Microphthalmia-associated transcription factor suppresses invasion by reducing intracellular GTP pools.

Melanoma progression is associated with increased invasion and, often, decreased levels of microphthalmia-associated transcription factor (MITF). Accordingly, downregulation of MITF induces invasion in melanoma cells; however, little is known about the underlying mechanisms. Here, we report for the first time that depletion of MITF results in elevation of intracellular GTP levels and increased amounts of active (GTP-bound) RAC1, RHO-A and RHO-C. Concomitantly, MITF-depleted cells display larger number of invadopodia and increased invasion. We further demonstrate that the gene for guanosine monophosphate reductase (GMPR) is a direct MITF target, and that the partial repression of GMPR accounts mostly for the above phenotypes in MITF-depleted cells. Reciprocally, transactivation of GMPR is required for MITF-dependent suppression of melanoma cell invasion, tumorigenicity and lung colonization. Moreover, loss of GMPR accompanies downregulation of MITF in vemurafenib-resistant BRAFV600E-melanoma cells and underlies the increased invasion in these cells. Our data uncover novel mechanisms linking MITF-dependent inhibition of invasion to suppression of guanylate metabolism.

Pharmacological targeting of guanosine monophosphate synthase suppresses melanoma cell invasion and tumorigenicity.

Malignant melanoma possesses one of the highest metastatic potentials among human cancers. Acquisition of invasive phenotypes is a prerequisite for melanoma metastases. Elucidation of the molecular mechanisms underlying melanoma invasion will greatly enhance the design of novel agents for melanoma therapeutic intervention. Here, we report that guanosine monophosphate synthase (GMPS), an enzyme required for the de novo biosynthesis of GMP, has a major role in invasion and tumorigenicity of cells derived from either BRAF(V600E) or NRAS(Q61R) human metastatic melanomas. Moreover, GMPS levels are increased in metastatic human melanoma specimens compared with primary melanomas arguing that GMPS is an attractive candidate for anti-melanoma therapy. Accordingly, for the first time we demonstrate that angustmycin A, a nucleoside-analog inhibitor of GMPS produced by Streptomyces hygroscopius efficiently suppresses melanoma cell invasion in vitro and tumorigenicity in immunocompromised mice. Our data identify GMPS as a powerful driver of melanoma cell invasion and warrant further investigation of angustmycin A as a novel anti-melanoma agent.

Unrepairable DNA double-strand breaks initiate cytotoxicity with HSV-TK/ganciclovir.

The herpes simplex virus thymidine kinase (HSV-TK) is the most widely used suicide gene in cancer gene therapy due to its superior anticancer activity with ganciclovir (GCV) compared with other HSV-TK substrates, such as 1-ß-D-arabinofuranosyl thymine (araT). We have evaluated the role of DNA damage as a mechanism for the superiority of GCV. Using γ-H2AX foci as an indicator of DNA damage, GCV induced ≥ sevenfold more foci than araT at similar cytotoxic concentrations. The number of foci decreased after removal of either drug, followed by an increase in Rad51 foci indicating that homologous recombination repair (HRR) was used to repair this damage. Notably, only GCV produced a late and persistent increase in γ-H2AX foci demonstrating the induction of unrepairable DNA damage. Both drugs induced the ATR damage response pathway, as evidenced by Chk1 activation. However, GCV resulted in greater activation of ATM, which coincided with the late induction of γ-H2AX foci, demonstrating the presence of DNA double-strand breaks (DSBs). The increase in DSBs after Rad51 induction suggested that they occurred as a result of a failed attempt at HRR. These data demonstrate that the late and unrepairable DSBs observed uniquely with GCV account for its superior cytotoxicity and further suggest that inhibition of HRR will enhance cytotoxicity with HSV-TK/GCV.

MLH1 deficiency enhances tumor cell sensitivity to ganciclovir.

