Protein kinase C zeta isoform is critical for proliferation in human glioblastoma cell lines.

Previous studies have confirmed that proliferation in glioblastoma cell lines can be blocked by non-isoform specific protein kinase C (PKC) inhibitors, e.g calphostin C, staurosporine. However, the exact mechanism of PKC involvement is poorly understood. The aim of this study was to explore the role of specific PKC isoforms in the aberrant growth of glioblastoma. Identification of the isoform(s) critical for proliferation in glioblastoma would present a better target for the design of chemotherapeutic strategies. To this end, we screened expression on PKC isoforms in four human glioblastoma cell lines both when proliferating and in a quiescent state using western assays. PKC isoforms alpha, beta, betaII and zeta were found to be expressed in all cell lines. PKC epsilon was detected in three out of four cell lines and PKC eta was detected in one out of four cell lines. Quiescence of growth resulted in down-regulation of PKC epsilon. We examined the role of these isoforms by studying the effect of PKC isoform-specific inhibitors bisindolylmaleimide-I and Gö6976 on proliferation in a panel of four human glioblastoma cell lines. Inhibition of PKC alpha and epsilon had no effect on proliferation, suggesting that previous studies targeting PKC alpha may not be of therapeutic benefit. More significantly, it was shown that inhibition of PKC zeta blocked proliferation. This suggests that the inhibition of PKC zeta may be an important chemotherapeutic target for arresting growth in glioblastoma.

Tamoxifen radiosensitization in human glioblastoma cell lines.

OBJECT: A combined tamoxifen and radiation therapy is being used in clinical trials to treat glioblastoma multiforme (GBM). The rationale behind this therapy is that tamoxifen is a radiosensitizer. However, the evidence for this is weak. The authors, therefore, examined the effect of combined radiation-tamoxifen therapy in three GBM cell lines of human origin. METHODS: The GBM cell lines were exposed to different concentrations (0.3-5 microg/ml) of tamoxifen and subsequently irradiated at varying doses (0.8-5 Gy). Tumor growth inhibition was measured using a proliferation assay. The interaction of tamoxifen and radiation therapies was quantified using the combination index method, which distinguishes whether a combined antitumor effect is synergistic, additive, or antagonistic. At high doses of tamoxifen or radiation there was significant inhibition of tumor cell proliferation. At low doses of either therapeutic agent, there was little effect. In one cell line, synergism occurred at high doses of tamoxifen and radiation. In the other two cell lines, an additive effect was observed. In only one of the three cell lines was there synergy between tamoxifen and radiation at doses that significantly inhibited tumor proliferation. CONCLUSIONS: Because synergy could not be demonstrated in all three cell lines at active dosages, the clinical combination of tamoxifen and radiation therapies may not be of benefit to all patients.

Adenovirus mediated gene therapy in a glioblastoma vaccine model; specific antitumor immunity and abrogation of immunosuppression.

Clinical trials are being performed using tumor genetically engineered to produce cytokines as a vaccine. The design of such a vaccine may be made more effective by further study using in-vitro as well as in-vivo models. We studied an in-vitro tumor 'vaccine' model in glioblastoma. We have demonstrated high efficiency transfection of the Interleukin-2 (IL-2) gene into glioblastoma cell lines using adenoviral vectors. Glioblastoma cell lines transduced with this vector could produce high levels of IL-2 for up to 2 weeks, long enough to elicit an antitumor immune response. We studied tumor/effector cell interactions using cytotoxicity assays coupled with flow cytometric analysis. Activation of CD8+ and expansion of CD3+/CD16+ effector cell subpopulations were observed, suggesting the generation of a specific anti-tumor response and the potential for systemic immunity. We demonstrated that glioblastoma produce immunosuppressive factors which reduce the antitumor response by peripheral blood effector cells. These immunosuppressive factors could be neutralized to improve antitumor response. A better understanding of tumor/effector cell interactions may improve the design of gene therapy trials.