Student Research: Hailing Lu

, , 2001
Faculty Advisor: Lucio G. Costa

The Effect of Inorganic Lead on DNA Synthesis in 1321N1 Human Astrocytoma Cells: Roles of Protein Kinase C and Miogen Activated Protein Kinases


Abstract

There is some limited evidence from epidemiological and experimental animal studies that exposure to lead may result in increased risk of gliomas. As lead is known to be a potent activator of protein kinase C (PKC), and gliomas are thought to be highly dependent on PKC for their proliferation, t his student was undertaken to investigate whether lead may act as a mitogen in these cells and the mechanistic pathway involved in this effect of lead. Human 1321N1 astrocytoma cells were exposed to lead acetate (100nM to 100 mM) for up to 24 hour. The treatment resulted in a concentration- and time- dependent increase in DNA synthesis, up to 427 (+ 50)% of control, as measured by incorporation of [methyl-3H]thymidine into cell DNA, without causing any cytotoxicity, as measured by LDH release. Flow cytometric analyses using Hoechst 33258 and bromodeoxyuridine showed that lead was able to stimulate the cell cycle transition from the G0/G1 phase to the S/G2 phase, resulting in increased percentage of cells in the latter phase (34.8% vs. 2.0% in control). Western blot analyses showed that lead induced translocation of PKCa, but not PKCe or PKCz from the cytosolic to the particulate fraction, with a concomitant increase in PKC enzyme activity. Prolonged exposure to lead (100mM, 24h) caused down-regulation of PKCa, but not of PKCe. The effect of lead on DNA synthesis was mediated through PKC as evidenced by the finding that two PKC inhibitors, GF109203X and staurosporine, inhibited lead-induced DNA synthesis, with IC50s of 754 (+120) nM and 0.41 (+0.08) nM, respectively; and that down regulation of PKC through prolonged treatment with 12-0-tetradecanoylphorbol 13-acetate (TPA, 200 ng/ml, 24hr) also blocked the effect of lead on DNA synthesis. Further experiments using a pseudosubstrate peptide targeting classical PKCs, and selective down regulation of specific PKC isofoRMS indicated that the effect of lead on DNA synthesis was mediated by PKCa. Altogether, these results suggest that lead stimulates DNA synthesis in human astrocytoma cells by a mechanism that involves activation of PKCa. Then we investigated if lead may activate the mitogen-activated protein kinase (MAPK) cascade through PKC activation. We found that exposure to lead acetate resulted in concentration- and time-dependent activation of MAPK (ERK1/2), as shown by increased phosphorylation and increased kinase activity. This effect was significantly reduced by PKC-specific inhibitor GF109203X, by down-regulation of PKC with TPA, and by a pseudosubstrate to PKCa. Lead was also able to activate MAPK kinase (MEK1/2), and this effect was mediated by PKC. Two MEK inhibitors, PD98059 and UO126, blocked lead-induced MAPK activation, and inhibited lead-induced DNA synthesis, as measured by incorporation of [methyl-3H]thymidine into cell DNA. The 90 KDa ribosomal S6 protein kinase, p90RSK, a substrate of MAPK, was also activated by lead in a PKC- and MAPK-dependent manner. These results indicate that in human astrocytoma cells, lead activates the MEK-MAPK(ERK1/2)- p90RSK signal transduction pathway sequentially, in a PKCa-dependent manner, leading to an increase in DNA synthesis. These findings may be relevant to the observation that lead may act as a promoter for brain tumors, most notably astrocytomas.