Suicide gene therapy with herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) is notable for producing multi-log cytotoxicity in a unique pattern of delayed cytotoxicity in S-phase. As hydroxyurea, a ribonucleotide reductase inhibitor that activates mismatch repair, can increase sensitivity to GCV, we evaluated the role of MLH1, an essential mismatch repair protein, in GCV cytotoxicity. Using HCT116TK (HSV-TK-expressing) colon carcinoma cells that express or lack MLH1, cell-survival studies demonstrated greater GCV sensitivity in the MLH1-deficient cells, primarily at high concentrations. This could not be explained by differences in GCV metabolism, as the less sensitive MLH1-expresssing cells accumulated more GCV triphosphate and incorporated more of the analog into DNA. SiRNA suppression of MLH1 in U251 glioblastoma or SW480 colon carcinoma cells also enhanced sensitivity to high concentrations of GCV. Studies in a pa nel of yeast deletion mutants confirmed the results with MLH1, and further suggested a role for homologous recombination repair and several cell-cycle checkpoint proteins in GCV cytotoxicity. These data suggest that MLH1 can prevent cytotoxicity with GCV. Targeting mismatch repair-deficient tumors may increase efficacy of this suicide gene therapy approach to cancer treatment.

GCV phosphates are transferred between HeLa cells despite lack of bystander cytotoxicity.

The role of gap junctional intercellular communication (GJIC) in bystander killing with herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) was evaluated in U251 cells expressing a dominant-negative connexin 43 cDNA (DN14), and in HeLa cells, reportedly devoid of connexin protein. These cell lines both exhibited 0% GJIC when assayed by Lucifer Yellow fluorescent dye microinjection. Bystander cytotoxicity was still apparent in 50:50 cocultures of DN14 and HSV-TK-expressing U251 cells, but not in 50:50 cocultures of HeLa cells. However, the sensitivity of HeLa HSV-TK-expressing cells to GCV decreased nearly 100-fold (IC90=109 microM) when cocultured with bystander cells compared to results in 100% cultures of HSV-TK-expressing cells (IC90=1.2 microM). A more sensitive flow cytometry technique to measure GJIC over 24 h revealed that the DN14 and HeLa cells exhibited detectable levels of communication (29 and 23%, respectively). Transfer of phosphorylated GCV to HeLa bystander cells occurred within 4 h after drug addition, and GCV triphosphate (GCVTP) accumulated to 213+/-84 pmol/10(6) cells after 24 h. In addition, GCVTP levels were decreased in HSV-TK-expressing cells in coculture (867+/-33 pmol/10(6) cells) compared to 100% cultures of HSV-TK-expressing cells (1773+/-188 pmol/10(6) cells). The half-life of GCVTP in the HSV-TK-expressing cells was approximately four times that measured in the bystander cells (12.3 and 3.1 h, respectively). These data suggest that the lack of bystander cytotoxicity in HeLa cocultures is due to low transfer of phosphorylated GCV and a rapid half-life of GCVTP in the bystander cells. Thus, GCV phosphate transfer to non-HSV-TK-expressing bystander cells may mediate either bystander cell killing or sparing of HSV-TK-positive cells, depending upon the cell specific drug metabolism.

Hydroxyurea significantly enhances tumor growth delay in vivo with herpes simplex virus thymidine kinase/ganciclovir gene therapy.

We have previously demonstrated with several cell lines in vitro that hydroxyurea (HU) synergistically enhances ganciclovir (GCV)-mediated cytotoxicity in bystander cells. In this study, we evaluated the role of DNA synthesis inhibition on enhanced bystander killing and assessed whether addition of HU would improve the efficacy of the HSV-TK/GCV system in vivo. Compared with GCV treatment alone, addition of HU resulted in increased DNA synthesis inhibition and delayed progression through S phase following removal of drug. In a xenograft tumor model, 1:10 and 1:1 mixtures of HSVtk- and LacZ-expressing SW620 cells were injected s.c. in the flanks of nude mice and treated i.p. (100 mg/kg GCV, 1500 mg/kg HU) daily for 5 days. Tumors from mice treated with GCV alone grew rapidly and increased to 10 times their initial size in 15.7 +/- 1.8 and 16.0 +/- 0.9 days for 1:10 and 1:1 mixtures, respectively. However, when both GCV and HU were administered in combination, a single complete tumor regression was observed in both the 1:10 and 1:1 groups. In the remaining mice treated with GCV/HU, it took 23.2 +/- 2.1 (1:10) and 26.4 +/- 3.8 days (1:1) to obtain a similar 10-fold increase in tumor size.

Radiosensitization by gemcitabine in p53 wild-type and mutant MCF-7 breast carcinoma cell lines.

