Biomedical subjects
A L Block
Publications and source records attributed to A L Block.
Platelet-activating factor activates HIV promoter in transfected SH-SY5Y neuroblastoma cells and MOLT-4 T lymphocytes.
Transfected gene constructs comprising the long terminal repeat (LTR) sequence of the human immunodeficiency virus (HIV) genome spliced to an assayable reporter gene have made possible the evaluation of a lipid mediator, platelet-activating factor (PAF), as a potential HIV transcriptional regulatory molecule. We assessed the activation of the HIV LTR promoter sequence linked to the chloramphenicol acetyltransferase (CAT) reporter gene (HIV-CAT) by PAF in both a human neural (SH-SY5Y neuroblastoma) and a human leukocytic (MOLT-4 T-lymphocyte) cell line. PAF activated expression of the HIV-CAT construct in both the SH-SY5Y and MOLT-4 T-cell lines. PAF-induced CAT activity was approximately six to seven times higher in the SH-SY5Y cells than in the MOLT-4 cells. Preincubation of cells with the specific PAF antagonist BN 52021 completely inhibited CAT expression in both cell lines. The biologically inactive PAF precursor lyso-PAF did not activate CAT expression. Assays for CAT mRNA demonstrated an increase after PAF treatment, an effect that was completely inhibited by BN 52021, and which was not elicited by lyso-PAF. These results show that PAF represents a potential cellular mediator evoking the expression of the HIV genome.
Morphine-induced transactivation of HIV-1 LTR in human neuroblastoma cells.
Infection by human immunodeficiency virus (HIV) is followed in many cases by a clinically quiescent or latent phase that appears to continue as long as host antiviral defense is intact. This has raised the possibility that certain host susceptibility factors (i.e., environmental cofactors) might influence the progression of the disease. In this study we demonstrate that morphine can function to activate HIV/LTR-CAT fusion gene (HIV-long terminal repeat-chloramphenicol acetyltransferase) when transfected into undifferentiated human SH-SY5Y neuroblastoma cells. The stimulatory effect of morphine is amplified in SH-SY5Y cells that have been induced to differentiate first with phorbol 12-myristate 13-acetate (PMA) and is much less in cells differentiated with retinoic acid (RA). Morphine does not appreciably activate HIV/LTR-CAT expression in human MOLT-3 and other T cells. Morphine activation of HIV/LTR-CAT in the SH-SY5Y cells is not reversible by naltrexone and appears to involve a Fos/Jun signaling system. Our results suggest that narcotics such as morphine may lead to activation of latent HIV infection. This may be particularly important in tissues, such as brain, which can host latent HIV infection and which is uniquely damaged in patients with acquired immunodeficiency syndrome (AIDS) as evidenced by neuronal degeneration and dementia. We also predict that these findings may have important implications for the pathogenesis of AIDS, particularly in opiate drug abusers.
Platelet-activating factor stimulates a fos/jun/AP-1 transcriptional signaling system in human neuroblastoma cells.
Platelet-activating factor (PAF) elicits a rapid and transient activation of the proto-oncogenes c-fos and c-jun in SH-SY5Y neuroblastoma cells, but only to a minor extent in Molt-4 T-lymphocytes. This effect is inhibited by pretreatment of cells with the PAF antagonist BN 52021, suggesting the involvement of a specific receptor. Moreover, PAF treatment can activate gene expression through an AP-1 element, and we propose that genomic trans-activation may occur in target genes containing a functional AP-1 transcription sequence. These results may further understanding of the molecular mechanisms by which PAF contributes to long-term phenotypic changes in the nervous system.
Induction of macrophage-like differentiation of HL-60 leukemia cells by tumor necrosis factor-alpha: potential role of fos expression.
