Fig. 3. Metabolism of ganciclovir. Chemical structure of the acyclic nucleoside ganciclovir (GCV), which is monophosphorylated by the enzyme herpes simplex thymidine kinase (HSV-TK). Further phosphorylation by cellular kinases leads to the creation of the toxic metabolite ganciclovir-triphosphate.

Glioma cells transduced and selected to express HSV-TK are 5000-fold more sensitive to GCV than their nontransduced counterparts (37), suggesting that transduction leads to enough HSV-TK expression to phosphorylate enough GCV to inhibit mammalian DNA polymerase. Thus, the poor affinity of mammalian DNA polymerase relative to herpes DNA polymerase for GCV does not impede chemosensitization upon transfection with HSV-TK. Because GCV's effects are limited to DNA, it targets replicating cells, much like the S-phase-specific chemotherapeutic agents.

Efficacy in vivo was demonstrated in a study in which rats harboring intracranial gliosarcoma tumors were treated with intratumoral implantation of a fibroblast packaging cell line secreting HSV-TK-expressing retroviral vectors, followed by intraperitoneal GCV treatment. Treated animals survived more than twice as long as controls (38).

HSV-TK/GCV is commonly used in cancer gene therapy because of its bystander effect. When 10% of murine sarcoma cells express HSV-TK, GCV eradicates the entire mixed population (39). The percentage of cells expressing HSV-TK has to be higher in vivo—subcutaneous tumors have to possess at least 50% HSV-TK-positive cells to be eliminated by GCV treatment (39).

HSV-TK/GCV's bystander effect requires cell-to-cell contact—the HSV-TK bystander effect can be abrogated when HSV-TK-expressing cells are separated from wild-type cells by a filter membrane (39). Whereas HSV-TK cells treated with [3H]GCV contain mostly GCV monophosphate and very little di- and triphosphate, bystander cells contain mostly GCV triphosphate (40). Taken together, these experiments suggest that the bystander effect results from cell-to-cell contact allowing transfer of GCV-triphosphate from HSV-TK+ cells to nontransduced cells—GCV-triphosphate is too polar to cross cell membranes, and thus cell-to-cell contact is required for its transfer. This contact occurs via gap junctions, a hypothesis supported by the fact that the magnitude of the bystander effect correlates with the extent of gap junction-mediated intercellular coupling (41).

There are also two nonspecific explanations for the in vivo HSV-TK bystander effect that are used to explain several in vivo cancer gene therapy bystander effects. First, transduction of dividing endothelial cells may lead to GCV-mediated death of the endothelial cells that comprise the neovasculature of the tumor, causing death of nontansduced and transduced tumor cells (42). Second, the bystander effect may result from an immune response against a nonhuman enzyme like HSV-TK, leading to diffuse cell death affecting neighboring nontransduced cells; or prodrug-mediated death of transduced tumor cells may liberate tumor antigens, generating an immune response that can then target transduced and nontransduced tumor cells (43,44). This hypothesis may explain the observation that eradication of localized tumor deposits in immunocompetent animals sometimes results in the simultaneous immunemediated regression of anatomically distant metastases, an effect dubbed the distant bystander effect (45). However, whereas the immune system may be an ally in HSV-TK gene therapy, since the bystander effect in vivo is not significantly enhanced relative to that in culture, the role of the immune response in eradicating a single tumor mass may not be substantial, which is noteworthy since brain tumors can cause immunosuppression and many brain tumor patients are treated with dexamethasone.

Although GCV is a prodrug, the doses required for tumor eradication have been slightly toxic in some animal studies. Studies using HSV-TK gene therapy in rodents have found that 150 mg GCV/kg body weight/d was required for complete tumor elimination, and this dose produced some treatment-related mortality (46). Improvements in HSV-TK/GCV gene therapy have been achieved via random sequence mutagenesis of the HSV-TK nucleoside binding site, generating mutant HSV-TK enzymes that display increased phosphoryla-tion of GCV (47).

Tumor cells transduced to express HSV-TK and treated with the antiviral agent acyclovir (ACV) display enhanced sensitivity to radiation in culture and in vivo (48). ACV sensitized cells to radiation regardless of whether its administration preceded or followed radiation. Radiation enhancement when ACV preceded radiation could occur because DNA that has incorporated ACV may be more susceptible to radiation-induced strand breakage; whereas ACV administered following radiation might sensitize cells by inhibiting the poly-merase activity required for repair of radiation-induced DNA damage.

