medullary collecting duct cells (human). Nucleotide sequences of EPO-R cDNAs from renal cells were identical to those of erythroid precursor cells. Specific 125I-EPO binding revealed a single class of high- to intermediate-affinity EPO-Rs in each tested cell line (kD 96 pm to 1. 4 nm; Bmax 0.3 to 7.0 fmol/mg protein). Western blots of murine proximal tubular cell membranes revealed an EPO-R protein of approximately 68 kDa. EPO stimulated DNA synthesis and cell proliferation dose dependently. CONCLUSION: This is the first direct demonstration, to our knowledge, that renal cells possess EPO-Rs through which EPO stimulates mitogenesis. This suggests currently unrecognized cytokine functions for EPO in the kidney, which may prove beneficial in the repair of an injured kidney while being potentially detrimental in renal malignancies. (+info)
COOH-terminal domain of p53 modulates p53-mediated transcriptional transactivation, cell growth, and apoptosis.
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The tumor suppressor protein p53 contributes to the control of cell cycle checkpoints and stress-induced apoptosis and is frequently mutated in many different types of human cancers. The COOH terminus of p53 modulates the transcriptional and apoptotic activities of the protein. Although COOH-terminal mutants of p53 are uncommon, we proposed that these p53 mutants nevertheless contributed to the selective clonal expansion of the cancer cells. Therefore, we analyzed the tumor-derived p53 COOH-terminal domain (CTD) mutants (352D/H, 356G/W, 342-stop, 360-del, and 387-del) functionally. The results have revealed that all mutants have impaired apoptotic activity when compared with wild-type p53. However, some of these mutants still transcriptionally transactivate p21Waf/Cip1 and inhibit cell growth. Interestingly, of the tumor-derived CTD mutants, oligomerization-defective mutant 342-stop was the only one that did not exhibit sequence-specific DNA binding or failed to transactivate p21Waf1/Cip1, Bax, and IGF-BP3 transcriptionally. The failure to inhibit cell growth by this tumor-derived CTD mutant supports the hypothesis that p53 sequence-specific transcriptional transactivity to p21Waf1/Cip1 is correlated with induction of cell cycle arrest and that the p53 transcriptional transactivity requires oligomerization of the p53 protein. These and other data indicate that the CTD of p53 is an important component of p53-mediated apoptosis and cell growth arrest and that inactivation of the apoptotic function, but not the inhibition of growth, is an important step during human tumorigenesis. (+info)
Bombesin stimulates adhesion, spreading, lamellipodia formation, and proliferation in the human colon carcinoma Isreco1 cell line.
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The neuropeptide bombesin and its mammalian homologue, gastrin-releasing peptide (GRP), enhance proliferation in some but not all human tumor cell lines. The pathophysiological relevance of the bombesin/GRP receptor (GRP-R), which is expressed in 30% of human colon tumor cell lines and in 24-40% of native tumors, has not been clearly assessed at this time. We studied the effects of bombesin in the recently characterized human colon carcinoma Isreco1 cell line. Competitive reverse transcription-PCR showed a high GRP-R mRNA level in Isreco1 cells, and binding studies confirmed the expression of bombesin/GRP-subtype receptors (Kd = 0.42 nM; Bmax = 18,000 sites/cell). Exposure to bombesin resulted in an increase of intracellular calcium concentrations. Bombesin (1 nM) induced cell spreading at 24 h (21.7+/-1.6% versus 6.4+/-0.8% in control cells; P<0.01) and markedly increased the formation of lamellipodia. In addition, adhesion of Isreco1 cells to collagen I-coated culture dishes was stimulated in the presence of 1 nM bombesin (69+/-6% versus 42+/-1% in control cells; P<0.01). Finally, bombesin significantly increased [3H]thymidine uptake by Isreco1 cells in a dose-dependent manner, with a first significant response at 0.1 nM and a maximal effect at 100 nM bombesin (192.2+/-9.7% of control). These results clearly indicate that bombesin exerts morphological, adhesive, and proliferative effects on Isreco1 cells, suggesting that expression of the bombesin/GRP-R may contribute to the malignant properties of colon carcinoma cells. (+info)
Analysis of the stimulation-inhibition paradox exhibited by lymphocytes exposed to concanavalin A.
