Protein kinase C␦ (PKC␦) is involved in the apoptosis of various cells in response to diverse stimuli. In this study, we characterized the role of PKC␦ in the apoptosis of C6 glioma cells in response to etoposide. We found that etoposide induced apoptosis in the C6 cells within 24 to 48 h and arrested the cells in the G 1 /S phase of the cell cycle. Overexpression of PKC␦ increased the apoptotic effect induced by etoposide, whereas the PKC␦ selective inhibitor rottlerin and the PKC␦ dominant-negative mutant K376R reduced this effect compared to control cells. Etoposide-induced tyrosine phosphorylation of PKC␦ and its translocation to the nucleus within 3 h was followed by caspase-dependent cleavage of the enzyme. Using PKC chimeras, we found that both the regulatory and catalytic domains of PKC␦ were necessary for its apoptotic effect. The role of tyrosine phosphorylation of PKC␦ in the effects of etoposide was examined using cells overexpressing a PKC␦ mutant in which five tyrosine residues were mutated to phenylalanine (PKC␦5). These cells exhibited decreased apoptosis in response to etoposide compared to cells overexpressing PKC␦. Likewise, activation of caspase 3 and the cleavage of the PKC␦5 mutant were significantly lower in cells overexpressing PKC␦5. Using mutants of PKC␦ altered at individual tyrosine residues, we identified tyrosine 64 and tyrosine 187 as important phosphorylation sites in the apoptotic effect induced by etoposide. Our results suggest a role of PKC␦ in the apoptosis induced by etoposide and implicate tyrosine phosphorylation of PKC␦ as an important regulator of this effect.

In this study we examined the cytotoxic effect of ZnO nanoparticles on various human cancer and normal cells. We found that the ZnO nanoparticles exerted a cytotoxic effect on the human glioma cell lines A172, U87, LNZ308, LN18, and LN229, whereas no cytotoxic effect was observed on normal human astrocytes. Similarly, the ZnO nanoparticles induced cell death in breast and prostate cancer cell lines while no major effect was observed in the respective normal breast and prostate cell lines. Using the fl uorescent dye 2,7-dichlorofl uorescein diacetate, we found that treatment of the glioma cells with ZnO nanoparticles induced a large increase in the generation of reactive oxygen species (ROS) and treatment of the cells with N-acetyl cysteine decreased the cytotoxic effect of the ZnO nanoparticles. In contrast, a smaller effect on ROS generation was observed in the normal astrocytes. These results suggest that ZnO nanoparticles may be employed as a selective cytotoxic agent for the eradication of cancer cells.

Protein kinase C␦ (PKC␦) inhibits proliferation and decreases expression of the differentiation marker glutamine synthetase (GS) in C6 glioma cells. Here, we report that distinct, specific tyrosine residues on PKC␦ are involved in these two responses. Transfection of cells with PKC␦ mutated at tyrosine 155 to phenylalanine caused enhanced proliferation in response to 12-phorbol 12-myristate 13-acetate, whereas GS expression resembled that for the PKC␦ wild-type transfectant. Conversely, transfection with PKC␦ mutated at tyrosine 187 to phenylalanine resulted in increased expression of GS, whereas the rate of proliferation resembled that of the PKC␦ wild-type transfectant. The tyrosine phosphorylation of PKC␦ and the decrease in GS expression induced by platelet-derived growth factor (PDGF) were abolished by the Src kinase inhibitors PP1 and PP2. In response to PDGF, Fyn associated with PKC␦ via tyrosine 187. Finally, overexpression of dominant negative Fyn abrogated the decrease in GS expression and reduced the tyrosine phosphorylation of PKC␦ induced by PDGF. We conclude that the tyrosine phosphorylation of PKC␦ and its association with tyrosine kinases may be an important point of divergence in PKC signaling.

We characterized the expression and function of the endoplasmic reticulum protein GRP78 in glial tumors. GRP78 is highly expressed in glioblastomas but not in oligodendrogliomas, and its expression is inversely correlated with median patient survival. Overexpression of GRP78 in glioma cells decreases caspase 7 activation and renders the cells resistant to etoposide- and cisplatin-induced apoptosis, whereas silencing of GRP78 decreases cell growth and sensitizes glioma cells to etoposide, cisplatin, and gamma-radiation. Thus, GRP78 contributes to the increased apoptosis resistance and growth of glioma cells and may provide a target for enhancing the therapeutic responsiveness of these tumors.

In this study, we examined the role of protein kinase C (PKC)-E in the apoptosis and survival of glioma cells using tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-stimulated cells and silencing of PKCE expression. Treatment of glioma cells with TRAIL induced activation, caspasedependent cleavage, and down-regulation of PKCE within 3 to 5 hours of treatment. Overexpression of PKCE inhibited the apoptosis induced by TRAIL, acting downstream of caspase 8 and upstream of Bid cleavage and cytochrome c release from the mitochondria. A caspase-resistant PKCE mutant (D383A) was more protective than PKCE, suggesting that both the cleavage of PKCE and its down-regulation contributed to the apoptotic effect of TRAIL. To further study the role of PKCE in glioma cell apoptosis, we employed short interfering RNAs directed against the mRNA of PKCE and found that silencing of PKCE expression induced apoptosis of various glioma cell lines and primary glioma cultures. To delineate the molecular mechanisms involved in the apoptosis induced by silencing of PKCE, we examined the expression and phosphorylation of various apoptosis-related proteins. We found that knockdown of PKCE did not affect the expression of Bcl2 and Bax or the phosphorylation and expression of Erk1/2, c-Jun-NH 2 -kinase, p38, or STAT, whereas it selectively reduced the expression of AKT. Similarly, TRAIL reduced the expression of AKT in glioma cells and this decrease was abolished in cells overexpressing PKCE. Our results suggest that the cleavage of PKCE and its down-regulation play important roles in the apoptotic effect of TRAIL. Moreover, PKCE regulates AKT expression and is essential for the survival of glioma cells. (Cancer Res 2005; 65(16): 7301-9)

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A critical problem in the development and implementation of stem cell-based therapy is the lack of reliable, noninvasive means to image and trace the cells post-transplantation and evaluate their biodistribution, final fate, and functionality. In this study, we developed a gold nanoparticle-based CT imaging technique for longitudinal mesenchymal stem cell (MSC) tracking within the brain. We applied this technique for noninvasive monitoring of MSCs transplanted in a rat model for depression. Our research reveals that cell therapy is a potential approach for treating neuropsychiatric disorders. Our results, which demonstrate that cell migration could be detected as early as 24 h and up to one month post-transplantation, revealed that MSCs specifically navigated and homed to distinct depression-related brain regions. We further developed a noninvasive quantitative CT ruler, which can be used to determine the number of cells residing in a specific brain region, without tissue destruction or animal scarification. This technique may have a transformative effect on cellular therapy, both for basic research and clinical applications.

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