Tumor necrosis factor-alpha (TNF-alpha) is a macrophage-derived cytokine elicited during cellular responses to various microbial infections. TNF-alpha exerts direct cytotoxicity toward some tumor cells in vitro and produces hemorrhagic tumor necrosis in vivo. In human promyelocytic HL-60 leukemia cells, human recombinant TNF-alpha (rTNF-alpha) exhibits a small early proliferative effect (within 48 h), followed by marked cytostatic activity at 96 h after the addition of rTNF-alpha. Cytostasis is contiguous with an induction of cell differentiation along the monocyte/macrophage lineage. The cell proliferation effects and the induction of the differentiated phenotype are preceded by an approximate 5-fold increase in c-fos mRNA levels within 90 min after rTNF-alpha treatment of log phase HL-60 cells. Nuclear in vitro transcription assays indicate that the effect of rTNF-alpha on c-fos mRNA abundance is controlled at the transcriptional level. We have also used a postembedding immunocolloidal gold electron microscopy technique to localize and semiquantitate pp55c-fos proto-oncoprotein levels in the nucleus of both control and rTNF-alpha-treated HL-60 leukemia cells. In response to rTNF-alpha, we have observed a rapid and transient accumulation of pp55c-fos in discrete nuclear substructures within 2 h after treatment. C-fos staining appears in clusters, which are preferentially localized over semi-condensed chromatin and interchromatin granules. These results suggest that pp55c-fos is involved in the signal transduction system initiated by rTNF-alpha during the induction of HL-60 differentiation.
Epidermal growth factor induction of cellular proliferation and protooncogene expression in growth-arrested rat H4IIE hepatoma cells: role of cyclic adenosine monophosphate.
The precise molecular events involved in growth factor-mediated cell proliferation in eukaryotes have not been entirely elucidated. Identification and characterization of the itnracellular molecular signaling systems linking growth factor function with nuclear events would provide insight into the regulatory mechanisms governing eukaryotic cell growth. In this report, we demonstrate that serum-deprived rat H4IIE hepatoma cells enter a quiescent state and remain viable in the absence of serum for up to 7 days. These cells can be stimulated to transverse the cell cycle and proliferate in response to epidermal growth factor (EGF) after a 24-h lag phase. We were able to completely mimic the mitogenic effects of EGF with 8-p-chlorophenylthio-cAMP (8-CPT-cAMP) but only partially with N6-(Bu)2-cAMP. EGF and 8-CPT-cAMP together induce a synergistic increase in H4IIE hepatoma cell proliferation. The calcium ionophore A23187 and the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate had little effect on H4IIE cell proliferation. EGF treatment led to a rapid and transient increase of intracellular cAMP concentration. Both 8-CPT-cAMP and EGF were also equally effective in causing a rapid and transient induction of c-fos and c-myc protooncogene mRNA levels when added to growth-arrested H4IIE cells while A23187, N-(Bu)2-cAMP, and 4 beta-phorbol 12-myristate 13-acetate were significantly less effective. Both EGF and 8-CPT-cAMP affect protooncogene expression in growth-arrested rat H4IIE hepatoma cells primarily at the transcriptional level. Localization and semi-quantification of nuclear pp55c-fos and 63 (kilodalton)-myc protooncoproteins by immunocolloidal gold electron microscopy revealed that EGF and/or 8-CPT-cAMP treatment of quiescent H4IIE hepatoma cells led to a marked and rapid nuclear accumulation of these proteins in discrete nuclear substructures. Cummulatively, these results suggest that cAMP participates in the intracellular signaling system mediating the mitogenic and protooncogene inducing effects of EGF on growth-arrested rat H4IIE hepatoma cells.
Comparison of DNA interstrand cross-linking and strand breakage by 1,3-bis(2-chloroethyl)-1-nitrosourea in polyamine-depleted and control human adenocarcinoma cells.
