Paul D. Boucher

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.

Hydroxyurea Induces Bystander Cytotoxicity in Cocultures of Herpes Simplex Virus Thymidine Kinase–Expressing and Nonexpressing HeLa Cells Incubated with Ganciclovir

Suicide gene therapy with the herpes simplex virus thymidine kinase (HSV-TK) cDNA and ganciclovir can elicit cytotoxicity to transgene-expressing and nonexpressing bystander cells via transfer of ganciclovir phosphates through gap junctions. HeLa cells do not exhibit bystander cytotoxicity, although we showed recently that they transfer low levels of ganciclovir phosphates to bystander cells. Here, we attempted to induce bystander cytotoxicity using hydroxyurea, an inhibitor of ribonucleotide reductase, to decrease the endogenous dGTP pool, which should lessen competition with ganciclovir triphosphate for DNA incorporation. Addition of hydroxyurea to cocultures of HSV-TK-expressing and bystander cells synergistically increased ganciclovir-mediated cytotoxicity to both cell populations while producing primarily an additive effect in cultures of 100% HSV-TK-expressing cells. Whereas HSV-TK-expressing cells in coculture were approximately 50-fold less sensitive to ganciclovir compared with cultures of 100% HSV-TK-expressing cells, addition of hydroxyurea restored ganciclovir sensitivity. Quantification of deoxynucleoside triphosphate pools showed that hydroxyurea decreased dGTP pools without significantly affecting ganciclovir triphosphate levels. Although hydroxyurea significantly increased the ganciclovir triphosphate:dGTP value for 12 to 24 hours in HSV-TK-expressing and bystander cells from coculture (1.4- to 4.9-fold), this value was increased for <12 hours (2.5-fold) in 100% HSV-TK-expressing cells. These data suggest that the prolonged increase in the ganciclovir triphosphate:dGTP value in cells in coculture resulted in synergistic cytotoxicity. Compared with enhancement of bystander cytotoxicity through modulation of gap junction intercellular communication, this strategy is superior because it increased cytotoxicity to both HSV-TK-expressing and bystander cells in coculture. This approach may improve clinical efficacy.

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 μM) when cocultured with bystander cells compared to results in 100% cultures of HSV-TK-expressing cells (IC90=1.2 μM). 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/106 cells after 24 h. In addition, GCVTP levels were decreased in HSV-TK-expressing cells in coculture (867±33 pmol/106 cells) compared to 100% cultures of HSV-TK-expressing cells (1773±188 pmol/106 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.

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.

807. Addition of HU to GCV Induces Bystander Cytotoxicity in Co-Cultures of HSVTK-Expressing and Non-Expressing HeLa Cells

Expression of the herpes simplex virus type I thymidine kinase (HSVTK) gene in tumor cells sensitizes them to the antiviral agent ganciclovir (GCV). Incubation with GCV results not only in cytotoxicity from cells that express the transgene, but neighboring bystander cells as well. Bystander cytotoxicity has been attributed to the transfer of phosphorylated GCV metabolites from HSVTK-expressing to non-expressing cells through protein channels known as gap junctions. We have previously reported good bystander cytotoxicity in the U251 glioblastoma and SW620 colon carcinoma cell lines, which have 84% and 39% gap junction intercellular communication (GJIC), respectively. However, in the HeLa cell line (23% GJIC), no bystander cytotoxicity resulted from incubating a co-culture of HSVTK-expressing and bystander HeLa cells with GCV. Further analysis revealed that lack of bystander cytotoxicity in these cells was not due to an inability to transfer phosphorylated GCV, but resulted from a low level of transfer along with a rapid half-life of GCVTP in bystander cells (3.1 hrs) when compared to the HSVTK-expressing cells (12.3 hrs). We hypothesized that reduction of the endogenous competitor for GCVTP incorporation into DNA, dGTP, would increase levels of GCV incorporation into DNA resulting in the induction of bystander cytotoxicity. To that end, the effect of hydroxyurea (HU), a ribonucleotide reductase inhibitor, on GCV cytotoxicity in cultures of 100% HSVTK-expressing HeLa cells and co-cultures of HSVTK-expressing and bystander HeLa cells was examined. Isobologram analysis of the interaction between GCV and HU demonstrated an additive-antagonistic effect on 100% cultures of HSVTK-expressing HeLa cells, while exhibiting a synergistic effect on the HSVTK-expressing and bystander cells in a co-culture. To determine the mechanism for the synergistic cytotoxicity demonstrated in each of the populations from the co-culture when incubated with GCV and HU, GCVTP and dNTP pools were quantified and the results were compared to 100% cultures of HSVTK-expressing HeLa cells. The results demonstrated a significant increase in the GCVTP:dGTP values in cultures of 100% HSVTK-expressing cells (>2.5 fold) and in co-cultures of HSVTK-expressing cells (>1.4 fold) and bystander cells (>1.8 fold) at 4 hours. However, at 12 hours the GCVTP:dGTP value for 100% cultures of HSVTK-expressing HeLa cells was at control levels (1.3 0.5 fold) while the values for HSVTK-expressing cells (>2.7 fold) and bystander cells (>4.9 fold) in co-culture were still significantly higher. These data suggest that the prolonged increase in the GCVTP:dGTP value in co-cultures incubated with GCV and HU leads to an increase in the levels of GCVTP incorporated into DNA resulting in synergistic cytotoxicity.

