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Quaternary structures of recombinant, cellular, and serum forms of thymidine kinase 1 from dogs and humans.

ABSTRACT: BACKGROUND: Thymidine kinase 1 (TK1) is a salvage enzyme involved in DNA precursor synthesis, and its expression is proliferation dependent. A serum form of TK1 has been used as a biomarker in human medicine for many years and more recently to monitor canine lymphoma. Canine TK1 has not been cloned and studied. Therefore, dog and human TK1 cDNA were cloned and expressed, and the recombinant enzymes characterized. The serum and cellular forms of canine and human TK1 were studied by size-exclusion chromatography and the level of TK1 protein was determined using polyclonal and monoclonal anti-TK1 antibodies. RESULTS: Canine TK1 phosphorylated the thymidine (dThd) analog 3'-azido-thymidine (AZT) as efficiently as it did dThd, whereas AZT phosphorylation by human TK1 was less efficient than that of dThd. Dog TK1 was also more thermostable and pH tolerant than the human enzyme. Oligomeric forms were observed with both enzymes in addition to the tetrameric and dimeric forms. Cellular TK1 was predominantly seen in dimeric and tetrameric forms, in the case of both dog TK1 from MDCK cells and human TK1 from CEM cells. Active serum TK1 was found mainly in a high molecular weight form, and treatment with a reducing agent shifted the high molecular weight complex to lower molecular weight forms with reduced total activity. Western blot analysis demonstrated a polypeptide of 26 kDa (dog) and 25 kDa (human) for cellular and serum TK1. There was no direct correlation between serum TK1 activity and protein level. It appears that a substantial fraction of serum TK1 is not enzymatically active. CONCLUSIONS: These results suggest that the serum TK1 protein differs from cellular or recombinant forms, is more active in high molecular weight complexes, and is sensitive to reducing agents. The results presented here provide important information for the future development and use of serum TK1 as a diagnostic biomarker in human and veterinary medicine.

SUBMITTER: Hanan S

PROVIDER: S-EPMC3411398 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Quaternary structures of recombinant, cellular, and serum forms of thymidine kinase 1 from dogs and humans.

Hanan Sharif S Jagarlamudi Kiran Kumar KK Liya Wang W Ellen He H Staffan Eriksson E

BMC biochemistry 20120628

<h4>Background</h4>Thymidine kinase 1 (TK1) is a salvage enzyme involved in DNA precursor synthesis, and its expression is proliferation dependent. A serum form of TK1 has been used as a biomarker in human medicine for many years and more recently to monitor canine lymphoma. Canine TK1 has not been cloned and studied. Therefore, dog and human TK1 cDNA were cloned and expressed, and the recombinant enzymes characterized. The serum and cellular forms of canine and human TK1 were studied by size-ex ...[more]

PMID: 22741536

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Project description:BackgroundDirectly targeting therapeutic suicide gene to a solid tumor is a hopeful approach for cancer gene therapy. Treatment of a solid tumor by an effective vector for a suicide gene remains a challenge. Given the lack of effective treatments, we constructed a bifidobacterial recombinant thymidine kinase (BF-rTK) -ganciclovir (GCV) targeting system (BKV) to meet this requirement and to explore antitumor mechanisms.MethodsBifidobacterium (BF) or BF-rTK was injected intratumorally with or without ganciclovir in a human colo320 intestinal xenograft tumor model. The tumor tissues were analyzed using apoptosis antibody arrays, real time PCR and western blot. The colo320 cell was analyzed by the gene silencing method. Autophagy and necroptosis were also detected in colo320 cell. Meanwhile, three human digestive system xenograft tumor models (colorectal cancer colo320, gastric cancer MKN-45 and liver cancer SSMC-7721) and a breast cancer (MDA-MB-231) model were employed to validate the universality of BF-rTK + GCV in solid tumor gene therapy. The survival rate was evaluated in three human cancer models after the BF-rTK + GCV intratumor treatment. The analysis of inflammatory markers (TNF-α) in tumor indicated that BF-rTK + GCV significantly inhibited TNF-α expression.ResultsThe results suggested that BF-rTK + GCV induced tumor apoptosis without autophagy and necroptosis occurrence. The apoptosis was transduced by multiple signaling pathways mediated by FasL and TNFR2 and mainly activated the mitochondrial control of apoptosis via Bid and Bim, which was rescued by silencing Bid or/and Bim. However, BF + GCV only induced apoptosis via Fas/FasL signal pathway accompanied with increased P53 expression. We further found that BF-rTK + GCV inhibited the expression of the inflammatory maker of TNF-α. However, BF-rTK + GCV did not result in necroptosis and autophagy.ConclusionsBF-rTK + GCV induced tumor apoptosis mediated by FasL and TNFR2 through the mitochondrial control of apoptosis via Bid and Bim without inducing necroptosis and autophagy. Furthermore, BF-rTK + GCV showed to repress the inflammation of tumor through downregulating TNF-α expression. Survival analysis results of multiple cancer models confirmed that BF-rTK + GCV system has a wide field of application in solid tumor gene therapy.

Dataset Information

Quaternary structures of recombinant, cellular, and serum forms of thymidine kinase 1 from dogs and humans.

Publications

Quaternary structures of recombinant, cellular, and serum forms of thymidine kinase 1 from dogs and humans.

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