The nucleoside analogue 2',2'-difluoro-2'-deoxycytidine (dFdCyd) is a potent radiosensitizer in several solid tumor cell lines. Radiosensitization has correlated with the dFdCyd-mediated decrease in dATP levels and is S-phase specific. Previous studies suggested that a cell line that was unable to progress through S phase after dFdCyd and radiation was not radiosensitized apparently because of the expression of wild-type p53. We have extended these results by using the MCF-7 human breast carcinoma cell line (wild-type p53) and the MCF-7/Adr subline (mutant p53) to determine whether p53 status affected radiosensitization or cell cycle progression after dFdCyd and radiation treatment. Both cell lines were sensitive to nanomolar concentrations of dFdCyd and showed significant radiosensitization, with radiation enhancement ratios of 1.6-1.8 after a 24-h exposure to either the IC(10) or IC(50) for dFdCyd. Nucleotide pool analysis demonstrated a >85% reduction in dATP pools in both cell lines within 8 h after drug addition. Both cell lines accumulated in S phase after a 24-h incubation with dFdCyd. After subsequent irradiation, MCF-7/Adr cells continued to progress through the cell cycle for at least 72 h. MCF-7 cells progressed for at least 24 h, and then exhibited a G(1) block at 48 h after drug and radiation treatment. These results demonstrate that a wild-type p53 cell line can be radiosensitized by dFdCyd, presumably because it was able to deplete dATP levels and progress through the cell cycle for at least 24 h after drug and radiation treatment.

Radiation concurrent with gemcitabine for locally advanced head and neck cancer: a phase I trial and intracellular drug incorporation study.

PURPOSE: To examine the feasibility and dose-limiting toxicity (DLT) of once-weekly gemcitabine at doses predicted in preclinical studies to produce radiosensitization, concurrent with a standard course of radiation for locally advanced head and neck cancer. Tumor incorporation of gemcitabine triphosphate (dFdCTP) was measured to assess whether adequate concentrations were achieved at each dose level. PATIENTS AND METHODS: Twenty-nine patients with unresectable head and neck cancer received a course of radiation (70 Gy over 7 weeks, 5 days weekly) concurrent with weekly infusions of low-dose gemcitabine. Tumor biopsies were performed after the first gemcitabine infusion (before radiation started), and the intracellular concentrations of dFdCTP were measured. RESULTS: Severe acute and late mucosal and pharyngeal-related DLT required de-escalation of gemcitabine dose in successive patient cohorts receiving dose levels of 300 mg/m(2)/wk, 150 mg/m(2)/wk, and 50 mg/m(2)/wk. No DLT was observed at 10 mg/m(2)/wk. The rate of endoscopy- and biopsy-assessed complete tumor response was 66% to 87% in the various cohorts. Tumor dFdCTP levels were similar in patients receiving 50 to 300 mg/m(2) (on average, 1.55 pmol/mg, SD 1.15) but were barely or not detectable at 10 mg/m(2). CONCLUSION: A high rate of acute and late mucosa-related DLT and a high rate of complete tumor response were observed in this regimen at the dose levels of 50 to 300 mg/m(2), which also resulted in similar, subcytotoxic intracellular dFdCTP concentrations. These results demonstrate significant tumor and normal tissue radiosensitization by low-dose gemcitabine. Different regimens of combined radiation and gemcitabine should be evaluated, based on newer preclinical data promising an improved therapeutic ratio.

Evidence for iterative ratcheting of receptor-bound hsp70 between its ATP and ADP conformations during assembly of glucocorticoid receptor.hsp90 heterocomplexes.

hsp90 and hsp70 are essential components of a five-protein system, including also the nonessential cochaperones Hop, hsp40, and p23, that assembles glucocorticoid receptor (GR).hsp90 heterocomplexes and causes the simultaneous opening of the steroid binding pocket to access by steroid. The first event in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent binding of hsp70 to the GR, which primes the receptor for subsequent ATP-dependent activation by hsp90 [Morishima, Y., Murphy, P. J. M., Li, D. P., Sanchez, E. R., and Pratt, W. B. (2000) J. Biol. Chem. 275, 18054-18060]. Here, we demonstrate that, during the priming step, ATP-bound hsp70 is converted to GR-bound hsp70 that is approximately 1/3 in the ADP- and approximately 2/3 in the ATP-dependent conformation. In the second step, hsp90, which is provided in the non-nucleotide-bound state, is converted to GR-bound hsp90 in the ATP-dependent conformation. The ATPase activity of hsp70 is K(+)-dependent, and the priming step is K(+)-dependent. Surprisingly, the subsequent hsp90-dependent step, which is rate-limiting for receptor activation, is also potassium-dependent. This suggests that GR-bound hsp70 is also converted from the ATP-dependent to the ADP-dependent conformation while it cooperates with hsp90 to activate steroid binding activity. Because the priming step requires both sustained high levels of ATP and YDJ-1 for optimal activity and because both steps require potassium, we predict that receptor-bound hsp70 undergoes iterative ratcheting between its ATP- and ADP-dependent conformations in opening the hydrophobic steroid binding pocket.

