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PubMed2002

Pharmacokinetics and organ distribution of N-methanocarbathymidine, a novel thymidine analog, in mice bearing tumors transduced with the herpes simplex thymidine kinase gene.

PURPOSE: The conformationally rigid nucleoside, N-methanocarbathymidine [(N)-MCT] exerts a potent antiproliferative effect both in vitro and in vivo against murine colon cancer cells (MC38) expressing the herpes simplex virus thymidine kinase gene (MC38/HSV-tk). Metabolic studies have revealed that high levels of (N)-MCT triphosphate accumulate in transduced cells and are incorporated into DNA, resulting in cell death. The objective of the present study was to assess the pharmacokinetic profile of (N)-MCT in C57BL/6 mice bearing nontransduced MC38 and MC38/HSV-tk tumors. METHODS: Male black C57BL/6 mice bearing subcutaneous tumors derived from wildtype and HSV-tk-transduced MC38 murine colon cancer cells in the left and right flank, respectively, were treated i.p. with radiolabeled (N)-MCT (100 mg/kg). Mice were killed at each of the predetermined times after drug administration. Blood, urine, tumors and various organs and tissues were obtained for measurement of drug levels. RESULTS: Plasma and tissue concentrations of (N)-MCT peaked at 0.25-0.5 h. The major pharmacokinetic parameters calculated for (N)-MCT in plasma were: T(1/2)beta 4.7 h, AUC 147 micro g.h/ml, CL 0.69 l/kg per h. The penetration of (N)-MCT into brain and testes was slow. Between 4 and 24 h after drug administration, the levels of (N)-MCT measured in HSV-tk-expressing tumors were significantly higher than in wildtype tumors. HPLC analysis of methanolic extracts of plasma and urine obtained at various times after drug administration revealed no (N)-MCT metabolites in the plasma, and the compound was secreted unchanged in the urine. CONCLUSIONS: After i.p. injection into mice, (N)-MCT was rapidly absorbed and distributed in all organs examined. No drug metabolites were detectable in plasma and the compound was secreted unchanged in urine. These results are essential for the future development and in postulating the most efficient use of (N)-MCT in the HSV-tk enzyme prodrug system for gene therapy approaches for the treatment of cancer.

Adenocarcinoma↗

PubMed1986

Zwitterionic 3'-O-acyl derivatives of thymidine 5'-phosphate as potential sources of intracellular thymidine 5'-phosphate in cells in culture.

A convenient route is described for attachment of acyl groups CO(CH2)nN(Et)2(CH2)mNH(Et)2 (n = 3, m = 2; n = 4, m = 2-4), CO(CH2)nN(Et)2(CH2)mNEt3 (n = 4, m = 2-4), or CO(CH2)4N(CH2CH2)3N(CH2)nCH3 (n = 1 or 9) to O-3' of thymidine 5'-phosphate (TMP). The compounds are prototypes of 5'-nucleotide derivatives in which the two anionic charges could become partially masked in intramolecular anionic-cationic interactions and which might be able to diffuse into mammalian cells to furnish intracellular antimetabolite 5'-nucleotides by hydrolytic loss of a dicationic 3'-O-acyl group. At pH 7.6, 37 degrees C, hydrolyses of the 3'-ester linkages were pseudo first order with t1/2 values in the range 28-85 h. Paper chromatography in n-PrOH-H2O at pH 7.6 showed that type 1 or 2 derivatives were equally or slightly less hydrophobic than TMP (Rf 0.24), whereas the n-decyl type 3 compound (Rf 0.66) was markedly more hydrophobic, apparently because chain branching in group 3 is less than in 1 or 2. A sensitive and specific assay was developed for liberation of intracellular TMP in cultured mouse L fibroblasts in which synthesis of TMP and, hence, of DNA was suppressed by a combination of aminopterin and 5'-amino-5'-deoxythymidine (5'-NH2-dT). The TMP derivatives (100 microM) stimulated DNA synthesis, but omission of 5'-NH2-dT increased stimulation 6.5-fold, suggesting that stimulation occurred via degradation of the derivatives to dT. In confirmation, derivatives of type 2 (n = 4, m = 2 or 4) (100 microM) or type 3 (n = 9) (200 microM), in the presence of aminopterin, did not stimulate DNA synthesis in the LM(TK-) strain of L cells, which is genetically deficient in dT kinase. TMP (1 mM) stimulated DNA synthesis 2-3-fold and appeared to enter LM(TK-) cells without dephosphorylation, because dT (1 mM) gave no stimulation. If TMP is assumed to enter solely by passive diffusion, the inactivity of the TMP derivatives can be ascribed in part to their 2-fold higher molecular weight which can be expected to reduce flux through natural membranes ca. 16-fold.

