Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Patent
1984-01-05
1987-01-20
Nucker, Christine M.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
435 5, 435 29, 435 34, C12Q 148
Patent
active
046379770
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method of determining the levels of the isoenzyme dTk (deoxythymidine kinase) in human or animal body fluids or cell samples. The invention further relates to the use of said method for the diagnosis and prognostics of diseases dominated by ATP (adenosinetriphosphate) mediated dTk activity, such as cancers, tumours and certain viral infections as well as of diseases characterized by CTP (cytidinetriphosphate) mediated dTk activity, such as HSV (Herpes Simplex Virus) type 1 and type 2 and VZV (Varicells Zoster Virus) infections. The invention also relates to the use of said method for dTk isoenzyme typing.
BACKGROUND OF THE INVENTION
The enzyme deoxythymidine kinase (dTk) provides the eucaryotic cell with a means for utilizing deoxythymidine (dT), which is not an intermediate in the thymidylate synthesis de novo. For this reason dTk is considered a salvage enzyme, introducing dT into the DNA metabolism. As the major dTk form of mammalian cell is only present during cell division, the dTk has been denominated scavenger enzyme.
Three different cellular isoenzymes in human cells have been described. The cytosolar dTk, called dTk-F, which occurs in optimal amounts in dividing cells (stages Gl to S) (Bello, Exptl. Cell Res. 89: 263, 1974; Littlefield, Biochim. Biophys. Acta. 115: 398, 1966) and is more or less absent in resting cells. In humans this enzyme is coded for in chromosome 17 near the galactokinase locus. The second cellular isoenzyme is the mitochondrial, denominated dTk-A, which is present in the mitochondrial matrix. The activity of this dTk remains relatively constant during the different cell stages (Adelstein et al; Develop. Biol. 216: 537, 1971), and dTk-A is coded for by chromosome 16. The third dTk, a minor activity called dTk-B, has only been reported in continuous cell lines HeLa and KB and is said to be confined to the inside of the mitochondrial membrane (reviewed by Kit, Pharmacol. Ther. 4: 501, 1979).
The three cellular dTks differ in biochemical properties. The dTk-F and dTk-B, which are quite similar, are distinguished, besides as to localization, by isoelectric focusing, having different pI, and by electrophoretic mobility. In contrast to dTk-F and dTk-B, the dTk-A accepts cytidinetriphosphate (CTP) as a phosphate donor and is not as sensitive as the others to dTTP (deoxythymidinetriphosphate) feedback inhibition. The dTk-A also phosphorylates deoxycytidine (dC) and is inhibited by dCTP (reviewed by Kit, Pharmacol. Ther. (4: 501, 1979).
With regard to viruses, specific isoenzymes, coded by the viral genome, have been shown in the cell after infection with viruses from the Herpes group and the Pox group. Enzymatically the human virus specific dTks resemble dTk-A, except for the vaccinia dTk which cannot utilize CTP as phosphate donor and not either can phosphorylate deoxycytidine. This dTk is easily distinguished from the human cellular dTks by electrophoresis (Kit et al., Progr. Med. Virol. 21: 13, Karger Basel 1975). Both the HSV dTks and the VZV dTk have a broader spectrum of possible phosphate donors and accept different pyrimidines and pyrimidine analogues as substrates (Cheng et al, Biochim. Biophys. Acta. 452: 370, 1976, and J. Virol. 31: 172, 1979). The competitive blocking of dTk isoenzyme mediated dT conversion to dTmp (deoxythymidinemonophosphate) exerted by the dT-analogue 2'-deoxy-5-iodourine (IUdR), has been known for a long time. The use of radio-labeled IUdR directly as a substrate to gain high sensitivity in assays of viral dTks was shown by us (Gronowitz & Kallander, Infec. Immun. 29: 425, 1980).
As mentioned dTk-F occurrence in cells is coupled to cell proliferation, and it is more or less absent in the differentiated cell (Munch-Petersen & Tyrsted, Biochim. Biophys. 478: 364, 1977). Studies of dTk activity in transplantable mouse tumours have revealed high dTk activities with correlation to growth rate (Bresnick et al, Cancer Res. 29: 1969, and Cancer Res. 31: 743, 1971). Recent reports have demonstrated enhanced dTk-F in periph
REFERENCES:
patent: 4302535 (1981-11-01), Skopek et al.
Infection and Immunity, vol. 36, pp. 30-37, published Apr. 1982 (Kallander, C. F. R. et al.) "Human Serum Antibodies to Carcella-Zoster Virus Thymidine Kinase).
Infection and Immunity, vol. 29, pp. 425-434, published Aug. 1980, (Gronowitz, J. S., Kallander C. F. R.) "Optimized Assay for Thymidine Kinase and its Application to the Detection of Antibodies Against Herpes Simplex Virus Type 1- and 2-Induced Thymidine Kinase).
Journal of Biological Chemistry, vol. 254, pp. 10747-10753, published Nov. 10, 1979 (Chen, M. S. et al.) "Kinetic Studies of Herpes Simplex Virus Type 1-Encoded Thymidine and Thymidylate Kinase, a Multifunctional Enzyme".
Biochimica et Biophysica Acta, vol. 452, pp. 370-381, published 1976 (Cheng Y-C) "Deoxythymidine Kinase Induced in HeLa TK-Cells by Herpes Simplex Virus Type 1 and Type 2".
J. Med. Chem., vol. 23, pp. 962-964 published 1980 (Nakayama, C. et al.) "Thymidine Phosphorylase, Substrate Specificity for 5-Substituted 2-Deozyuridines".
Chemical Abstracts, vol. 96 (1982), Abstract No. 138542s, Izotoptechnike 1981, 24 (3), 109-118.
Gronowitz Jan S.
Kallander Clas F. R.
Nucker Christine M.
Wieder Stephen C.
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