Metabolism of 4'-thio-beta-D-arabinofuranosylcytosine in CEM cells.

Because of the excellent in vivo activity of 4'-thio-beta-D-arabinofuranosylcytosine (T-araC) against a variety of human solid tumors, we have studied its metabolism in CEM cells to determine how the biochemical pharmacology of this compound differs from that of beta-D-arabinofuranosylcytosine (araC). Although there were many quantitative differences in the metabolism of T-araC and araC, the basic mechanism of action of T-araC was similar to that of araC: it was phosphorylated to T-araC-5'-triphosphate (T-araCTP) and inhibited DNA synthesis. The major differences between these two compounds were: (i) T-araC was phosphorylated to active metabolites at 1% the rate of araC; (ii) T-araCTP was 10- to 20-fold more potent as an inhibitor of DNA synthesis than was the 5'-triphosphate of araC (araCTP); (iii) the half-life of T-araCTP was twice that of araCTP; (iv) the catalytic efficiency of T-araC with cytidine deaminase was 10% that of araC; and (v) the 5'-monophosphate of araC was a better substrate for deoxycytidine 5'-monophosphate deaminase than was the 5'-monophosphate of T-araC. Of these differences in the metabolism of these two compounds, we propose that the prolonged retention of T-araCTP is a major factor contributing to the activity of T-araC against solid tumors. The data in this study represent another example of how relatively small structural changes in nucleoside analogs can profoundly affect the biochemical activity.

The role of cell cycle progression in radiosensitization by 2',2'-difluoro-2'-deoxycytidine.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdCyd) has been shown to be a potent radiosensitizer in tumor cells both in vitro and in vivo. We evaluated the ability of dFdCyd to enhance the radiosensitivity of two human glioblastoma cell lines. The results demonstrated that U251 cells were more sensitive to the cytotoxicity of dFdCyd, and that dFdCyd was able to radiosensitize these cells. In contrast, D54 cells were more resistant to the cytotoxic effect of dFdCyd, and no radiosensitization occurred at any concentration of dFdCyd tested. Because radiosensitization by dFdCyd has been correlated with its ability to deplete dATP pools through inhibition of ribonucleotide reductase by dFdCyd diphosphate, we evaluated the metabolism of dFdCyd in both cell lines. At equitoxic concentrations of dFdCyd, both cell lines accumulated similar levels of the cytotoxic metabolite, dFdCyd triphosphate, as well as similar levels of dFdCyd monophosphate in DNA. In U251 cells, radiosensitizing concentrations of dFdCyd (10 or 25 nM; IC10 or IC50) depleted dATP by approximately 80% within 4 h. In contrast, 80 nM (IC50) was unable to deplete dATP by >30% within 4 h in D54 cells. Higher concentrations of dFdCyd or hydroxyurea, an inhibitor of ribonucleotide reductase that depleted dATP >90%, also did not produce radiosensitization in D54 cells. D54 cells were not resistant to radiosensitization because bromodeoxyuridine was able to induce radiosensitization. Because D54 cells express wild-type p53, whereas U251 cells express a mutant p53, the effect of dFdCyd and ionizing radiation on cell cycle progression was evaluated. Radiation alone produced a G1 block in D54 cells and a transient G2-M block in U251 cells. After a 24 h incubation with dFdCyd alone or in combination with ionizing radiation, U251 cells readily accumulated in S-phase, which remained elevated for at least 72 h, consistent with previous results in other mutant p53 cell lines. In addition, radiation enhanced the ability of dFdCyd to induce S-phase-specific cell death in U251 cells. In contrast, D54 cells showed a G1 block after dFdCyd and radiation exposure, with fewer cells in S-phase for at least 48 h after drug washout/irradiation. Furthermore, treatment with dFdCyd and/or radiation did not increase the amount of S-phase-specific cell death in D54 cells compared with control cells. These results suggest that the G1 block in D54 cells resulting from wild-type p53 induction prevented radiosensitization by dFdCyd.

