Thymidine kinase
From Wikipedia, the free encyclopedia
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thymidine kinase 1, soluble
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| Identifiers | |
| Symbol(s) | TK1 |
| Entrez | 7083 |
| OMIM | 188300 |
| RefSeq | NM_003258 |
| UniProt | P04183 |
| Other data | |
| EC number | 2.7.1.21 |
| Locus | Chr. 17 q23.2-25.3 |
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thymidine kinase 2, mitochondrial
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| Identifiers | |
| Symbol(s) | TK2 |
| Entrez | 7084 |
| OMIM | 188250 |
| RefSeq | NM_004614 |
| UniProt | O00142 |
| Other data | |
| EC number | 2.7.1.21 |
| Locus | Chr. 16 [1] |
Thymidine kinase TK, is an enzyme, a phosphotransferase (a kinase): 2'-deoxythymidine kinase, ATP-thymidine 5'-phosphotransferase, EC 2.7.1.75. It can be found in most living cells. It is present in two forms in mammalian cells, TKI and TKII. Certain viruses also have genetic information for expression of viral thymidine kinases. Thymidine kinase catalyses the reaction: Thd + ATP = TMP + ADP, where Thd is deoxythymidine, ATP is (energy rich) adenosine 5’-triphosphate, TMP is deoxythymidine 5’-phosphate and ADP is adenosine 5’-diphosphate. Thymidine kinases have a key function in the synthesis of DNA and thereby in cell division, as they are part of the unique reaction chain to introduce deoxythymidine into the DNA. Deoxythymidine is present in the body fluids as a result of degradation of DNA from food and from dead cells. Thymidine kinase is required for the action of many antiviral drugs. It is used to select hybridoma cell lines in production of monoclonal antibodies. In clinical chemistry it is used as a proliferation marker in the diagnosis, control of treatment and follow-up of malignant disease, mainly of hematological malignancies.
[edit] History
The incorporation of thymidine in DNA was demonstrated around 1950. Somewhat later, it was shown that this incorporation was preceded by phosphorylation, and around 1960 the enzyme responsible was purified and characterized. It was shown that higher organisms have two isoenzymes, that are chemically very different, TKI and TKII. The former was first found in fetal tissue, the second was more abundant in adult tissue, and initially they were termed fetal and adult thymidine kinase. Soon it was shown that TKI was present in the cytoplasm only in anticipation of cell division (cell cycle dependent), while TKII was located in mitochondria and cell cycle independent. The genes of the two types were localized in the mid seventies. The gene for TKI was cloned and sequenced. The corresponding protein has a molecular weight of about 25 kD. Genes for virus specific thymidine kinases have been identified in Herpes simplex virus, Varicella zoster virus and Epstein-Barr virus.
[edit] Physiological context
Deoxythymidine monophosphate, the product of the reaction catalysed by thymidine kinase, is in turn phosphorylated to deoxythymidine diphosphate and further to deoxythymidine triphosphate. The triphosphate is included in a DNA string, a reaction catalysed by a DNA polymerase (enzyme) and a complementary DNA string (or an RNA string in the case of reverse transcriptase, an enzyme present in retrovirus). Deoxythymidine monophosphate is produced by the cell in two different reactions: either by phosphorylation of deoxythymidine as described above or by methylation of deoxyuridine monophosphate, that is a product of other metabolic pathways unrelated to thymidine. The second route is used by the cell under normal conditions and it is sufficient to supply deoxythymidine monophosphate for DNA repair. When a cell prepares to divide, a complete new set-up of DNA is required, and the requirement for building blocks, including deoxythymidine triphosphate increases. Cells prepare for cell division by making some of the enzymes required during the division. They are not normally present in the cells and are downregulated and degraded afterwards. Such enzymes are called salvage enzymes. Thymidine kinase I is such a salvage enzyme while thymidine kinase II is not cell cycle dependent.
[edit] Thymidine kinase for identification of dividing cells
The first indirect use of thymidine kinase in biochemical research was the identification of dividing cells by incorporation of radiolabeled thymidine and subsequent measurement of the radioactivity or autoradiography to identify the dividing cells. For this purpose tritiated thymidine is included in the growth medium. In spite of errors in the technique it is still used to determine the growth rate of malignant cells and to study the activation of lymphocytes in immunology.