Recent evidence from our laboratory and from others suggested that pretreatment with alpha-difluoromethylornithine (DFMO) sensitizes some human and rodent tumor cell lines to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Many human tumor cells are resistant to chloroethylnitrosourea-induced DNA interstrand cross-linking and cell kill due to their high levels of the DNA repair protein O6-alkylguanine DNA alkyltransferase. We therefore investigated DFMO-mediated sensitization to BCNU in BCNU-sensitive and -resistant cells. Colony formation assays were used to compare BCNU cytotoxicity in DFMO-pretreated and control cultures of two colon tumor lines, HT-29 cells, which have high alkyltransferase levels and thus are BCNU-resistant, and BE cells, which are deficient in this repair capacity and thus are BCNU-sensitive. Polyamine depletion significantly enhanced BCNU cytotoxicity only for the repair-proficient HT-29 cell line. BE cells were 40-fold more sensitive to BCNU than were HT-29 cultures. However, in BE cells, no effect of polyamine depletion was found on cellular response to BCNU treatment at 72 h after DFMO treatment. Reverse-phase high-performance liquid chromatography assays of polyamine concentrations in cell extracts verified that DFMO produced comparable degrees of polyamine depletion for both cell lines. DNA alkaline elution analysis was used to monitor BCNU-induced formation of DNA single strand breaks, DNA interstrand cross-links, and DNA-protein cross-links. Equal concentrations of BCNU produced similar levels of strand breaks and DNA-protein cross-links in DFMO-pretreated and control cultures for both cell lines. These data suggest that DNA in polyamine-deficient HT-29 and BE cells is not more accessible to BCNU than is DNA in controls. No DNA interstrand cross-links were detected in either DFMO-pretreated or control HT-29 cells after BCNU treatment. Further, in BE cells which accumulate BCNU-induced DNA interstrand cross-links, no increase in the measureable levels of cross-links resulted from polyamine deficiency. Our observations suggest that mechanisms other than increased DNA interstrand cross-link formation may be mediating the enhanced efficacy of BCNU in polyamine-deficient HT-29 cell cultures. Our findings may also imply that cellular targets for BCNU other than DNA damage may be responsible for DFMO-induced chemosensitization in the repair-proficient cells.
Experimental parameters and a biological standard for acridine orange detection of drug-induced alterations in chromatin condensation.
We investigated a number of sample-preparative parameters for use of flow cytometry to detect chromatin condensation in cells stained with acridine orange after DNA in situ is partially denatured by acid treatment. Stability and data reproducibility for both control and drug-treated ME-180 and HT-29 cells were assessed over: a range of cell concentrations in 2.56 X 10(-5) M acridine orange; 15 days of storage in fixative; various times between RNase digestion and staining; and increasing times between staining and analysis. Listmode data for red and green fluorescence were collected and mean fluorescence intensities of G1, S, and G2 subpopulations of HT-29 and ME-180 cells were computed. These were normalized to data from HeLa-S3 cells and fluorescent microspheres to control for inter-experiment variations in staining and instrumental parameters, respectively. The normalized red and green fluorescence data were used to calculate alpha 1 for G1 cells [alpha t = red fluorescence/(total fluorescence)]. Exponentially growing HeLa-S3 cells were a very consistent and reproducible biological standard to control for fixation and staining variability. Mean fluorescence intensities of control and difluoromethylornithine-treated (i.e., polyamine depleted) cells remained stable and reproducible across all tested ranges for cell concentration, storage in fixative, and time after RNase digestion. This technique can thus be used to evaluate difluoromethylornithine-induced changes in chromatin condensation of samples stored for as long as 2 weeks and analyzed all on 1 day.
Effects of DFMO-induced polyamine depletion on human tumor cell sensitivity to antineoplastic DNA-crosslinking drugs.
