The oncogenesis of human gliomas has been described to occur through the accumulation of genetic damage that eventually leads to the activation of proto-oncogenes and/or the loss of function of tumor suppressor genes. This hypothesis is based on previous studies that have described amplification of epidermal growth factor receptor gene and losses of sequences associated with chromosomes 9, 10, and 17 in gliomas. This project is directed at the localization and identification of a putative glioblastoma-associated tumor suppressor (GTS) gene located on chromosome 10. Several investigations have demonstrated that deletion of chromosome 10 alleles are the most frequent genetic alteration observed in glioblastomas (GBMs). To further examine this observation, we have re- inserted human chromosome 10 into two glioblastoma cell lines by micro-cell mediated chromosomal transfer and have demonstrated a dramatic loss of the tumorigenic phenotype of the hybrid cells. The hybrid cells exhibit a loss of their ability to grow in soft agarose, a decreased saturation density in monolayer, and failure to form tumor in nude animals. The utilization of this functional method demonstrates the presence of a GTS gene on chromosome 10. Our long-term goal to isolate the GTS gene involves the use of two non-mutually exclusive approaches: to fragment chromosome 10, and to re-insert the various defined pieces into GBM cells to regionally locate the GTS gene by assaying for functional tumor suppression. Our efforts will particularly be directed towards the region 10q22 to 10qter, where preliminary evidence suggests the GTS gene may reside. Additionally, subtractive hybridizations will be performed on cDNA expression libraries to identify genes which are differentially expressed as a result of re- insertion of chromosome 10 into GBM cells. The differentially expressed gene products will then be characterized for their gene structure, expression patterns, sequence, and gene structure, in order to eventually examine their functional roles. Furthermore, these two approaches can be combined to identify genes which map to specific chromosomal regions of interest and are differentially expressed. These results should increase our understanding of the specific genetic alterations involved in glial oncogenesis and may then provide us with new diagnostic markers or specific targets for therapeutic intervention in human gliomas.
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