Acylation↗

PubMed1984

Thymidylate synthetase-deficient mouse FM3A mammary carcinoma cell line as a tool for studying the thymidine salvage pathway and the incorporation of thymidine analogues into host cell DNA.

A thymidylate (dTMP) synthetase-deficient murine mammary carcinoma cell line (FM3A/TS-), auxotrophic for thymidine (dThd), proved extremely useful for studying the dependence of cell growth on the exogenous supply of dThd, the relation between cell growth and DNA synthesis, and the ability of a series of 25 5-substituted 2'-deoxyuridines (dUrd) to substitute for dThd in sustaining cell growth. FM3A/TS-cells did not proliferate unless dThd was supplied to the cell culture medium. The 5-halogenated dUrd derivatives 5-chloro-dUrd, 5-bromo-dUrd and 5-iodo-d Urd also sustained FM3A/TS- cell growth. The extents of incorporation of [methyl-3H]dThd and 5-iodo-[6-3H]dUrd into DNA were closely correlated with their stimulatory effects on FM3A/TS- cell growth. This suggests that the stimulatory effects of the dUrd analogues on the growth rate of FM3A/TS- cells may be considered as evidence for their incorporation into host cell DNA. Based on this premise it is postulated that, in addition to 5-chloro-dUrd, 5-bromo-dUrd, 5-iodo-dUrd and dThd itself, the following dThd analogues are also incorporated into FM3A/TS- cell DNA (in order of the extent to which they are incorporated): 5-hydroxy-dUrd greater than 5-propynyloxy-dUrd greater than 5-ethyl-dUrd greater than 5-ethynyl-dUrd approximately 5-vinyl-dUrd. Thus, the dTMP synthetase-deficient FM3A/TS- cell line represents a unique system to dissociate the de novo and salvage pathways of dTMP biosynthesis and to distinguish those dUrd analogues that are incorporated into DNA from those that are not.

Animals↗

PubMed1981

Herpes simplex virus thymidine kinase activity of thymidine kinase-deficient Escherichia coli K-12 mutant transformed by hybrid plasmids.

A hybrid plasmid (pAGO) that contains the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in the form of a 2-kilobase-pair (kbp) Pvu II fragment inserted at the Pvu II site of plasmid pBR322 was used to transform TK- Escherichia coli K-12 strain KY895. pAGO-transformed KY895 cells exhibited partially restored ability to incorporate [3H]dThd into DNA and an HSv-1-specific TK activity. Bacteria cured of plasmid pAGO (or transformed by plasmid pBR322) did not show enhanced incorporation of [3H]dThd into DNA or HSV-1 TK activity. Plasmid pMH1A was derived from pAGO by deletion of 2067 bp of DNA sequence from pBR322 and 105 bp from the HSV-1 TK gene. E. coli K-12 strain KY895 cells transformed by pMH1A did not show enhanced incorporation of [3H]dThd into bacterial DNA, although pMH1A DNA isolated from transformed KY895 cells, like pAGO DNA, did transform TK- mouse fibroblast [LM(TK-)] cells to the TK+ phenotype. The expression of HSV-1 TK activity by E. coli K-12 suggests that intervening sequences may be absent from the coding region of HSV-1 tk or that the coding region of the gene possesses short intervening sequences which do not disrupt the translational reading frame.

Animals↗

PubMed1999

Interaction of the recombinant herpes simplex virus type 1 thymidine kinase with thymidine and aciclovir: a kinetic study.