Monitoring intratumoral prodrug-activating enzyme expression after gene therapy allows for decisions regarding whether repeated transduction of the tumor is necessary and also helps time prodrug delivery to coincide with maximal transgene expression. The demonstration of noninvasive imaging of HSV-TK gene expression using the substrate [131I]-labeled 2'-fluoro-2'-deoxy-1-P-D-arabinofuranosyl-5-iodo-uracil combined with a clinical gamma camera, single photon emission computed tomography (SPECT), or positron emission tomography (49) has led to investigations seeking novel HSV-TK substrates with high imaging sensitivity.

As of September 2001, 62 clinical trials using adenoviruses or retroviruses expressing HSV-TK to treat mesothelioma, ovarian cancer, glioblastoma, breast cancer, melanoma, multiple myeloma, and astrocytoma have been proposed (3). A total of 603 patients have been enrolled, and major tumor regressions have been observed in 13% of patients for whom sufficient information is available.

Cytosine Deaminase/5-Fluorocytosine

5-Fluorocytosine (5-FC), an agent used to treat infections by fungi such as Candida and Cryptococcus neoformans, is a prodrug converted into the active agent 5-fluorouracil (5FU) by the CD enzyme, which is uniquely expressed in certain fungi and bacteria. Whereas 5-FC is nontoxic to humans because of the lack of cellular CD expression, 5-FU is used to treat colon, pancreatic, and breast cancers. The toxic effects of 5-FU are mediated by three of its intracellular metabolites: 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP), 5-fluoro-2'-deoxyuridine-5'-triphosphate (FdUTP), and 5-fluorouridine-5'-triphosphate (FUTP) (52). FdUMP inhibits the enzyme thymidylate synthetase, which converts deoxyuridylate (dUMP) into thymidylate (dTMP). Because thymidylate synthetase is the only source of de novo thymidylate synthesis, a cell treated with 5-FU ultimately becomes deficient in deoxythymidine-5'-triphosphate (dTTP), leading to the incorporation of both uridine triphosphate and FdUTP into DNA. Uracils in DNA are normally removed, but the lack of dTTP leaves them unreplaced and leaves the DNA strand nicked. The nicked DNA cannot be replicated, leading to cell death. 5-FU also targets RNA via incorporation of FUTP into all three types of RNA, where it inhibits mRNA polyadenylation, tRNA methylation, and processing of rRNA precursors. If RNA-directed effects led to toxicity, 5-FU could prove toxic against the 85-95% of the cells in a malignant glioma that are not proliferating at any given time (53), but 5-FU could also prove toxic against permanently arrested cells such as neurons. However, 5-FU's established efficacy in chemotherapy suggests that 5-FU's RNA-directed effects contribute minimally to cytotoxicity in vivo (Fig. 4).

Rodent gliosarcoma cells expressing the E. coli cytosine deaminase gene become 77-fold more sensitive to 5-FC in culture (54). In addition, mice whose CD+ tumors were eliminated by 5-FC resist subsequent rechallenge with unmodified wild-type tumor (55). Tumor cells expressing CD may present CD peptides on class I MHC, where they could serve as superantigens, leading to polyclonal activation of T cells (56).

CD/5-FC has a stronger bystander effect than HSV-TK/GCV (57). In culture, a mixture containing 33% CD-expressing cells displayed a dose-response curve identical to cultures containing 100% CD-expressing cells. Some nude mice subcutaneous tumors containing just 4% CD-expressing cells displayed no detectable tumor after 3 wk of 5-FC treatment, and all tumors containing only 2% CD-expressing cells displayed significant tumor regressions after 5-FC treatment. In contrast to HSV-TK/GCV, cell-to-cell contact does not appear to be required for the CD/5-FC bystander effect. Because high-performance liquid chromatography analysis detected 5-FU in the medium of cultured CD-expressing cells after 5-FC treatment, the bystander effect appears to result from 5-FU exiting CD-expressing cells and entering wild-type cells by facilitated diffusion. This mechanism could render the CD/5-FC strategy more effective than nh2

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