Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-delta subspecies.

Abstract

Several lines of CHO cells stably overexpressing protein kinase C (PKC) subspecies to various extents were established by the DNA-mediated transfer. Upon treatment with phorbol 12-myristate 13-acetate, the growth of the cells expressing the PKC-6 subspecies was markedl inhibited, whereas cell lines expressing PKC-a, PKC-IH, and PKC-C subspecies were not sign tly affected. Flow cytometric analysis indicated that all cell les overexpressing PKC-8 subspecies accumulated in G2/M phase in response to phorbol 12-myristate 13-acetate. In these arrete cells, dikaryons were predominant, implying that phorbol ester-induced inhibition of cell division is specific to telophase. These results suggest PKC-6 subspecies may play a role in the normal cell cycle progression.Protein kinase C (PKC) appears to play crucial roles in signal transduction leading to cell growth and differentiation. This enzyme was first shown to be activated by diacylglycerol generated by the receptor-mediated hydrolysis of inositol phospholipids and has been identified as a major receptor of tumor-promoting phorbol esters (1). Molecular cloning and biochemical studies have revealed that the PKC family is a large family, consisting ofat least eight subspecies, a, 3PI, /11, y, 8, E, A, and q (PKC-L) in mammalian'tissues (2-5). These subspecies show subtly different enzymological properties and distinct tissue distribution. Molecular genetic approaches have also been used to explore the role of this PKC family in cell proliferation by expressing defined enzyme subspecies, such as PKC-a, PKC-P, or PKC-y, in mammalian cells (6-11). In some cell lines stably overproducing PKC subspecies, growth promotion such as enhanced growth rates, increased saturation densities, and anchorageindependent growth has been observed (6,7,9,10 MATERIALS AND METHODS Construction of Expression Plasmids. cDNAs for rat PKC-a, -/311, -8, and -C subspecies were excised from plasmids pTB755, pTB708, pTB808, and pTB949, respectively (14-17), by complete or partial digestion with EcoP. These cDNAs were then separately inserted downstream of the Abelson murine leukemia virus long terminal repeat in plasmid pTB399 (18) by replacing the interleukin 2 sequence. The long terminal repeat-PKC cDNA segments between Sal I and Cla I sites ofthese plasmids were transferred to the same sites of plasmid pTB348 (18), which carried the hamster dihydrofolate reductase (DHFR) cDNA under the control of the simian virus 40 early promoter. The resulting expression plasmids for the a, . 3II,8, and ; subspecies are referred to as pTB789, pTB705, pTB1322, and pTB1324, respectively. All the procedures were done as described (19).Transfection, Selection, and Gene Amplification. DHFR-CHO cells (20) were transfected with each of the expression constructs by the calcium phosphate-DNA coprecipitation method (21). DHFR+ transformants were selected by colony formation in Dulbecco's modified Eagle's medium (DMEM) containing 10%6 (vol/vol) dialyzed fetal bovine serum and proline (35 ,Ag/ml). Gene amplificatio...

Protein expression of the α, γ, δ and ε subspecies of protein kinase C changes as C6 glioma cells become contact inhibited and quiescent in the presence of serum

“…Involvement of PKC in the regulation of cell growth is well established [19,20] and in C6 cells PKC activity changes with different stages of the cell cycle being highest in Go [21]. However, the role of individual PKC subspecies in the regulation of cell growth is poorly understood [6][7][8] though PKC ~ is a RAF-1 kinase [4] which provides one route to activation of certain early response genes. Here we show that PKC activity and protein expression of the ~ and ~ subspecies of PKC increases as C6 cells become growth-arrested by contact inhibition in the presence of serum while protein expression of the y and e subspecies decreases, these subspecies being barely detected in contact-inhibited quiescent C6 cells.…”

“…The c and n PKC subspecies are activated by phorbol esters [1,2]. Some PKC subspecies such as ~, 6 and ~" are widely expressed whereas the 7, r/and 0 subspecies have a restricted distribution [1,2,5]. The role of individual PKC subspecies in growth regulation is not well defined though PKC a activates Raf-1 by direct *Corresponding author.…”

“…Over-expression of c and n group PKC subspecies in cells causes contrasting effects on growth rate and cell morphology (summarised in [6,7]) while the downregulation-sensitive PKC subspecies (cz,/3, 6, e) regulate cyclin-dependent kinases in endothelial cells [8].…”

Increased protein kinase Cδ in mammary tumor cells: relationship to transformation and metastatic progression

“…Overexpression, knockout and inhibitor studies have all been used to evaluate the roles of individual PKCs in cell growth and to demonstrate that isozyme functions are unique rather than overlapping (reviewed in Jaken, 1997). For example, overexpressing PKCe in normal ®broblasts increased anchorage-dependent and -independent growth, whereas overexpressing PKCa and d inhibited growth (Cacace et al, 1993;Mischak et al, 1993;Watanabe et al, 1992;Yamaguchi et al, 1995). In contrast, overexpressing PKCa in MCF7 mammary tumor cells increased anchorage-dependent and independent growth (Ways et al, 1995).…”

Identification of the primary growth response gene, ST2/T1, as a gene whose expression is differentially regulated by different protein kinase C isozymes

“…For example, PKC-c~ and -~, but not PKC-]3II, -e, -t/or -5, mediate differentiation of myeloid 32D cells to mature macrophages [23]. What is more, overexpression of PKC-e in NIH/3T3 cells leads to oncogenic transformation [24,25], whereas overexpression of PKC-J inhibits proliferation in different cell systems [24,26]. The highly diverse biological actions of PKC clearly call for a determination of the specific biochemical functions of each PKC isoform.…”