Herpes Simplex Virus type 1 thymidine kinase (HSV 1 TK) is a key target for antiviral therapy and it phosphorylates a broad spectrum of nucleosides and nucleotides. We report the results from kinetic and inhibition experiments with HSV 1 TK, and show that there is a preferred, but not exclusive, binding order of substrates, i.e. dT binds prior to ATP. Furthermore, the results provide new informations on the mechanism of binding suggesting that HSV1 TK undergoes conformational changes during the catalytic cycle.

Acyclovir↗

PubMed1971

Transfer of thymidine kinase to thymidine kinaseless L cells by infection with ultraviolet-irradiated herpes simplex virus.

L cells lacking thymidine kinase (TK) activity (Ltk(-) cells) have been stably transformed to a TK-positive phenotype by infection with ultraviolet-irradiated herpes simplex virus (HSV-UV). The highest frequency of the Ltk(-) to Ltk(+) transformation observed in these experiments was approximately 10(-3), whereas no measurable transformation was observed (less than 10(-8)) in the absence of HSV-UV infection. Cell lines of HSV-transformed Ltk(+) cell lines contain 7 to 24 times as much TK activity as do the parental Ltk(-) cells, and they have been maintained in culture for a period exceeding 8 months. The kinetics of thermal inactivation of the TK activity derived from an Ltk(+) HSV-transformed cell line and the TK activity from Ltk(-) cells lytically infected with infectious HSV are similar. Both of these TK activities are much more thermolabile than the TK activity present in wild-type L cells. A mutant strain of HSV which does not induce TK activity during lytic infection does not cause the Ltk(-) to Ltk(+) transformation. These data suggest that either an HSV TK gene has been transferred to Ltk(-) cells or that an HSV gene product has caused the expression of a previously repressed cellular enzyme.

Animals↗

PubMed1998

Thymidine phosphorylase moderates thymidine-dependent rescue after exposure to the thymidylate synthase inhibitor ZD1694 (Tomudex) in vitro.

The inhibition of de novo thymidine (dThd) synthesis by the novel folate-based thymidylate synthase (TS) inhibitor ZD1694 (Tomudex) can achieve tumor cell-specific cytotoxicity in vivo. However, nucleosides in the surrounding microenvironment of tumors may be used by the salvage pathway to regenerate any depleted pools, thus providing an efficient mechanism through which to circumvent the ZD1694-dependent toxicity. Anabolism of dThd to dTMP by dThd kinase (TK) is the first committed step in the dThd salvage pathway. However, dThd phosphorylase (dThdPase) can compete with TK by catalyzing the reversible phosphorolytic cleavage of dThd to thymine and deoxyribose 1-phosphate and rendering the salvaged dThd metabolically unavailable. Both TK and dThdPase are up-regulated in some tumors, and their relative importance is not fully defined. We have studied the influence of dThdPase expression on the capacity of exogenous dThd to reverse ZD1694-dependent growth inhibition and have shown that both intra- and extracellular dThdPase activity can effectively moderate dThd-rescue. This suggests that tumor levels of dThdPase may be an important factor in the outcome of ZD1694 therapy.

Antimetabolites, Antineoplastic↗

PubMed1992

Location and characterization of the bovine herpesvirus type 4 thymidine kinase gene; comparison with thymidine kinase genes of other herpesviruses.

The location and nucleotide sequence of the bovine herpesvirus type 4 (BHV-4) thymidine kinase (TK) gene was determined. The coding region of the TK gene is 1335 nucleotides long and corresponds to a polypeptide of 445 amino acids. Comparison of TK amino acid sequences of BHV-4 and 16 herpesvirus TKs reveals a greater homology to those of the gammaherpesviruses EBV and specially HVS, than to those of alphaherpesviruses. The open reading frames detected in the vicinity of TK gene were homologous to the corresponding ones in other herpesviruses.

Amino Acid Sequence↗

PubMed1987

Fowlpox virus thymidine kinase: nucleotide sequence and relationships to other thymidine kinases.

