Protein kinase C inhibitors arrest the C6 glioma cell cycle at a mid-G1 phase restriction point: Implications for the antiproliferative action of valproate

Abstract

Abstract-The teratogenic mechanism(s) of valproate (VPA) have been suggested to anse through inhibition of proliferation coupled with differentiation at a mid-G I phase restriction point in the cell cycle. As protein kinase C (PKC) plays a pivotal role in cell proliferation and differentiation, the effect of inhibitors specific for the catalytic and regulatory domains on transit through the G I phase of the cell cycle was determined. Calphostin C and bisindolylmaleimide GF 109203X produced a dose-dependent decrease in proliferation of C6 glioma with approximate 50% inhibitory concentration values of 10 nM and 111M, respectively. Flow cytometric analysis indicated proliferative arrest to be in the G I phase with the expected concomitant decrease of cells in the G2/M and S phases. Following release from drug-induced proliferative arrest, cells exhibited a synchronous entry into S phase as evidenced by an increase in [3H)thymidine incorporation after approximately 6-8 hr, indicating the restriction point to be in the mid-GI phase. Using mitotically synchronized cells continuously exposed to valproate (2 mM), PKC activity was found to be significantly reduced in the mid-G I phase (5.5 hr) but not at an earlier (2.5 hr) time point, implying VPA to exert its effect at a site upstream to the point of proliferative arrest at 5-6 hr into the G I phase which as yet, remains to be defined. er) 1998 Elsevier Science Ltd Abbreviations: ANOVA = analysis of variance; DMEM = Dulbecco's modified Eagle's medium; DMSO = dimethyl sulfoxide; IC so = concentration at which 50% inhibition is achieved; PBS = phosphate buffered saline; PKC = protein kinase C; PLC = inositol phospholipid-specific phospholipase C; VPA = valproic acid.

Antiproliferative action of valproate is associated with aberrant expression and nuclear translocation of cyclin D3 during the C6 glioma G1 phase

“…Significant levels of cyclin D3 were detected in the mid‐G1 phase in VPA‐exposed cells, whereas normally the expression of this protein is very low or undetectable at this time (Figs 3a and 1c). Furthermore, the most prominent increase in cyclin D3 expression occurred at 5–6 h after mitotic selection, precisely coinciding with the known restriction point of VPA in the G1 phase (Martin and Regan 1991; O'Brien and Regan 1998). In repeated experiments VPA did not reproducibly affect PCNA, cyclin D1, cdk4 or kip1/p27 expression in the mid‐G1 phase (Figs 3b–e).…”

“…The antiproliferative action of VPA is known to occur at a defined restriction point in the mid-G1 phase (5-6 h in C6 glioma) and similar IC 50 values are observed over a wide variety of cell types, including fibroblasts, glia and neuroblastoma (Regan 1985;Regan 1988, 1991;Regan et al 1990;Courage-Maguire et al 1997;O'Brien and Regan 1998;Skladchikova et al 1998;Walmod et al 1998). This VPA restriction point is distinguished from those induced by either serum or growth factor deprivation, which occur later in the G1 phase (Pardee 1989).…”

“…This VPA restriction point is distinguished from those induced by either serum or growth factor deprivation, which occur later in the G1 phase (Pardee 1989). VPA-arrested cells assume a differentiated phenotype, as evidenced by changes in cell-substratum adhesivity, morphology, motility and gene expression Regan 1988, 1991;Berezin et al 1996;O'Brien and Regan 1998;Walmod et al 1998). Moreover, the antiproliferative action of VPA is reversible upon drug washout at doses equivalent to the therapeutic range Regan 1988, 1991).…”

Antiproliferative actions of inhalational anesthetics: comparisons to the valproate teratogen

“…Although classical binding sites are unknown for VPA and inhalational anesthetics [11,15], they share many common molecular actions. Both are analogous in that they exhibit stereoisomerism, an antiproliferative action in the G1 phase of the cell cycle, and indirectly inhibit the regulatory domain of protein kinase C which plays a pivotal role in the regulation of cell proliferation and dierentiation and in malformations of development [1,7,12,18,21,22]. To explore this commonality further, in respect of the relative potential of iso¯urane, en¯urane and sevo¯urane to induce developmental malformations, we compared their antiproliferative actions to those of valproate, a known human teratogen.…”

Differential enantioselective effects of pentyl‐4‐yn‐valproate on spatial learning in the rat, and neurite outgrowth and cyclin D3 expression in vitro

“…Embryos of mice exposed to high doses of VPA reproducibly show exencephaly -a neural tube defect closely related to the neural tube malformations seen in human embryos exposed to VPA in utero (Kao et al 1981;Bjerkedal et al 1982;Robert and Guibaud 1982;Nau 1986a,b;Turner et al 1990). Although the molecular mechanisms underlying this in vivo teratogenicity are unknown, it is clear that they directly relate to the antiproliferative/pro-differentiative potency of VPA, which can be detected in vitro in various cell types (Regan 1985;Martin and Regan 1991;O'Brien and Regan 1998). This antiproliferative action occurs at a defined restriction point in the G1 phase and appears to be mediated by the alteration of temporal signal transduction events that are required for passage through the G1 phase of the cell cycle, including glycoprotein sialylation and cyclin D3 expression (Martin and Regan 1991;Bacon et al 1997Bacon et al , 2002.…”