Biomedical subjects

L A Karolak

Publications and source records attributed to L A Karolak.

3 recordsLinked to original sources

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↗

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↗

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↗