The thymidine kinase (TK) gene of fowlpox virus (FPV) is located in a 2.2-kb HindIII-ClaI fragment derived from a 5.5-kb EcoR1 fragment of the FPV genome. The TK gene was mapped to the region of a 700-bp XbaI fragment contained within this HindIII-ClaI fragment. Nucleotide sequence analysis of this region revealed an open reading frame of 183 codons. Identification of this region as the FPV TK gene was confirmed by its homology with the vaccinia virus TK at both the nucleotide and amino acid levels. The derived FPV TK polypeptide has a calculated molecular weight of 20,380 and is six amino acids larger than the vaccinia virus TK gene product. We have reported previously that the FPV TK gene operates in vaccinia virus without the requirement for a vaccinia virus promoter. The sequence homologies between the two TK promoters substantiated this observation. Northern blot analysis of RNAs from cells infected with a vaccinia virus recombinant expressing the FPV TK gene showed major (700 nucleotide) and minor (1000 nucleotide) transcripts from the FPV TK gene. The deduced amino acid sequence of the FPV TK has significant homology with the TKs from chicken, man, and three other poxviruses, but shows no homology with herpes simplex virus TK. Comparisons of the homologous sequences indicated that the "core" of the enzyme has probably evolved in poxviruses four times as quickly as in vertebrates. Characterization of the FPV TK gene may facilitate the construction of recombinant FPVs as vehicles for the delivery of vaccine antigens to poultry and other avian species.

Amino Acid Sequence↗

PubMed1985

Actions of exogenous heparan sulfate and hyaluronic acid on growth and thymidine incorporation of normal and transformed human fibroblasts. A comparison with the effects of high cell density and low serum concentration and a warning against thymidine incorporation as a measure of DNA synthesis.

Treatment of normal human (WI-38) cells with exogenous heparan sulfate (HS) reduced cell growth and incorporation of radio-isotope-labeled thymidine (TdR) into DNA. In spite that growth of their transformants (WI-38 CT-1) was enhanced by HS treatment, transformed cells also decreased in TdR incorporation thereby. This peculiar observation was explained by a reduction of TdR uptake, leading to a decrease in specific radioactivity of newly synthesized DNA. The changes in cell growth and TdR incorporation by HS treatment were revealed to be similar to the changes with increasing cell density rather than by serum starvation.

Biological Transport↗

PubMed1991

Escherichia coli thymidine kinase: nucleotide sequence of the gene and relationships to other thymidine kinases.

The thymidine kinase (TK)-encoding gene (tdk) of Escherichia coli is located at min 27 of the E. coli genetic map. Sequence analysis of this region revealed an open reading frame of 205 codons. Identification of this region as the E. coli tdk gene was confirmed by its similarity to other TK-encoding genes. The E. coli amino acid (aa) sequence showed significant similarity to the corresponding TK polypeptides of vertebrates and large DNA viruses, but showed no similarity to known herpes virus TK enzymes. Mapping of highly conserved positions among all sequences indicates the importance of these residues for catalytic activity and may facilitate further functional studies. Using a distance matrix method, the evolutionary relationships among the TK aa sequence of poxviruses, eukaryotes and prokaryotes were analyzed and a potential phylogenetic tree was established.

Amino Acid Sequence↗

PubMed2001

Specific recognition of cytosolic thymidine kinase in the human lung tumor by monoclonal antibodies raised against recombinant human thymidine kinase.

Anti-TK monoclonal antibodies (mAbs) were raised against recombinant human cytosolic thymidine kinase (rhTK) and characterized by Western immunoblotting, enzyme-linked immunosorbent assay (ELISA) and immunostaining of tumor cells. Twenty-three clones of TK mAbs were characterized to recognize specifically not only rhTK produced by Escherichia coli but also TK subunit of 25 kDa in human lung cancer. The anti-TK mAbs reacted specifically with cytosolic TK but not with mitochondrial TK. Only one clone of the mAbs inhibited the catalytic activity of TK. By solid phase sandwich enzyme immunoassay using these mAbs, we could quantitate the cytosolic TK content in tissues. Immunohistochemical staining analysis using one of the TK mAbs showed that human lung adenocarcinoma and squamous cell carcinoma exhibited much higher staining intensity than stromal cells. These mAbs are useful for biochemical studies on the regulation of human TK in proliferating cells such as tumor cells and for diagnosis of highly proliferating tumors.

