Scientific Officers: N.V. Shirsat, Ph.D. (Head), N.N. Hasgekar, Ph.D., J.J. Kayal, Ph.D.

Research Fellows :M.S. Moodbidri, M.N. Harmalkar, A.M. Gokhale, S.M. Sanghavi.

Malignant tumours of the central nervous system are a major cause of deaths resulting from cancer in children, and also in adults. Astrocytomas account for ~80% of all primary malignant brain tumours. Cancer being the result of deregulated proliferation and suppression of cell death mechanisms, we have been studying the molecular mechanisms underlying these phenomenon of astrocytes.

Studies on the mechanism of proliferation and differentiation of astrocytes (Lady Tata Memorial Trust)

Studies in the laboratory have shown that hexamethylene bisacetamide (HMBA) inhibits proliferation of C6 glioma cells, and the expression of the immediate early gene fra-1 is downregulated during HMBA-induced growth inhibition. Fra-1 over-expression, however, is found to inhibit proliferation and induce morphological differentiation of C6 glioma cells. Furthermore, over-expressed fra-1 enhances growth inhibitory effect of all the three differentiation inducers studied, viz. dbcAMP, staurosporine and HMBA. Fra-1 is a component of the AP-1 transcription factor. Fra-1 itself lacks C-terminal transactivation domain and has been shown to inhibit fos and jun-dependent transactivation. Effect of fra-1 on glioma cell proliferation is consistent with its proposed role as a negative regulator of AP-1 transcription activity. In addition to the growth inhibitory effect, fra-1 overexpression brings about spontaneous apoptosis by itself and complete apoptosis on treatment with dbcAMP or staurosporine. Apoptosis induction on Fra-1 over-expression has been documented so far for retinal photoreceptor cells. In retinal cells, fra-1 can substitute for fos in bringing about cell death. In C6 glioma cells, on the other hand, gel shift studies indicate that fos-jun heterodimer supports survival and proliferation, while fra-1-jun heterodimer brings about growth inhibition and apoptosis. The mechanism underlying fra-1-induced apoptosis is now being investigated. Fra-1 overexpression also results in drastic reduction in anchorage-independent growth and tumorigenicity of C6 cells. The effect of fra-1 gene expression on other astrocytoma cell lines is therefore being studied.

Studies on the mechanism of apoptosis of malignant astrocytes (Indian Council of Medical Research)

Tamoxifen is known to induce apoptosis specifically of only malignant astrocytes. This effect of tamoxifen is supposedly brought about by inhibition of protein kinase C, an effect distinct from its anti-oestrogenic action. In clinical trials, long term treatment with tamoxifen has been found to be effective in some cases of glioma. The mechanism of tamoxifen-induced apoptosis is therefore being investigated using C6 glioma cells stably transfected with a eukaryotic expression vector expressing bcl-2 under metallothionein promoter. Low bcl-2 expression offers protection against tamoxifen-induced apoptosis. Higher bcl-2 expression, on the other hand, accelerates apoptosis. Either low or high bcl-2 levels do not offer significant protection against radiation or staurosporine-induced apoptosis. Levels of bcl-2 and bcl-XL fall while those of proapoptotic bcl-Xs increase during tamoxifen-induced apoptosis. Among the IAP (inhibitor of apoptosis) family genes, XIAP levels decrease during tamoxifen-induced apoptosis of C6 glioma cells. Stress-activated protein kinases, viz. JNK1 and p38 kinase are found to be activated during tamoxifen-induced accelerated apoptosis of bcl-2 overexpressing clones. Inhibition of the activity of JNK1, but not p38 kinase, offers protection against the accelerated apoptosis. Phosphorylation of c-jun by JNK1 leading to increased AP-1 activity appears to be responsible for the accelerated cell death as dominant negative c-jun prevents cell death. The possibility of tamoxifen increasing radiation sensitivity by activating stress-activated protein kinases is being investigated. Fresh glioma tumour cells are being cultured. Radiation sensitivity and tamoxifen sensitivity of these cells is being determined by MTT assay. Marked heterogeneity in radiosensitivity and tamoxifen sensitivity has been observed in tumour specimens analysed so far. Expression of bcl-2 family members in glioma cells is being studied by RT-PCR analysis and correlated with in vitro radiation and tamoxifen sensitivity and clinical outcome.

Studies on teratocarcinoma-derived neural precursor cell lines

Two neural precursor cell lines (NT and JT) have been established and characterised from neurogenic transplantable teratocarcinomas developed in Swiss and C3H (Jax) mice respectively. These cell lines express Nestin, a neuroepithelial stem cell marker. NT cell line also expresses GFAP, a glial cell specific marker and NFP, a neuronal lineage marker. This bipotential cell line is an experimental equivalent to human medulloblastoma. Protein kinase C inhibitors have a remarkable effect on the proliferation of NT cells, while JT cells are resistant. NT cells die within 24 hrs of treatment with 10 nM staurosporine. The mechanism underlying staurosporine-induced apoptosis of NT cells is currently being investigated.

Role of Wnt and hedgehog genes in pathogenesis of medulloblastoma

Medulloblastoma is a highly malignant tumour of the cerebellum, and the most common childhood brain tumour. Recent studies show involvement of genes belonging to hedgehog and Wnt signaling cascades in pathogenesis of medulloblastoma. The aim of this project is to understand the mechanism of tumorigenesis in medulloblastoma by studying the expression of Wnt and hedgehog signaling cascade genes along with cell cycle regulators. About 10% of the sporadic medulloblastoma specimens studied so far show nuclear expression of Gli-1 and b-catenin indicating deregulated hedgehog and Wnt pathway respectively. Nuclear expression of both Gli-1 and b-catenin in most of the specimens is consistent with the known cross talk between these two pathways. Cyclin D1 - a gene targeted by the Wnt pathway appears to be expressed at a higher level in normal cells of the developing cerebellum rather than in tumour cells.