Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0017638 (glioma)
 30,880 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Herpes simplex virus thymidine kinase (HSVTK) with the guanosine analog ganciclovir (GCV) is currently the most widely used suicide gene/prodrug system for gene therapy of cancer. Despite the broad application of the HSVTK/GCV approach, phosphorylation of GCV to its active state is inefficient such that high, myelosuppressive doses of GCV are needed to observe an antitumor effect. One strategy used to overcome the poor substrate specificity of HSVTK towards GCV (Km = 45 microM) has been to create novel forms of TK with altered substrate preferences. Such mutant TKs have shown benefit and are currently in clinical use. We describe here a second strategy to increase the amount of intracellular triphosphorylated GCV by involving the second enzyme in the GCV activation pathway, guanylate kinase (GMK). As a means to overcome the bottleneck of prodrug activation from the monophosphate to the diphosphate, we sought to combine both the critical HSVTK and GMK activities together. In this report we describe the construction of a fusion or chimeric protein of HSVTK and guanylate kinase, show data that demonstrate it confers a approximately 175-fold decrease in IC50 compared to HSVTK alone in response to ganciclovir treatment in stably transfected C6 glioma cells and finally, we present biochemical evidence of a kinetic basis for this improved cell killing.
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PMID:A guanylate kinase/HSV-1 thymidine kinase fusion protein enhances prodrug-mediated cell killing. 1681 Jan 97

Herpes simplex virus thymidine kinase (HSVTK) with ganciclovir (GCV) is currently the most widely used suicide gene/prodrug system in cancer gene therapy. A major limitation in this therapy is the inefficient activation of GCV by HSVTK to its active antimetabolites. We described earlier two strategies to overcome this limitation: (1) generation of HSVTK mutants with improved GCV activation potential and (2) construction of a fusion protein encoding HSVTK and mouse guanylate kinase (MGMK), the second enzyme in the GCV activation pathway. As a means to further enhance GCV activation, two MGMK/HSVTK constructs containing the HSVTK mutants, mutant 30 and SR39, were generated and evaluated for their tumor and bystander killing effects in vitro and in vivo. One fusion mutant, MGMK/30, shows significant reduction in IC(50) values of approximately 12 500-fold, 100-fold, and 125-fold compared with HSVTK, mutant 30 or MGMK/HSVTK, respectively. In vitro bystander analyses show that 5% of MGMK/30-expressing cells are sufficient to induce 75% of tumor cell killing. In an xenograft tumor model, MGMK/30 displays the greatest inhibition of tumor growth at a GCV concentration (1 mg kg(-1)) that has no effect on wild-type HSVTK-, MGMK/HSVTK-, or mutant 30-transfected cells. Another fusion construct, MGMK/SR39, sensitizes rat C6 glioma cells to GCV by 2500-fold or 25-fold compared with HSVTK or MGMK/HSVTK, respectively. In vitro analyses show similar IC(50) values between cells harboring SR39 and MGMK/SR39, although MGMK/SR39 seems to elicit stronger bystander killing effects in which 1% of MGMK/SR39-transfected cells result in 60% cell death. In a xenograft tumor model, despite observable tumor growth inhibition, no statistical significance in tumor volume was detected between mice harboring SR39- and MGMK/SR39-transfected cells when dosed with 1 mg kg(-1) GCV. However, at a lower dose of GCV (0.1 mg kg(-1)), MGMK/SR39 seems to have slightly greater tumor growth inhibition properties compared with SR39 (P< or =0.05). In vivo studies indicate that both mutant fusion proteins display substantial improvements in bystander killing in the presence of 1 mg kg(-1) GCV, even when only 5% of the tumor cells are transfected. Such fusion mutants with exceptional prodrug converting properties will allow administration of lower and non-myelosuppressive doses of GCV concomitant with improved tumor killing and as such are promising candidates for translational gene therapy studies.
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PMID:Fusion enzymes containing HSV-1 thymidine kinase mutants and guanylate kinase enhance prodrug sensitivity in vitro and in vivo. 1976 47