Adenocarcinoma↗

PubMed2002

Establishment and use of a cell line expressing HSV-1 thymidine kinase to characterize viral thymidine kinase-dependent drug-resistance.

To understand the mechanisms of antiviral drug resistance and to have a system to examine the cytotoxicity of herpes simplex virus type 1 (HSV-1) inhibitors that are thymidine kinase (TK)-dependent, we have constructed a plasmid pFTK1 by inserting a DNA fragment containing the TK gene of HSV-1 strain F into the eukaryotic expression vector pcDNA3.1/His A. TK-deficient 143B cells were transfected with this vector and neomycin-resistant cells were selected. Cell survival in HAT medium and TK activity of the cell lysates were examined to ascertain HSV-1 TK expression. A cell line expressing the viral TK gene, FTK143B (FTK), was established and used for characterization of two laboratory-derived TK-deficient drug-resistant HSV-1 mutants of strain F. The antiviral activities of several drugs, mostly nucleoside analogues, were compared in the Vero, 143B and FTK cell culture systems. We showed that both mutant viruses lost their resistance to acyclovir and to other HSV-1 TK-dependent compounds in FTK cells but not in Vero and 143B cells. Significantly increased cytotoxicity of ganciclovir and (E)-5-(2-bromovinyl)-2'-deoxyuridine was also observed in the FTK cells. This HSV-1 TK gene-transfected cell model is a useful tool to rapidly determine HSV-1 drug resistance at the viral TK level.

Animals↗

PubMed2004

Discovery of bicyclic thymidine analogues as selective and high-affinity inhibitors of Mycobacterium tuberculosis thymidine monophosphate kinase.

Thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt) represents an attractive target for selectively blocking bacterial DNA synthesis. Hereby, we report on the discovery of a novel class of bicyclic nucleosides (10 and 11) and one dinucleoside (12), belonging to the most selective inhibitors of TMPKmt discovered so far.

Antitubercular Agents↗

PubMed2000

Herpes simplex virus thymidine kinase gene therapy in experimental rat BT4C glioma model: effect of the percentage of thymidine kinase-positive glioma cells on treatment effect, survival time, and tissue reactions.

Herpes simplex virus thymidine kinase (HSV-tk) gene transfer and ganciclovir (GCV) administration have been suggested for the treatment of malignant gliomas. To understand tissue responses and possible ways to improve the treatment effect, we studied tumor growth, tissue reactions, and survival time after HSV-tk/GCV treatment in a syngeneic BT4C rat glioma model by mixing various ratios of stably transfected HSV-tk-expressing BT4C-tk glioma cells with wild-type BT4C glioma cells (percentage of BT4C-tk cells: 0%, 1%, 10%, 30%, 50%, and 100%), followed by injection into BDIX rat brains (n = 79). With the exception of some animals with end-stage tumors, very little astroglia or microglia reactivity was detected in the wild-type tumors as analyzed by immunocytochemistry using glial fibrillary acid protein (GFAP)-, vimentin-, human histocompatibility leukocyte antigen-DR-, OX-42-, and CD68-specific monoclonal antibodies. After 14 days of GCV treatment, tumors induced with > or = 10% BT4C-tk cells showed a significant reduction in tumor size (P < .05) and prolonged survival time (P < .01). Astrogliosis, as indicated by a strong GFAP and vimentin immunoreactivity, was seen in the tumor scar area. GFAP and vimentin reactivity was already present after the GCV treatment in tumors induced with 1% BT4C-tk cells. Much less human histocompatibility leukocyte antigen-DR-positive microglia was seen in the treated animals, indicating low microglia reactivity and immunoactivation against the tumor. However, GCV-treated tumors were positive for apoptosis, indicating that apoptosis is an important mechanism for cell death in the BT4C-tk glioma model. Our results suggest that > or = 10% transfection efficiency is required for a successful reduction in BT4C glioma tumor size with HSV-tk/GCV treatment in vivo. Tissue reactions after 14 days of GCV treatment are characterized by astrogliosis and apoptosis, whereas microglia response and immunoactivation of the brain cells appear to play a minor role. Stimulation of the microglia response by gene transfer or other means might improve the efficacy of the HSV-tk/GCV treatment in vivo.

