Modulation by staurosporine of phorbol‐ester‐induced effects on growth and protein kinase C localization in a549 human lung‐carcinoma cells

Abstract

12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin 1 are activators of protein kinase C (PKC). TPA is a potent inhibitor of the growth of A549 cells, while bryostatin 1 exerts a weak antiproliferative effect upon this cell line. We tested the hypothesis that the PKC inhibitor staurosporine (STAU) can interfere with the effects of TPA or bryostatin 1 on A549 cells. STAU alone arrested A549 cell growth effectively with an IC50 of 0.65 nM as determined by cell counting after incubation for 96 hr. It also caused the release of lactate dehydrogenase from cells with an IC50 of 18.4 nM. On incubation with cells for up to 8 hr, STAU (100 nM) alone did not reduce thymidine incorporation into cells. However, it partially abrogated the inhibition of DNA synthesis caused by TPA or bryostatin 1 (10 nM). The IC50 for inhibition by STAU of the activity of PKC purified from A549 cells was 6.1 nM. Localization and levels of PKC were studied by Western blot and phorbol ester receptor binding analyses. STAU (100 nM) did not prevent the TPA-induced rapid redistribution of PKC to the cell membrane, but instead increased it by 25%. The PKC downregulation caused by TPA was not reduced in the presence of STAU. The results suggest that (i) PKC activation is involved in growth inhibition caused by TPA or bryostatin 1 in A549 cells, and (ii) subcellular localization or levels of PKC can be pharmacologically manipulated even under conditions of inhibited kinase function.

Comparison of ability of protein kinase C inhibitors to arrest cell growth and to alter cellular protein kinase C localisation

“…Our previous studies have shown that these three PKC isoenzymes are abundantly expressed in A549 cells (Stanwell et al, 1994). Consistent with its ability to attenuate the TPA-mediated change in distribution of phorbol ester binding sites (Bradshaw et al, 1992), staurosporine increased TPA-induced translocation of cPKC-c. Although the effect was not significant, as deduced from the densitometry readings, a definite trend was observed (P = 0.08) (Figure 4).…”

“…It inhibits PKC enzyme activity potently, but also causes the subcellular translocation of nPKCs (Kiley et al, 1992) and augments TPA-induced redistribution of phorbol ester binding sites (Bradshaw et al, 1992 One of the aims of this study was to explore any relationship between structure and activity among staurosporine and four of its analogues with respect to their effects on cell growth and on PKC. In both cell types the order of growthinhibitory potency was staurosporine > UCN-01 > CGP 41251 >RO 31 8220 >GF 109203X.…”

“…Activator-induced translocation of PKC is thought to position the enzyme in close proximity to its physiologically important-substrates. For example, in human A549 lung and MCF-7 breast carcinoma cells the tumour promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA) and the experimental anti-tumour agent bryostatin 1 provoke the rapid redistribution of PKC from the cytosol to the particulate and nuclear fractions (Issandou et al, 1988; (Bradshaw et al, 1992). In view of these findings we wished to elucidate whether the effect on the subcellular localisation of PKC is a general feature of PKC inhibitors or a specific property of staurosporine.…”

Expression of the antiapoptotic MCL1 gene product is regulated by a mitogen activated protein kinase-mediated pathway triggered through microtubule disruption and protein kinase C

“…The only di erence observed was that the e ects of TPA on MCL1 expression, as compared to those of the microtubule-disrupting agents, exhibited di erential sensitivity to the inhibitor: a concentration of 500 nM calphostin C was required to completely inhibit the increase in MCL1 seen with TPA (a 250 nM concentration producing partial inhibition; data to be published elsewhere), while a concentration of 100 nM calphostin C completely inhibited the increase in MCL1 seen with the microtubule-disrupting agents. Downregulation of PKC by preincubation with TPA (17610 710 M for 24 h; Basu et al, 1992;Bradshaw et al, 1992;Rinaudo et al, 1995) completely inhibited the increase in MCL1 produced by either TPA or microtubule-disrupting agents (data not shown). In sum, PKC appeared to participate in the increase in MCL1 produced by both types of inducing agents.…”

New Aspects of Regulatory Signaling Pathways and Novel Therapies in Pancreatic Cancer

“…The tetradecanylphorbol acetate (TPA) is a PKC activator with a variety of effects in different cells, including increased proliferation in fibroblasts, epidermal cells and several cancer cell lines [167]. In contrast, TPA causes growth arrest of other cancer cell lines [168], it potently inhibits proliferation of human pancreatic cancer cell lines [169]. Cyclins A, B, E and CDC2 seem to contribute to TPA-induced growth inhibition and G 2 /M arrest in pancreatic cancer cells; cyclins A and B are likely involved in TPA-induced G 2 /M arrest because both proteins are involved in S phase and G 2 /M transition during cell proliferation [170].…”