Synergistic enhancement of herpes simplex virus thymidine kinase/ganciclovir-mediated cytoxicity by hydroxyurea.

We have previously demonstrated (L. Z. Rubsam et al., Cancer Res., 59: 669-675, 1999) that low ganciclovir (GCV) triphosphate (TP) levels similar to cellular deoxynucleotide concentrations can induce multilog killing in cells stably expressing herpes simplex virus thymidine kinase (HSV-TK). In this study, we evaluated whether reducing the endogenous competitor of GCV-TP, dGTP, enhanced GCV-mediated cytotoxicity. In SW620 human colon carcinoma cells stably expressing HSV-TK, the addition of the ribonucleotide reductase inhibitor, hydroxyurea (HU), decreased cellular dGTP pools and simultaneously increased the accumulation of GCV-TP levels. The amount of GCV nucleotide transfer from HSV-TK-expressing to nonexpressing (bystander) cells was quantitated in physically separated pHook-expressing bystander cells. Elevation of the GCV-TP:dGTP ratio by HU resulted in increased levels of GCV nucleotides transferred from HSV-TK-expressing to bystander cells during a 24 h drug incubation and enhanced GCV monophosphate incorporation into DNA after drug removal. Isobologram analysis demonstrated that the combination of GCV and HU was additive in 100% HSV-TK cultures and synergistic in HSV-TK/bystander mixtures. IC50 values for GCV in 1:1 cocultures of HSV-TK-expressing and nonexpressing SW620 cells were reduced from 1.5 microM to 0.07 microM with 2 mM HU. A similar reduction was also observed with HT-29 cells and U251 cells. With 2 mM HU, IC50 values for GCV in 10:90, 5:95, and 1:99 SW620 HSV-TK-expressing and nonexpressing cocultures were reduced from 55 microM to 0.3 microM, 71 microM to 0.8 microM, and 118 microM to 7 microM, respectively. These results demonstrate the ability to pharmacologically enhance HSV-TK/GCV-mediated bystander killing and may have an important therapeutic impact.

Radiosensitization by gemcitabine.

Gemcitabine is a potent radiosensitizer in both laboratory studies and in the clinic. Initial laboratory studies showed that gemcitabine radiosensitizes a wide variety of rodent and human tumor cells in culture. Maximum radiosensitization occurs in cells that demonstrate concurrent redistribution into S phase and d-adenosine triphosphate pool depletion. Although the mechanism of sensitization is not yet clear, recent evidence from our laboratory suggests that gemcitabine lowers the threshold for radiation-induced apoptosis. Our preclinical data were used to design gemcitabine dose-escalation trials in combination with standard radiation for patients with unresectable head and neck cancer and pancreatic cancer. In head and neck cancer, we have found that gemcitabine doses far below the maximum tolerated dose for the drug when used alone significantly potentiate the toxicity of treatment. Comparatively, normal tissue sensitization has not been as marked in the treatment of pancreatic tumors. These findings have led us to conduct experiments using an animal model to improve the therapeutic index of treatment. We conclude that gemcitabine is a promising radiation sensitizer that will need to be developed cautiously if excessive normal tissue toxicity is to be avoided.

Comparison of the mechanism of cytotoxicity of 2-chloro-9-(2-deoxy-2- fluoro-beta-D-arabinofuranosyl)adenine, 2-chloro-9-(2-deoxy-2-fluoro- beta-D-ribofuranosyl)adenine, and 2-chloro-9-(2-deoxy-2,2-difluoro- beta-D-ribofuranosyl)adenine in CEM cells.

In an effort to understand biochemical features that are important to the selective antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine [Cl-F( upward arrow)-dAdo], we evaluated the biochemical pharmacology of three structurally similar compounds that have quite different antitumor activities. Cl-F( upward arrow)-dAdo was 50-fold more potent as an inhibitor of CEM cell growth than were either 2-chloro-9-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)adenine [Cl-F( downward arrow)-dAdo] or 2-chloro-9-(2-deoxy-2, 2-difluoro-beta-D-ribofuranosyl)adenine [Cl-diF( upward arrow downward arrow)-dAdo]. The compounds were similar as substrates of deoxycytidine kinase. Similar amounts of their respective triphosphates accumulated in CEM cells, and the rate of disappearance of these metabolites was also similar. Cl-F( upward arrow)-dAdo was 10- to 30-fold more potent in its ability to inhibit the incorporation of cytidine into deoxycytidine nucleotides than either Cl-F( downward arrow)-dAdo or Cl-diF( upward arrow downward arrow)-dAdo, respectively, which indicated that ribonucleotide reductase was differentially inhibited by these three compounds. Thus, the differences in the cytotoxicity of these agents toward CEM cells were not related to quantitative differences in the phosphorylation of these agents to active forms but can mostly be accounted for by differences in the inhibition of ribonucleotide reductase activity. Furthermore, the inhibition of RNA and protein synthesis by Cl-F( downward arrow)-dAdo and Cl-diF( upward arrow downward arrow)-dAdo at concentrations similar to those required for the inhibition of DNA synthesis can help explain the poor antitumor selectivity of these two agents because all cells require RNA and protein synthesis.

