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

Boucher, Paul D.; Ostruszka, Leo J.; Murphy, Patrick J.; Shewach, Donna S.

doi:10.1038/sj.gt.3301730

Cited by 23 publications

(21 citation statements)

References 30 publications

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“…We have previously demonstrated that introduction of a mechanistically based inhibitor designed to decrease the endogenous competitor (dGTP) for GCVTP incorporation into DNA synergistically enhanced bystander cytotoxicity in several different tumor cell lines. 11,44 This strategy was successful both in vitro and in vivo. This approach may form an important addition to clinical trials.…”

Section: Discussionmentioning

confidence: 99%

“…[40][41][42][43] We have demonstrated previously that cells with low gap junction intercellular communication (GJIC) can exhibit good bystander cytotoxicity both in vitro and in vivo. 8,11,44 These studies presented here further examine the role of GJIC in bystander cytotoxicity using two approaches. First, U251 glioblastoma cells, which exhibit high GJIC (480% communication between neighboring cells), were transfected with a dominant-negative connexin 43 (DNCx43) cDNA vector and the effect on bystander cytotoxicity was evaluated.…”

Section: Introductionmentioning

confidence: 99%

“…[4][5][6][7] Numerous studies have demonstrated excellent cell killing with GCV in HSV-TK-expressing tumor cells both in vitro and in vivo. 2,5,[8][9][10][11] The antitumor activity is the result of phosphorylation of GCV by HSV-TK to GCVMP. [12][13][14][15][16] Upon further phosphorylation by endogenous cellular kinases to its cytotoxic triphosphate form, GCVTP then competes with endogenous dGTP for incorporation into DNA.…”

Section: Introductionmentioning

confidence: 99%

“…22 In HSV-TK/ GCV therapy, bystander cytotoxicity has been effective at killing cocultures of HSV-TK-expressing and HSV-TKnon-expressing cells in vitro and at reducing tumor size in vivo. 10,11,[29][30][31][32][33][34][35][36] HSV-TK expression in as few as 10% of tumor cells in vivo resulted in tumor regression or a significant reduction in tumor growth when GCV was administered. Bystander cytotoxicity has been attributed to the transfer of phosphorylated GCV metabolites from HSV-TK-expressing cells to non-HSV-TK-expressing bystander cells, presumably via protein channels known as gap junctions.…”

Section: Introductionmentioning

confidence: 99%

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

Gentry

Boucher

et al. 2005

Gene Ther

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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 (IC 90 ¼ 109 mM) when cocultured with bystander cells compared to results in 100% cultures of HSV-TK-expressing cells (IC 90 ¼ 1.2 mM). 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 213784 pmol/10 6 cells after 24 h. In addition, GCVTP levels were decreased in HSV-TK-expressing cells in coculture (867733 pmol/10 6 cells) compared to 100% cultures of HSV-TK-expressing cells (17737188 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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Gentry

Boucher

et al. 2005

Gene Ther

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show abstract

“…24 The suicide genes encode nonmammalian enzymes that can convert nontoxic prodrugs into cellular toxic metabolites. The most widely used suicide gene is the HSVtk/GCV system that can convert prodrug GCV into phosphorated GCV.…”

Section: Discussionmentioning

confidence: 99%