Protein kinase C isozyme expression in phorbol ester-sensitive and -resistant EL4 thymoma cells.

To investigate whether differential protein kinase C isozyme expression in phorbol ester-sensitive and -resistant EL4 thymoma cells could account for the difference in phorbol ester responsiveness, we purified and characterized isozymes from the two cell lines. In both cell types, two peaks of protein kinase C activity were resolved on hydroxylapatite following DEAE-cellulose and phenyl-Superose chromatography. Western blot analysis showed that the first peak corresponded to protein kinase C-beta and the second to protein kinase C-alpha. Two-dimensional phosphotryptic mapping of the purified alpha and beta isozymes did not reveal any reproducible differences between sensitive and resistant EL4 cells. Nor were any differences between the cell types observed in the cytosolic versus membrane localization of alpha and beta protein kinase C. Northern blot analysis showed the expression of mRNA for protein kinase C-alpha, -beta, -delta and -epsilon in both cell lines, and the absence of mRNA for gamma or zeta. Although no major differences in expression of alpha, beta, or delta mRNA between sensitive and resistant EL4 cells were detectable, expression of protein kinase C-epsilon mRNA in resistant cells was only 20-25% of that in sensitive. Western blot analysis with anti-protein kinase C-epsilon antibodies showed the presence of the epsilon-isozyme in sensitive cells and the absence of detectable amounts in resistant cells. Although protein kinase C-epsilon constitutes only a small portion of the total protein kinase C in sensitive cells, the possibility is raised that decreased protein kinase C-epsilon expression may contribute to the failure of resistant EL4 cells to respond to phorbol esters.

Effects of cigarette smoke condensate and 12-O-tetradecanoylphorbol-13-acetate on gap junction structure and function in cultured cells.

The effects of cigarette smoke condensate (CSC) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on gap junction structure, quantity and function were investigated. Gap junction morphology was studied in rotary-shadowed freeze-fracture replicas of primary chick embryo hepatocytes. CSC (24 micrograms/ml) induced a strong decrease of gap junction areas; within 6 h the areas were reduced by greater than 60%. In the first 3 h of exposure, TPA (100 ng/ml) also reduced gap junction areas, but in the next 3 h a partial recovery was observed. Protoplasmic fracture face centre-to-centre particle spacings were used as a measure for gap junction coupling. CSC had a slow (although not significant) reducing effect on particle spacings, while TPA induced a reduction from 10.6 nm (control) to 10.0 nm within 3 h, indicating a reduction of coupling. Gap junctions were quantified in thin sections of cultured chick embryo hepatocytes, V79 fibroblasts, and co-cultivated hepatocytes and V79 cells. CSC did not influence gap junction numbers in any of these cultures, while TPA treatment caused a disappearance of gap junctions between hepatocytes and between hepatocytes and V79 cells in the first 12 h of cultivation. In the following 36 h a slow recovery could be observed. Gap junctions between V79 cells had already disappeared within 30 min. Metabolic co-operation between hepatocytes and hypoxanthine-guanine phosphoribosyltransferase-deficient V79 cells was quickly and continuously blocked by CSC over 27 h, whereas the phorbol ester induced a transient block. The dissimilar effects of these compounds on both gap junction structure and function indicate that they act via different mechanisms. The finding that CSC did not inhibit phorbol ester protein kinase C binding and did not activate this protein kinase in vitro supports this hypothesis.

Differential activation of protein kinase C isozymes by short chain phosphatidylserines and phosphatidylcholines.

To investigate the importance of the physical state of phospholipids for activation of protein kinase C, we have used short chain phospholipids, which, depending on their concentration, can exist as either monomers or micelles. We previously reported that short chain phosphatidylcholines (PC) can activate protein kinase C at concentrations that correlate with the critical micelle concentration of the activating lipid (Walker, J. M., and Sando, J. J. (1988) J. Biol. Chem. 263, 4537-4540). We have now expanded this work to short chain phosphatidylserine (PS) systems in order to examine the role of Ca2(+)-phospholipid interactions in the activation process. Short chain PS were synthesized from corresponding PC and purified by reverse-phase high pressure liquid chromatography. Use of the short chain system has revealed significant differences in the activation of type II and type III protein kinase C isozymes. The type II isozyme required Ca2+ in the presence of long chain PS vesicles; in the presence of the short chain phospholipid micelles (PC or PS), most of the activity was Ca2+ independent. Addition of diacylglycerol caused a small increase in type II activity in all phospholipid systems. In contrast, type III protein kinase C was Ca(+)-dependent in all of the lipid systems. The concentration of Ca2+ required to activate type III protein kinase C was independent of the phospholipid type despite large differences in the ability of these lipids to bind Ca2+. This isozyme required diacylglycerol only in the PC micelle system or with vesicles composed of long chain saturated PS. The presence of short chain PS micelles or long chain PS with unsaturated fatty acyl chains rendered this Ca2(+)-dependent protein kinase C virtually diacylglycerol independent. These results are consistent with a model in which type II protein kinase C requires Ca2+ primarily for membrane association, a requirement which is bypassed with the micelle system, whereas type III protein kinase C has an additional Ca2+ requirement for activity that does not involve Ca2(+)-phospholipid interactions.

Control of lymphokine production by protein kinase C.

As illustrated in Fig. 3, our understanding of lymphokine production is incomplete. Nuclear effects of PKC are probably mediated by phosphorylated substrates. Some of the substrates are themselves being identified as kinases and phosphatases, adding more complexity to the pathway. Use of resistant cell lines is one of the approaches that may help link early phosphorylation events with later transcriptional responses.

Modulating effects of naturally occurring indoles on SCE induction depend largely on the type of mutagen.

The modulating effects of pretreatment of cultured cells with indole-3-carbinol (I3C) and indole-3-acetonitrile (I3A) on the induction of sister-chromatid exchanges (SCEs) by mutagens from different chemical classes were investigated. Cultured primary chick embryo hepatocytes were treated for different periods with I3C (25 micrograms/ml) and with I3A (35 micrograms/ml). Treatment with I3C resulted in a 3-fold increase in ethoxyresorufine-O-deethylase (Erod) activity and a 2-fold increase in ethoxycoumarine-O-deethylase (Etco) activity. Treatment with I3A resulted in a 1.6-fold increase in Erod activity and a 2-fold increase in Etco activity. Pretreatment of cultured primary chick embryo hepatocytes with I3C resulted in a 30-45% decrease in the number of SCEs induced by benzo[a]pyrene (B(a)P) and dimethylnitrosamine (DMN) in co-cultured V79 Chinese hamster cells. No decrease in SCE induction was observed for 2-aminoanthracene (2AA) and the direct-acting alkylating agent ethyl methanesulphonate (EMS). In contrast, when dibromoethane (DBE) was tested pretreatment with I3C resulted in an increase in SCE induction. Pretreatment with I3A again resulted in a 20-40% decrease in SCE induction for B(a)P whereas no decrease was observed for DMN, 2AA and EMS. The results of this study indicate that the type of effect of indole pretreatment largely depends on the type of mutagen selected.