Animals↗

PubMed1989

Thymidine kinase synthesis is repressed in nonreplicating muscle cells by a translational mechanism that does not affect the polysomal distribution of thymidine kinase mRNA.

The molecular basis for replication-dependent expression of thymidine kinase (TK) activity (EC 2.7.1.21) was investigated in mouse skeletal muscle cells transformed with multiple copies of the chicken TK gene. When shifted to mitogen-depleted medium, proliferating myoblasts irreversibly withdraw from the cell cycle and commit to terminal differentiation. Early after commitment, postreplicative myocytes maintain nearly proliferative levels of TK mRNA but have greatly reduced levels of TK activity. Metabolic labeling studies with [35S]methionine indicated that the decrease in TK activity was associated with a 10-fold reduction in the rate of TK protein synthesis. Commitment had little effect on the stability or catalytic efficiency of TK protein. The decrease in TK synthetic rate in the continued presence of TK mRNA indicated that translation of TK mRNA was repressed in committed cells. The distribution of TK mRNA between ribonucleoprotein particles and polysomes was determined. In both proliferative cells and committed cells, TK mRNA levels were maximal in polysomes containing five to seven ribosomes. Thus, the synthesis of TK protein in nonreplicating muscle cells was inhibited by a translational mechanism that did not alter the average number of ribosomes engaged by TK mRNA.

Animals↗

PubMed1996

Overexpression of thymidine kinase mRNA eliminates cell cycle regulation of thymidine kinase enzyme activity.

Expression of thymidine kinase (TK) enzyme activity and mRNA is strictly S phase-specific in primary cells. In contrast, DNA tumor virus-transformed cells have enhanced and constitutive levels of TK mRNA during the whole cell cycle. Their TK protein abundance, however, still increases at the G1-S transition and stays high throughout G2 until mitosis. Therefore, post-transcriptional control must account for the decoupling of TK mRNA from protein synthesis in G1. To characterize the underlying mechanism, we studied the consequences of TK mRNA abundance on the cell cycle-dependent regulation of TK activity in nontransformed cells. Constitutive as well as conditional human and mouse TK cDNA vectors were stably transfected into mouse fibroblasts, which were subsequently synchronized by centrifugal elutriation. Low constitutive TK mRNA expression still resulted in a fluctuation of TK activity with a pronounced maximum in S phase. This pattern of cell cycle-dependent TK activity variation reflected the one in primary cell but is caused by post-transcriptional control. Increasing overexpression of TK transcripts after hormonal induction compromised this regulation. At the highest constant mRNA levels, regulation of enzyme activity was totally abolished in each phase of the cell cycle. These data indicate that post-transcriptional regulation of TK is tightly coupled to the amount of mRNA; high concentrations apparently titrate a factor(s) required for repressing TK production during G1 and presumably also G2.

3T3 Cells↗

PubMed1988

Evolution of the herpes thymidine kinase: identification and comparison of the equine herpesvirus 1 thymidine kinase gene reveals similarity to a cell-encoded thymidylate kinase.

We have identified the equine herpesvirus 1 (EHV-1) thymidine kinase gene (TK) by DNA-mediated transformation and by DNA sequencing. Alignment of the amino acid sequence of the EHV-1 TK with the TKs from 3 other herpesviruses revealed regions of homology, some of which correspond to the previously identified substrate binding sites, while others have as yet, no assigned function. In particular, the strict conservation of an aspartate within the proposed nucleoside binding site suggests a role in ATP binding for this residue. Comparison of 5 herpes TKs with the thymidylate kinase of yeast revealed significant similarity which was strongest in those regions important to catalytic activity of the herpes TKs, and, therefore we propose that the herpes TK may be derived from a cellular thymidylate kinase. The implications for the evolution of enzyme activities within a pathway of nucleotide metabolism are discussed.

Amino Acid Sequence↗