Cytotoxicity and accumulation of ganciclovir triphosphate in bystander cells cocultured with herpes simplex virus type 1 thymidine kinase-expressing human glioblastoma cells.

The ability of herpes simplex virus type 1 thymidine kinase (HSV-TK)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-TK-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs through the transfer of phosphorylated GCV, there is little direct proof that bystander cells can accumulate GCV nucleotides. We have studied the ability of U251 human glioblastoma cells expressing HSV-TK (U251tk cells) to induce cytotoxicity in neighboring U251 bystander cells that lack the viral kinase (U251beta gal cells) and evaluated whether this bystander cell killing is mediated by GCV nucleotides. The cytotoxicity studies demonstrated that the ratio of HSV-TK-expressing cells:bystander cells was important in determining the sensitivity of both cell types to GCV. U251tk cells cocultured with an equal number of U251beta gal cells (a 50:50 ratio) exhibited a sensitivity to GCV similar to that observed in the absence of bystander cells, with >99.8% cell kill at 1 microm GCV. However, in cultures with 10% U251tk cells and 90% bystander cells (a 10:90 ratio), 1 microM GCV decreased the survival of U251tk cells by only 54%. Strong bystander cell killing was observed at both ratios. In a 50:50 coculture of U251tk and U251beta gal cells, the survival of bystander cells was decreased by >99.5% with 3 microM GCV, whereas 30 microM GCV was required to effect a similar decrease in bystander cell survival when 90% of the culture consisted of U251beta gal cells. To determine whether this bystander cell killing may be mediated by GCV nucleotides, we developed a technique to separate the two cell populations after coculture. A U251 bystander cell line was developed from the parental cell line by transfection with the cDNA coding for green fluorescent protein (U251gfp cells), which permitted the separation of U251gfp cells from nonfluorescing U251tk cells by flow cytometry with cell sorting. With this technique, bystander cells were isolated in a viable state with >97% purity within 1 h after harvest, permitting analysis of the nucleotide pools for the presence of phosphorylated GCV. The results demonstrated that significant levels of the triphosphate of GCV (GCVTP) accumulated in bystander cells within 4 h of coculture, and this accumulation was dependent upon the percentage of HSV-TK-expressing cells as well as the concentration of GCV and the length of incubation. The proportion of GCVTP in bystander cells was consistently 50-80% of that in HSV-TK-expressing cells in the 50:50 or 10:90 cocultures, suggesting a facile transfer of phosphorylated GCV. However, the actual amount of GCVTP was as much as 8-fold lower in both the U251tk and U251beta gal cells cocultured at a ratio of 10:90 compared to those cocultured at a ratio of 50:50, which is consistent with the lesser effect on cell survival. When U251tk and U251gfp cells were cultured with 1-beta-D-arabinofuranosylthymine (araT), the 5'-triphosphate of araT accumulated in the bystander cells, demonstrating that the transfer of phosphorylated compounds between these cell types is not restricted to GCV nucleotides. However, the proportion of araT-5'-triphosphate in bystander cells compared to that in HSV-TK-expressing cells was lower than that for GCVTP, and the amount was not sufficient to decrease survival in the bystander population.

Superior cytotoxicity with ganciclovir compared with acyclovir and 1-beta-D-arabinofuranosylthymine in herpes simplex virus-thymidine kinase-expressing cells: a novel paradigm for cell killing.