We investigated the effect of pretreatment with difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, on the cytocidal responses of four human adenocarcinoma cell lines to two alkylating and crosslinking agents: chlorambucil and N,N',N"-triethylenethiophosphoramide (thiotepa). The cell lines studied included HuTu-80 (duodenum), HT-29 (colon), ME-180 (cervix), and A-427 (lung). A 48- to 72-h pretreatment with DFMO reduced intracellular putrescine and spermidine contents to less than 10% and less than 1% of control levels. This treatment also caused a 30%-70% decline in spermine content. Survival of control and DFMO-pretreated cells after treatment with chlorambucil or thiotepa was measured by a plating efficiency assay. For three of the four lines studied, the DFMO-induced partial polyamine depletion significantly protected cells from the lethal effects of chlorambucil. In ME-180 cultures alone, DFMO pretreatment did not alter the cytocidal efficacy of chlorambucil. Addition of exogenous putrescine to cultures of HuTu-80, HT-29, or A-427 24 h after DFMO addition but 24 h before treatment with chlorambucil reversed the polyamine depletion and its protective effects on chlorambucil-induced cell kill. In contrast to the above observations, DFMO and partial polyamine depletion had no effect on cell survival after thiotepa treatment for any of the cell lines investigated.
Altered cell cycle phase distributions in cultured human carcinoma cells partially depleted of polyamines by treatment with difluoromethylornithine.
We have studied the effects of partial polyamine depletion, induced by treatment with alpha-difluoromethylornithine (DFMO) on cell cycle phase distributions in five cultured human carcinoma cell lines. We used flow cytometry of cells stained with chromomycin-A3 and computer analysis to measure phase distributions of treated and control cultures. All five lines respond to 1-5 mM DFMO treatment with a total absence of measurable putrescine, a loss of greater than 90% of spermidine, and a 30-40% decline in spermine by 48 h after DFMO addition. The proliferation of all five lines is inhibited as well. Nonetheless, only four of the cell lines (HuTu-80, HT-29, MCF-7, and A-427) show a marked increase in the G1-phase fraction and decrease in the S-phase fraction as a consequence of DFMO treatment. Small, but significant, decreases in the G2-M populations of these cell lines also occurred after DFMO treatment. Exogenous putrescine (5-50 microM) reversed both the polyamine depletion and the perturbed phase distributions of DFMO-treated cultures but was without effect on phase distributions of cultures not treated with DFMO. The fifth cell line (ME-180) showed no effect of polyamine depletion on cell cycle phase distributions in DFMO-treated cultures and also no effect of exogenous putrescine on phase fractions of either control or DFMO-treated cells. These observations indicate that some human tumor cell lines are dependent upon adequate intracellular polyamine content for maintenance of cell cycle traverse. They also imply that human tumor cell lines are heterogeneous with regard to their cell cycle response to DFMO-induced polyamine deficiency.
Reduced cytocidal efficacy for adriamycin in cultured human carcinoma cells depleted of polyamines by difluoromethylornithine treatment.
We have studied the effect of pretreatment with difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, on the cytocidal responses of four human adenocarcinoma cell lines to Adriamycin (ADR). The cell lines utilized included HuTu-80 (duodenum), HT-29 (colon), ME-180 (cervix), and A-427 (lung). A 48-h DFMO pretreatment reduced putrescine and spermidine content to less than 10 and less than 1% of control levels and decreased spermine to between 70 and 30% of controls. Plating efficiency assays were used to generate ADR dose-response survival curves for DFMO-treated and control cultures. The DFMO pretreatment significantly protected human adenocarcinoma cells from the lethal effects of ADR. Addition of exogenous putrescine to the DFMO-treated cultures 24 h before treatment with ADR restored their cytocidal response to ADR to near control levels. Putrescine had no effect on cell survival in cultures that were not pretreated with DFMO. These observations suggest that DFMO-induced protection from ADR may be a specific consequence of DFMO-induced inhibition of polyamine biosynthesis. Alternatively, since ADR efficacy varies directly with cellular growth rates and DFMO inhibits proliferation, the protection may have resulted from DFMO-induced growth inhibition. Comparison of ADR uptake in DFMO-pretreated and control cells showed that the protection did not result from decreased intracellular accumulation of ADR.
Community hospital CME-Its proper place.
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Making your point: how to make your own projection pointer.
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Community hospital continuing medical education--a missed opportunity.
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Cartoons for cardiology: artistry and articulation.
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Continuing medical education in small community hospitals.
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Anatomy of a biomedical communications department: a major health care/education center requires mass production of high-quality instructional visuals.
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