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High doses of Concanavalin A (Con A), which normally inhibit T-lymphocyte stimulation as measured by increases in DNA synthesis, cause these lymphocytes to become committed to mitogenesis while also generating a dominant but reversible negative growth signal. The observed response to the stimulatory signal as measured by the rate of commitment to enter the S phase (i.e., the rate at which the stimulation becomes lectin independent) increases with lectin concentration even in the inhibitory range. The generation of this positive signal is prevented by treating the cells with colchicine. Cells that have become committed but are also simultaneously blocked from entering the S phase by the high doses of Con A can begin synthesizing DNA if the lectin is released by adding a competitive inhibitor of binding. Experiments done in agarose cultures in which lymphocytes are kept from contact with each other suggest that the reversible inhibitory signal is mediated by structures in the individual cells rather than as a result of agglutination. Continuously dividing cells of the lymphoid line P388 are also individually and reversibly inhibited by Con A. These findings are considered in terms of the relation of the inhibitory signal to the microtubular components of cell surface modulating assemblies made up of submembranous arrays of microtubules, microfilaments, and associated proteins. (+info)
Effect of sodium butyrate on lymphocyte activation.
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Butyrate, in relatively low concentrations, has been shown to induce synthesis of enzymes, cause changes in cell morphology, and inhibit growth of a variety of mammalian cells in tissue culture (reviewed in [1]). In this communication, we report our observations on the effect of butyrate on lymphocyte activation. Butyrate completely and reversibly inhibits mitogen-induced blast formation. We present evidence that it does not interfere with the binding of mitogens, that it does not inhibit a number of the "early" reactions involved in activation, and that it does not affect ongoing DNA synthesis for an extended period of time. However, butyrate rapidly inhibits any increase in the rate of DNA synthesis. (+info)
Involvement of p21 in the PKC-induced regulation of the G2/M cell cycle transition.
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Activation of protein kinase C (PKC) inhibits cell cycle progression at the G1/S and G2/M transitions. We found that phorbol 12-myristate 13-acetate (PMA) induced upregulation of p21, not only in MCF-7 cells arrested in the G1 phase as previously shown, but also in cells delayed in the G2 phase. This increase in p21 in cells accumulated in the G1 and G2/M phases of the cell cycle after PMA treatment was inhibited by the PKC inhibitor GF109203X. This indicates that PKC activity is required for PMA-induced p21 upregulation and cell cycle arrest in the G1 and G2/M phases of the cell cycle. To further assess the role of p21 in the PKC-induced G2/M cell cycle arrest independently of its G1 arrest, we used aphidicolin-synchronised MCF-7 cells. Our results show that, in parallel with the inhibition of cdc2 activity, PMA addition enhanced the associations between p21 and either cyclin B or cdc2. Furthermore, we found that after PMA treatment p21 was able to associate with the active Tyr-15 dephosphorylated form of cdc2, but this complex was devoid of kinase activity indicating that p21 may play a role in inhibition of cdc2 induced by PMA. Taken together, these observations provide evidence that p21 is involved in integrating the PKC signaling pathway to the cell cycle machinery at the G2/M cell cycle checkpoint. (+info)
Effects of nucleoside analog incorporation on DNA binding to the DNA binding domain of the GATA-1 erythroid transcription factor.
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We investigate here the effects of the incorporation of the nucleoside analogs araC (1-beta-D-arabinofuranosylcytosine) and ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl] guanine) into the DNA binding recognition sequence for the GATA-1 erythroid transcription factor. A 10-fold decrease in binding affinity was observed for the ganciclovir-substituted DNA complex in comparison to an unmodified DNA of the same sequence composition. AraC substitution did not result in any changes in binding affinity. 1H-15N HSQC and NOESY NMR experiments revealed a number of chemical shift changes in both DNA and protein in the ganciclovir-modified DNA-protein complex when compared to the unmodified DNA-protein complex. These changes in chemical shift and binding affinity suggest a change in the binding mode of the complex when ganciclovir is incorporated into the GATA DNA binding site. (+info)