1033. A Novel Mechanism of Synergistic Cytoxicity between Prodrugs, 5-FC and GCV in Double Suicide Gene Therapy

The simultaneous use of CD/5-FC and HSV-TK/GCV suicide gene protocols has resulted in enhanced antitumor activity in cultured tumor cell lines as well as in mouse models. It has been suggested that the CD/5-FC-mediated decrease in dTTP leads to lower thymidine levels and decreased competition for GCV phosphorylation by HSV-TK. In this study, we demonstrate that concurrent addition of prodrugs, 5-FC and GCV, was less efficacious than sequential addition of 5-FC followed by GCV treatment in human DU145 prostate carcinoma cells infected with an adenovirus containing a CD/HSV-TK fusion gene. The level of radiolabeled GCV-TP in cells treated simultaneously with 200 μg/ml 5-FC and 1.0μM GCV for 24 hr was 188 pmol/106 cells compared to 143 pmol/106 cells in cells treated with GCV alone. However, if the cells were pretreated 24 hr with 5-FC and then incubated 24 hr with GCV, GCV-TP levels were 2.6-fold higher compared to GCV treatment alone. [H3]GCV incorporation into DNA was also elevated more than 2-fold in pretreated cells compared to simultaneous addition. In addition, compared to GCV treatment alone, sequential addition of 5-FC and GCV resulted in an additional 60% growth inhibition, whereas only 15% inhibition was observed when both drugs were given together. We measured the effect of 5-FC pre-incubation on endogenous deoxynucleotide concentrations by incubating DU145 cells transduced with the CD/HSV-TK adenovirus with 200 μg/ml 5-FC over a 24 hr period. As expected, cellular dTTP levels were gradually depleted over the 24 hr incubation (from 82 to 11 pmol/106 cells). However, the level of dGTP also decreased (from 9 to 1 pmol/106 cells) concurrently with the dTTP pool decrease over this time period. This reduction of dTTP and dGTP concentrations was not observed when CD/HSV-TK-expressing DU145 cells were treated over 24 hr with both 5-FC and GCV simultaneously. The results from these experiments allowed us to propose and test a novel hypothesis for the synergistic interaction between CD/5-FC and HSV-TK/GCV treatments. We suggest that the CD/5-FC-mediated reduction of dTTP concentrations results in concurrent decrease of dGTP levels due to allosteric regulation of ribonucleotide reductase. Since dGTP is the endogenous competitor of GCV-TP, a depleted dGTP pool at the time of GCV addition can result in increased GCV incorporation into DNA and cell kill. In fact, including increasing amounts of deoxyguanosine during the 5-FC pre-incubation reverses the depletion of endogenous dGTP, reduces the amount of GCV incorporated into DNA and negates the GCV-mediated cytotoxicity associated with 5-FC pre-incubation in DU145 cells expressing CD and HSV-TK. Understanding this mechanistic interaction between these two enzyme/prodrug systems may help recognize better strategies for creating more efficacious clinical protocols.