Abstract
F9 teratocarcinoma is a useful model for studying early embryogenesis since these cells can differentiate into primitive or parietal endoderm under the influence of retinoic acid or retinoic acid and cyclic AMP, respectively. We have found that three isoforms of protein kinase C (PKC alpha, -beta, and -gamma) were expressed in undifferentiated stem cells. When the cells were treated with retinoic acid either alone or in the presence of cAMP for 120 h, PKC alpha mRNA and protein levels increased, whereas those of PKC beta and PKC gamma became undetectable. These changes began within 24 h of drug treatment and were complete by 48-72 h. In order to determine the functional significance of the induction of PKC alpha during F9 differentiation, we established two stable transfectants that overexpressed PKC alpha protein between 4- and 5-fold compared to wild type cells. Characterization of these cell lines revealed an altered pattern of expression of some of the markers of F9 differentiation. The clone that had the highest amount of PKC alpha protein constitutively expressed mRNA for type IV collagen and c-Jun, which are not normally expressed until 24-48 h of treatment with differentiation agents. In the other overexpressing clone, these markers were induced much faster than in wild type cells. The growth rate of both overexpressing clones was less than wild type cells, while the expression of the PKC beta protein in these clones was similar to the levels found in differentiated F9 cells. However, other markers of differentiation, including the cellular morphology and levels of pST6-135 and c-myc RNA, responded to agents identically in both wild type and PKC-alpha-overexpressing clones. Therefore, overexpression of PKC alpha is not sufficient to induce full differentiation of F9 cells. However, our data suggest that certain pathways that lead to the expression of differentiation-dependent genes are regulated by PKC alpha protein levels.
Highlights
From the $Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118 and the §Department of Molecular Biology, Yokohama Citv Uniuersity School of Medicine, 3-9,Fuku-ura, Kamazawa-Ku
F9 teratocarcinoma is a usefulmodel for studying lowing the expression of several specific markers
When the cellswere treated with dependent enzyme, whichis activated by diacylglycerol and is retinoic acid either alone or in the presence of CAMPfor believed to be involvedin regulating a wide variety of cellular
Summary
Proteins isolated from the clones YK504-1 and YK504-25 aand We have shown that PKCp is down-regulated during Fdi9fclone selected for G418 resistance from F9 cells transfected ferentiation (Figs.1and 2).We examined the expresonly with pSVneo. Clone YK504-1 expressed PKCa at a level sion of the PKCp protein iwn ild type, neomycin-resistant, and. 4.4-fold higher than that of the neomycin-resistant clone as bothPKCm-overexpressingcloneswasdecreased by 82-909'0 determined by scanning laser densitometry.RA treatment did compared to either untreated neomycin-resistant or wild type not increase PKCa levels in YK504-25 cells but did increase cells (Fig. 6). The control clone had almost type IV collagen [2] occur somewhat later in the differentiation a 10-fold increase in the levelof PKCa. pathway and require new protein synthesis for induction of Characterimtion of PKCa-overexpressing Cell Lines-Both their mRNAs. Q p e IV collagen mRNA was induced by about
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