The therapeutic combination of the herpesvirus simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and the prodrug, ganciclovir (GCV), has found great utility for the treatment of many types of cancer. After initial phosphorylation of GCV by HSV-1 TK, cellular kinases generate the toxic GCV-triphosphate metabolite that is incorporated into DNA and eventually leads to tumor cell death. The cellular and pharmacological mechanisms by which metabolites of GCV lead to cell death are still poorly defined. To begin to address these mechanisms, different mutated forms of HSV-1 TK at residue Gln-125 that have distinct substrate properties were expressed in mammalian cell lines. It was found that expression of the Asn-125 HSV-1 TK mutant in two cell lines, NIH3T3 and HCT-116, was equally effective as wild-type HSV-1 TK for metabolism and sensitivity to GCV, bystander effect killing and induction of apoptosis. The major difference between the two enzymes was the lack of deoxypyrimidine metabolism in the Asn-125 TK-expressing cells. In HCT-116 cells expressing the Glu-125 TK mutant, GCV metabolism was greatly attenuated, yet at higher GCV concentrations, cell sensitivity to the drug and bystander effect killing were diminished but still effective. Cell cycle analysis, 4 ,6 -diamidine-2 -phenylindoledihydrochloride staining, and caspase 3 activation assays indicated different cell death responses in the Glu-125 TK-expressing cells as compared with the wild-type HSV-1 TK or Asn-125 TK-expressing cells. A mechanistic hypothesis to explain these results based on the differences in GCV-triphosphate metabolite levels is presented.Delivery and expression of herpesvirus thymidine kinase (HSV-1 TK) 1 in combination with ganciclovir (GCV) has shown great clinical promise as a gene therapy of different cancers (1-4). GCV is a prodrug that must be initially phosphorylated by HSV-1 TK and then cellular kinases to the toxic triphosphate form, GCVTP, that incorporates into cellular DNA and may act as an inhibitor of DNA polymerase ␦ (5-7). The basis for the original clinical trials was the ability of 10% or less HSV-1 TK-expressing cells to mediate a bystander effect whereby non-TK-expressing cells also became sensitive to GCV killing (1,8,9). In vitro, the primary mechanism of the bystander effect has been determined to be the gap junction mediated transfer of GCV metabolites to neighboring non-HSV-1 TK-expressing cells (10 -13). In response to GCV phosphorylation and/or metabolite transfer, most cell types have been reported to undergo apoptosis, which appears to be the cellular mechanism by which both the HSV-1 TK-expressing cells and bystander cells ultimately die (5,11,14). Recently, GCV has been reported to induce S-and G 2 /M phase cell cycle arrest in HSV-1 TK-expressing cells (5,(15)(16)(17), and these changes were associated with modulation of Cdc2/cyclin B activities (16) and increased levels of cyclin B1 (15). In vivo, it is clear that initial HSV-1 TK/GCV tumor cell killing results in a complex inflammatory stimulation of...
Herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) gene therapy can induce apoptosis in tumor cells that are normally resistant to this type of cell death, although the cellular mechanisms by which this occurs remain to be elucidated. Human colon tumor cell lines expressing HSV-TK were treated with GCV or four other inducers of apoptosis: butyrate, camptothecin (CPT), Taxol (paclitaxel), or 7-hydroxystaurosporine (UCN-01). Over a 2-4 day treatment period with GCV or the other four drugs, protein levels of the apoptosis agonist Bak increased 1.5-to 3-fold, whereas a corresponding decrease in the levels of the apoptosis antagonist, Bcl-X L , was observed in butyrate-, CPT-, and 7-hydroxystaurosporine (UCN-01)-treated cells. GCV and paclitaxel treatments resulted in increased levels of Bcl-X L . In two-drug combinations with GCV plus one of the four other drugs, increased tumor cell killing was found with GCV plus UCN-01 or with some GCV/butyrate combinations; the other two tested combinations were largely antagonistic. The GCV/UCN-01 and GCV/butyrate combinations resulted in increased Bak and decreased Bcl-X L protein levels, while the GCV/CPT and GCV/paclitaxel combinations resulted in increased levels of both proteins. The results highlight the potential for new combination therapies of HSV-TK/GCV and chemotherapeutic drugs that result in increased tumor cell apoptosis for future treatments of colon cancer.
We have previously demonstrated that altered exocrine pancreatic stimulus-secretion coupling is associated with ovariectomy and chronic estradiol administration. To elucidate possible mechanisms underlying those effects we examined the ability of chronic administration of different doses of estradiol to regulate the CCK signal transduction pathway in isolated rat pancreatic acini. Doses of estradiol ranging from 0.5 to 119 μg/day were administered to ovariectomized rats for 18 days. Ovariectomy was associated with enhanced CCK-stimulated pancreatic amylase release, whereas estradiol dose dependently decreased the magnitude of CCK-stimulated amylase release. Ovariectomy was also associated with enhanced CCK receptor numbers on acinar cell membranes. Estradiol administration was associated with dose-dependent decreases in CCK receptor numbers. Neither ovariectomy nor estradiol administration affected CCK receptor affinity. Moreover, estradiol administration was associated with increased expression of the α-subunit of the heterotrimeric G protein Gq/11(Gαq/11). Recent findings (H. Ohnishi, S. A. Ernst, D. I. Yule, C. W. Baker, and J. A. Williams. J. Biol. Chem. 272: 16056–16061, 1997) demonstrate that Gαq/11may exert a tonic inhibitory effect on pancreatic enzyme release. In view of these findings, the increased expression of Gαq/11 induced by estradiol likely contributes to the inhibition of pancreatic enzyme release. We conclude that the effect of estradiol to decrease pancreatic secretion is mediated through regulation of CCK receptor density and Gαq/11 expression.
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