Enzyme-prodrug therapy using ganciclovir and herpes simplex virus-thymidine kinase (HSV-TK) has demonstrated excellent antitumor activity in many different types of malignant cells. Previously, we noted that ganciclovir was substantially more cytotoxic than other HSV-TK substrates. Therefore, we embarked on a study to determine the basis for the superior cytotoxicity of ganciclovir. In U251tk human glioblastoma cells that stably express HSV-TK, ganciclovir elicited a >4 log cell kill instead of the < or =1.5 log cell kill mediated by two other HSV-TK substrates, 1-beta-D-arabinofuranosylthymine (araT) and acyclovir. Study of the metabolism of these drugs demonstrated that acyclovir was poorly phosphorylated to its active triphosphate with DNA incorporation below the limit of detection, which may explain the < 1 log cell kill in these cells. Lower levels of ganciclovir triphosphate accumulated compared with araT triphosphate (araTTP) under conditions that induced < or =1 log cell kill (67 versus 1235 pmol/10(7) cells, respectively), and the half-life for the triphosphate of ganciclovir was shorter than that of araT (terminal half-lives of 15 and 41 h, respectively). Incorporation of ganciclovir monophosphate into DNA was less than that of araT monophosphate, and both analogues were retained in DNA for > or =48 h. Thus, the superior cytotoxicity of ganciclovir was not due to enhanced metabolism to active forms. Highly cytotoxic concentrations of ganciclovir produced only weak inhibition of DNA synthesis. This allowed cells to proceed through S and G2-M phases during and after drug exposure, resulting in a doubling of cell number by 48 h after drug washout. As they attempted to progress through the cell cycle a second time, ganciclovir-treated cells accumulated in early S-phase and remained there until cell death, suggesting that ganciclovir incorporation in the DNA template was important for cytotoxicity. In contrast, strong inhibition of DNA synthesis by araTTP prevented cells from traversing the cell cycle for at least 12 h after drug washout, when the active metabolite was largely degraded araT-treated cells were unable to divide for at least 72 h after drug exposure, at which point the surviving cells displayed a normal cell cycle distribution pattern. Based on the results presented here, we propose a novel paradigm in which the ability of ganciclovir to incorporate into DNA without inhibiting progression through S-phase, combined with high cytotoxicity for incorporated ganciclovir monophosphate, produces multilog cytotoxicity.

Metabolism in human cells of the D and L enantiomers of the carbocyclic analog of 2'-deoxyguanosine: substrate activity with deoxycytidine kinase, mitochondrial deoxyguanosine kinase, and 5'-nucleotidase.

The carbocyclic analog of 2'-deoxyguanosine (CdG) has broad-spectrum antiviral activity. Because of recent observations with other nucleoside analogs that biological activity may be associated the L enantiomer rather than, as expected, with the D enantiomer, we have studied the metabolism of both enantiomers of CdG to identify the enzymes responsible for the phosphorylation of CdG in noninfected and virally infected human and duck cells. We have examined the enantiomers as substrates for each of the cellular enzymes known to catalyze phosphorylation of deoxyguanosine. Both enantiomers of CdG were substrates for deoxycytidine kinase (EC 2.7.1.74) from MOLT-4 cells, 5'-nucleotidase (EC 3.1.3.5) from HEp-2 cells, and mitochondrial deoxyguanosine kinase (EC 2.7.1.113) from human platelets and CEM cells. For both deoxycytidine kinase and mitochondrial deoxyguanosine kinase, the L enantiomer was the better substrate. Even though the D enantiomer was the preferred substrate with 5'-nucleotidase, the rate of phosphorylation of the L enantiomer was substantial. The phosphorylation of D-CdG in MRC-5 cells was greatly stimulated by infection with human cytomegalovirus. The fact that the phosphorylation of D-CdG was stimulated by mycophenolic acid and was not affected by deoxycytidine suggested that 5'-nucleotidase was the enzyme primarily responsible for its metabolism in virally infected cells. D-CdG was extensively phosphorylated in duck hepatocytes, and its phosphorylation was not affected by infection with duck hepatitis B virus. These results are of importance in understanding the mode of action of D-CdG and related analogs and in the design of new biologically active analogs.

Differential ganciclovir-mediated cytotoxicity and bystander killing in human colon carcinoma cell lines expressing herpes simplex virus thymidine kinase.

