PubMed Health⌕ Search

Biomedical subjects

L M Obeid

Publications and source records attributed to L M Obeid.

64 records · Page 4Linked to original sources
PubMed1993

Selectivity of ceramide-mediated biology. Lack of activity of erythro-dihydroceramide.

Ceramide is emerging as a putative second messenger mediating effects of extracellular agents on cell growth and differentiation (Okazaki, T., Bielawska, A., Bell, R. M., and Hannun, Y. (1990) J. Biol. Chem. 265, 15822-15831) and programed cell death (Obeid, L. M., Linardic, C. M., Karolak, L. A., and Hannun, Y. A. (1993) Science 259, 1769-1771). In this study, the eight stereoisomers of C2-ceramide and dihydroceramide were synthesized, and their cellular activity was investigated. The four stereoisomers of C2-ceramide were active in inhibition of cell growth and induction of apoptosis with modest differences in potency. On the other hand, with C2-dihydroceramide only the threo compounds were active in these assays whereas the erythro compounds were totally inactive. Thus, of the two naturally occurring molecules, the analog of D-erythro-ceramide (with the 4-5 trans double bond) was active, whereas the analog of D-erythro-dihydroceramide was inactive. These results demonstrate the specificity of ceramide action and suggest that the introduction of the double bond is critical for imparting the biochemical and biological activity of ceramide.

Apoptosis↗
PubMed1993

Tumor necrosis factor-alpha (TNF-alpha) signal transduction through ceramide. Dissociation of growth inhibitory effects of TNF-alpha from activation of nuclear factor-kappa B.

Tumor necrosis factor-alpha (TNF-alpha) exerts pleiotropic biologic effects. Although TNF-alpha appears to activate a number of signal transduction pathways, the role of second messengers in mediating the different effects of TNF-alpha are not well defined. In this study, we investigated the role of ceramide as an intracellular mediator of TNF-alpha action. In Jurkat T cells, TNF-alpha caused early activation of the sphingomyelin cycle with peak hydrolysis of sphingomyelin observed at 30 min following addition of TNF-alpha. In this cell line, TNF-alpha caused potent activation of nuclear factor-kappa B (NF-kappa B) and exerted potent cytostatic/cytocidal activity. C2-ceramide mimicked the effects of TNF-alpha on cell growth in a dose-dependent manner, but C2-ceramide was unable to induce activation of NF-kappa B under multiple conditions investigated. C2-ceramide, however, enhanced activation of NF-kappa B in response to TNF-alpha with peak effects observed at a concentration of C2-ceramide of 5 microM. Thus, ceramide functions as a selective mediator of the cytostatic/cytotoxic effects of TNF-alpha and plays a positive feedback role in activation of NF-kappa B. TNF-alpha signaling, therefore, involves multiple second-messenger pathways that function independently or coordinately to transduce distinct functions of TNF-alpha.

Base Sequence↗
PubMed1993

Selective regulation of expression of protein kinase C beta isoenzymes occurs via alternative splicing.

The mechanisms involved in regulating the selective expression of protein kinase C (PKC) isoenzymes are poorly understood. Two human B lymphoblastoid cell lines, IM-9 and BJA-B, exhibited differential expression of the two alternatively spliced products of the PKC beta gene, PKC beta I and beta II. The IM-9 cell line expressed 3-4-fold more PKC beta II protein than the BJA-B cell line, whereas the BJA-B cell line expressed 2-3-fold more PKC beta I protein. This differential expression was found to be regulated at the mRNA level. Comparison of PKC beta I and beta II messages in poly(A)+ mRNA and total cellular RNA revealed that selective polyadenylation was not involved. The messages for PKC beta I and beta II had comparable half-lives in both cell lines, ruling out differential message stability. In addition, similar ratios of PKC beta I and beta II messages in cytosolic and nuclear fractions suggested that differential mRNA transport was not involved. In the IM-9 cell line, the predominance of mature PKC beta II message as well as that of a larger message spliced to PKC beta II provided evidence that the differential expression of PKC beta II was regulated at the level of mRNA splicing. In the BJA-B cell line, equal amounts of mature PKC beta I and beta II message and the absence of the larger message suggested that the splicing of the PKC beta gene product can be regulated to produce altered ratios of PKC beta I and beta II. Implications of these studies on the differential expression of PKC isoenzymes and their roles in biology are discussed.

