Mechanistic studies on ganciclovir cytotoxicity in cultured glioma cells that express the herpes simplex virus thymidine kinase gene.

Transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene to cancer cells followed by ganciclovir (GCV) treatment has resulted in dramatic tumor regressions in animals, prompting clinical trials of HSV-TK/GCV gene therapy. Cells expressing HSV-TK can phosphorylate GCV to GCV triphosphate (TP) which interferes with cellular DNA synthesis. Initial studies showed that GCV was substantially more cytotoxic (IC\sb{99} = 0.3-0.5 $\mu$M) than related analogs, thymine arabinoside (araT) and acyclovir (ACV) (IC\sb{99}'s $>$1,000 $\mu$M) in C6 rat and U251 human glioma cells that express HSV-TK (C6BSTK & U251tk). To understand the basis for the unique cytotoxicity of GCV, this dissertation compared the metabolism and DNA effects of GCV, ACV, and araT in U251tk and C6BSTK cells. The superior cytotoxicity of GCV was not due to better metabolism compared to araT since CCVTP levels were 18-fold lower than araTTP levels (67 vs. 1235 pmols/10\sp7 cells) at equitoxic conc., the half-life of GCVTP was shorter than araTTP (11 vs. 41 hr), and less GCV incorporated into DNA. Also, both drugs incorporated into DNA primarily at internal positions. Interestingly, GCV weakly inhibited DNA synthesis compared to araT, and consequently cells doubled after a 24 hr exposure to 1 $\mu$M GCV (IC\sb{99.9}), while 100 $\mu$M araT (IC\sb{90}) inhibited growth for 3 days. After completing one cycle of DNA replication, CCV treated cells accumulated in early S-phase and remained there until cell death, indicating that GCV incorporation in the DNA template was important for cytotoxicity. One reason for the success of HSV-TK/GCV therapy is that limited expression of HSV-TK in a tumor is sufficient to kill the entire population with GCV, most likely due to transfer of GCV nucleotides from HSV-TK positive cells to HSV-TK negative bystander cells. Using a technique to identify GCVTP in bystander cells, it was observed that GCVTP levels increased with GCV conc. and exposure to U251tk cells, corresponding to bystander killing. Impressively, in 1:1 mixtures, 24 hr GCV (1-10 $\mu$M) treatments resulted in similar levels of GCVTP (150-1800 pmol/10\sp7 cells) in HSV-TK positive and negative cells. In summary, these studies provide insight on the cytotoxic mechanism of GCV in 100% HSV-TK positive cells and in co-cultures of cells that lack and express HSV-TK. This information may contribute to the design of protocols which enhance GCVTP anti-tumor activity.Ph.D.BiochemistryBiological SciencesCellular biologyHealth and Environmental SciencesOncologyPharmacologyPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/130812/2/9811172.pd