The two human colon carcinoma cell lines HT-29 and SW620, which stably express herpes simplex virus thymidine kinase (HSV-TK), are sensitized to the cytotoxic effects of the antiviral drug ganciclovir (GCV). Compared with HT-29 cells, SW620 cells were more sensitive to lower GCV concentrations (<1 microM), accumulated GCV triphosphate more rapidly, and incorporated higher levels of GCV into DNA. Following a 24-hr exposure to 10 microM GCV, bystander killing was as much as sixfold greater in SW620 cells than HT-29 cells. This bystander effect was dependent on the level of HSV-TK expression, the number of cells expressing HSV-TK, and the overall confluency of the cells. However, bystander killing did not correlate with gap junctional intercellular communication as determined by microinjection of Lucifer Yellow fluorescent dye. SW620 cells were coupled to <3% adjacent cells (compared with >50% for HT-29 cells), but were still able to transfer phosphorylated GCV to bystander cells as soon as 4 hr after drug was added. These results emphasize the importance of cell-specific metabolism in HSV-TK/GCV-mediated cytotoxicity and may suggest a novel mechanism for bystander killing.

Kinetic analysis of human deoxycytidine kinase with the true phosphate donor uridine triphosphate.

Deoxycytidine kinase is the rate-limiting process in the activation for several clinically important antitumor agents. Previous studies have focused on deoxycytidine (dCyd) and adenosine triphosphate (ATP) as substrates for this enzyme. In view of recent data indicating that uridine triphosphate (UTP) is the physiologic phosphate donor for this enzyme, a study of the kinetic properties of dCyd kinase with dCyd and UTP was undertaken. The results presented here demonstrate that UTP and ATP produce kinetically distinguishable differences in nucleoside phosphorylation by dCyd kinase. At high dCyd concentrations, dCyd kinase exhibited substrate activation with ATP. In contrast, in the presence of UTP, substrate inhibition was observed at concentrations of dCyd greater than 3 microM. Inhibition by dCyd was noncompetitive with respect to UTP and could not be reversed by a 200-fold increase in UTP concentration, indicating that the inhibition was not due to dCyd binding at the nucleotide binding site. The kinetic mechanism for dCyd kinase was determined with dCyd and UTP as substrates. UTP was the preferred phosphate donor with a true Km value of 1 microM compared to 54 microM with ATP, resulting in a 50-fold greater substrate efficiency for UTP. Although the double-reciprocal plots with UTP produced parallel lines, initial velocity plots with other phosphate donors and product inhibition studies indicated that dCyd kinase formed a ternary complex with its substrates. The parallel lines with UTP were apparently due to a low dissociation constant for UTP, which was calculated as more than 13-fold lower than its Km value. Analysis of product inhibition studies indicated that dCyd kinase followed an ordered A-B random P-Q reaction sequence, with UTP as the first substrate to bind. In contrast, previous results demonstrated a random bi-bi sequence for dCyd kinase in the presence of ATP. The combined results indicate that the enzyme can follow a random bi-bi reaction sequence, but with UTP as the phosphate donor, the addition of nucleotide prior to dCyd is strongly preferred. The noncompetitive substrate inhibition, which was independent of UTP concentration, indicates that high concentrations of dCyd promote addition of the nucleoside prior to UTP, resulting in a lower velocity.

Delayed radiosensitization of human colon carcinoma cells after a brief exposure to 2',2'-difluoro-2'-deoxycytidine (Gemcitabine).

We have shown that 2',2'-difluoro-2'-deoxycytidine (dFdCyd; Gemcitabine), a deoxycytidine analogue, is a potent radiation sensitizer when cells are exposed to it continuously for >16 h in low concentrations (in the range of 10 nM). However, the most common method of clinical administration is by short-term infusion (30-90 min). Therefore, we wished to determine under what conditions dFdCyd could produce radiosensitization after a relatively brief exposure to drug. We hypothesized that the long half-life of the phosphorylated metabolites of dFdCyd would produce long-lasting dNTP pool perturbation, particularly dATP pools, leading to radiosensitization hours or even days after the drug was removed from the medium. We tested this hypothesis by exposing HT29 human colon cancer cells for 2 h to clinically relevant concentrations of dFdCyd, removing the drug from the medium, and assessing radiation sensitivity up to 72 h later. We found that 100 nM dFdCyd, which was noncytotoxic, radiosensitized HT29 cells up to 48 h after drug removal. During this period, there was an increase in the S phase population, whereas by 72 h after drug removal, the cell cycle distribution resembled that seen under control conditions. dATP pools remained depleted throughout the 72-h period after drug treatment. This study supports the hypothesis that radiosensitization occurs in cells that are replicating DNA in the presence of perturbed dNTP pools. Furthermore, they may be useful in the design of rational clinical trials using dFdCyd as a radiation sensitizer.