Alternative Splicing↗
PubMed1993

Programmed cell death induced by ceramide.

Sphingomyelin hydrolysis and ceramide generation have been implicated in a signal transduction pathway that mediates the effects of tumor necrosis factor-alpha (TNF-alpha) and other agents on cell growth and differentiation. In many leukemic cells, TNF-alpha causes DNA fragmentation, which leads to programmed cell death (apoptosis). C2-ceramide (0.6 to 5 microM), a synthetic cell-permeable ceramide analog, induced internucleosomal DNA fragmentation, which was inhibited by zinc ion. Other amphiphilic lipids failed to induce apoptosis. The closely related C2-dihydroceramide was also ineffective, which suggests a critical role for the sphingolipid double bond. The effects of C2-ceramide on DNA fragmentation were prevented by the protein kinase C activator phorbol 12-myristate 13-acetate, which suggests the existence of two opposing intracellular pathways in the regulation of apoptosis.

Apoptosis↗
PubMed1993

Selective regulation of expression of protein kinase C (PKC) isoenzymes in multidrug-resistant MCF-7 cells. Functional significance of enhanced expression of PKC alpha.

The multidrug resistance (MDR) phenotype induces cross-resistance to many chemotherapeutic agents in cancer cells. Protein kinase C (PKC) has been implicated in the regulation of the MDR phenotype. In order to determine the role of specific PKC isoenzymes in regulating the MDR phenotype, the expression and activity of PKC isoenzymes in the human breast cancer cell line, MCF-7-WT, and an MDR subline, MCF-7-MDR, were examined. The MDR phenotype was associated with a 10-fold increase in calcium-dependent PKC activity as well as a 10-fold decrease in calcium-independent activity was due to a selective increase in the activity was due to a selective increase in the expression of PKC alpha as determined by Western blot analysis and hydroxylapatite chromatography. This increase in expression of PKC alpha was regulated at the message level as demonstrated by Northern blot analysis. The decrease in calcium-independent activity was caused by a decrease in the expression of PCK delta and epsilon. The significance of the increase in PKC alpha expression was then demonstrated by a commensurate 11-fold increase in the basal and stimulated phosphorylation of the myristolated alanine-rich C kinase substrate. Phosphorylation of P-glycoprotein, the cellular mediator of the MDR phenotype, was increased > 20-fold in the unstimulated MCF-7-MDR cell line and its phosphorylation was further increased 2-fold in response to phorbol 12-myristate 13-acetate. These changes paralleled the increases in P-glycoprotein pump function and the MDR phenotype underscoring the role for PKC alpha in regulating P-glycoprotein phosphorylation and function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters.

The expression of the beta isoenzyme for protein kinase C is regulated developmentally and in response to inducers of cell differentiation (such as phorbol esters and 1 alpha,25-dihydroxyvitamin D3). The 5' segment of the gene for protein kinase C beta was cloned from a human leukocyte genomic library in EMBL3 bacteriophage. This segment of the gene (greater than 54 kilobases in length) encompassed the coding sequence for the amino-terminal regulatory domain of the enzyme, the 5'-untranslated region, and the 5'-flanking region. Initiation of transcription was identified by S1 nuclease analysis and confirmed by RNase protection analysis at 197 base pairs 5' of the initiator ATG. Sequence analysis of the 5'-flanking region revealed it to be extremely G+C-rich (> 80%) with many features of a CpG island. Comparison of sequence with known cis-regulatory motifs disclosed a number of potential regulatory elements including an octamer binding motif at -76, Sp1-binding sites at -94 and -63, E boxes at -110, -26, and +18, an AP-1 site at -442, and an AP-2 site at -330. To demonstrate promoter activity, a 630-base pair fragment extending from -587 to +43 was subcloned in front of a promoterless luciferase gene. This fragment was able to drive the expression of luciferase in transient transfections of human hematopoietic cells. Deletion analysis demonstrated that a fragment -111 to +43 was necessary and sufficient for promoter activity; this fragment did not contain TATA or CAAT motifs. The promoter was stimulated 8-20-fold by phorbol esters accounting for the previously observed transcriptional activation of protein kinase C beta. This phorbol ester responsiveness was conferred by the basal promoter (-111 to +43) and was independent of the AP-1 site. These results define a novel mechanism of protein kinase C autoregulation at a transcriptional level.

Animals↗
PubMed1991

5-HT1A and histamine H1 receptors in HeLa cells stimulate phosphoinositide hydrolysis and phosphate uptake via distinct G protein pools.

Regulation of phosphate uptake was studied in a HeLa cell line after transfection with DNA encoding the human 5-HT1A receptor. In these cells, 5-HT stimulates sodium-dependent phosphate uptake via protein kinase C activation. Endogenous histamine H1 receptors (739 +/- 20 fmol/mg protein) were identified with [3H]pyrilamine. Histamine (i) stimulated phosphoinositide hydrolysis (EC50 = 8.6 +/- 4.1 microM), (ii) activated protein kinase C (2.4-fold increase in activity), and (iii) increased phosphate uptake (EC50 = 3.2 +/- 1.8 microM) by increasing maximal transport (Vmax(basal) = 6.2 +/- 0.3 versus Vmax(histamine) = 9.1 +/- 0.4) without changing the affinity of the transport process for phosphate. Prolonged treatment with 16 microM phorbol 12-myristate 13-acetate completely blocked protein kinase C activation and markedly attenuated the stimulation of phosphate uptake induced by histamine, establishing that 5-HT and histamine stimulate phosphate uptake through the common pathway of protein kinase C activation. The linkages of the histamine H1 and 5-HT1A receptors to G protein pools were assessed in two ways. (i) The stimulation of phosphoinositide hydrolysis, protein kinase C activity, and phosphate uptake associated with histamine were insensitive to pertussis toxin, whereas those associated with 5-HT were very sensitive to pertussis toxin. (ii) The stimulation of phosphoinositide hydrolysis, protein kinase C activity, and phosphate uptake induced by histamine and 5-HT were additive. These findings suggest that distinct receptor types can stimulate phosphoinositide hydrolysis, protein kinase C, and phosphate uptake in an additive fashion through distinct pools of G proteins in a single cell type.

Biological Transport↗
PubMed1990

Transcriptional regulation of protein kinase C by 1,25-dihydroxyvitamin D3 in HL-60 cells.

Vitamin D3 treatment of the human promyelocytic cell line, HL-60, is accompanied by an increase in phorbol ester receptor number (Martell, R. E., Simpson, R. U., and Taylor, J. M. (1987) J. Biol. Chem. 262, 5570-5575). In this study, the mechanism and significance of vitamin D3-induced changes in protein kinase C levels were investigated. Treatment of HL-60 cells with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) resulted in a 2-3-fold increase in phorbol dibutyrate binding at 24 h. This was accompanied by a 4.2-fold increase in steady state mRNA levels for the beta isoenzyme of protein kinase C and by a 3.8-fold increase in its transcriptional rate as determined from nuclear run-off studies. Protein kinase C alpha mRNA, which was approximately 15% of the beta isoenzyme levels, showed similar increases in mRNA and transcriptional rates in response to 1,25-(OH)2D3. Protein kinase C gamma mRNA was not detected. The increases in protein kinase C levels were accompanied by increases in activation of the enzyme by phorbol esters. More importantly, 1,25-(OH)2D3 caused a 1.5-2.0-fold increase in the endogenous phosphorylation of protein kinase C substrates independent of exogenous activators or endogenous formation of diacylglycerol. This is the first report of transcriptional activation of protein kinase C. This mechanism of up-regulation of protein kinase C may explain the increased activity of protein kinase C in vitamin D3-treated HL-60 cells, and may constitute an important mechanism for the long term modulation of the protein kinase C pathway of cell regulation independent of diacylglycerol generation.

Caenorhabditis elegans Proteins↗