[edit] Thymidine kinase for drug design
Some drugs are specifically directed against dividing cells. They can be used against tumour and viral disease, as the diseased cells proliferate much more frequently than normal cells. The mechanism most often used is phosphorylation of a thymidine analogue by thymidine kinase. The monophosphate is further phosphorylated to the corresponding triphosphate and incorporated in the growing DNA chain, where it may stop the growth of the chain as it is chemically unable to bind a further base, or because it makes the resulting DNA chain defective. Several HIV drugs belong to this class including AZT. Some antiviral drugs make use of the wide specificity of viral thymidine kinase than that of human thymidine kinases. For treatment of viral diseases a prodrug is used, that in itself is not toxic, but is converted to a toxic drug by phosphorylation by viral thymidine kinase, while the human thymidine kinase, with its more narrow specificity, is unable to phosphorylate. In this way only the cells infected by the virus will be exposed to the drug. Such drugs are Acyclovir and Ganciclovir whose monophosphates are further phosphorylated in infected cells to highly toxic triphosphates. Such drugs are only effective against virus from the herpes group with their own thymidine kinase. The sensitivity of cells containing herpesvirus thymidine kinase gene as a “suicide gene” has also been used for experimental treatment of cancer. In animal models thymidine kinase was introduced into tumour cells via a herpes simplex viral vector. Later the animals were treated with the prodrug Ganciclovir. In animals treated with the drug, the tumours disappeared while in control animals the tumour continued to grow. When the thymidine kinase gene was introduced only part of the tumour cells received the gene, but tumour cells without the gene still died. This is called the “bystander effect”, and results from the diffusion of the active enzyme to neighboring cells. The viral vector can also diffuse to nearby cells as well. As little as 10% of the cells must contain the viral thymidine kinase gene to kill the tumour. A similar use of the thymidine kinase makes use of the presence in some tumour cells of substances not present in normal cells (tumor markers). Such tumor markers are for instance CEA (Carcino Embryonic Antigen) and AFP (Alpha Feto Protein). The genes for these tumor markers may be used as promoter genes for thymidine kinase. Thymidine kinase will then be activated in cells expressing the tumour marker but not in normal cells, and treatment with for example Ganciclovir will kill the tumour cells but not normal cells. Introduction of herpesvirus thymidine kinase into tumour cells may be problematic, as the transfection requires injection of modified retrovirus. The introduction of live retroviruses for medical purposes may cause as many problems as it solves. Another use of the “suicide gene” concept is to use the herpesvirus thymidine kinase gene as a safety system in immunotherapy. For instance, it has been suggested to treat HIV positive patients by harvesting their own T-lymphocytes, expanding a population directed to HIV where the herpesvirus thymidine kinase gene has been introduced as a safety system. It is hoped that the T-lymphocytes will kill HIV and HIV-infected cells. If problems occur when these T-lymphocytes are re-infused in the patient the infused cells can easily be killed by infusion of Ganciclovir.
[edit] Thymidine kinase for selection of hybridomas
Hybridomas are cells obtained by fusing tumour cells (which can divide infinitely) and immunoglobulin producing lymphocytes. Hybridomas can be expanded to produce large quantities of immunoglobulins with a given unique specificity (monoclonal antibodies). One problem is to single out the hybridomas from the large excess of unfused cells after the cell fusion. One common way to solve this is to use thymidine kinase negative cell lines for the fusion. The thymidine kinase negative cells are obtained by growing the cell line in the presence of thymidine analogues, that kill the thymidine kinase positive cells. The negative cells can then be expanded and used for the fusion. After fusion, the cells are grown in a medium with methotrexate that blocks the de novo synthesis of thymidine monophosphate. The unfused cells from the thymidine kinase deficient cell line die as they have no source of thymidine monophosphate. The lymphocytes eventually die as they are not “immortal”. Only the hybridomas that have “immortality” from their cell line ancestor and thymidine kinase from the lymphocyte survive. Those that produce the desired antibody are then selected and cultured to produce the monoclonal antibody.
[edit] Thymidine kinase in clinical chemistry
Thymidine kinase is a salvage enzyme, and therefore only present in anticipation of cell division. Therefore it will only be set free to the circulation from cells undergoing division. The enzyme is not set free from cells undergoing normal division where the cells have a special mechanism to degrade the proteins no longer needed after the cell division. In normal subjects the amount of thymidine kinase in serum or plasma is therefore very low. Tumour cells release enzyme to the circulation, probably in connection with the disruption of dead or dying tumour cells. The thymidine kinase level in serum therefore serves as a measure of malignant proliferation, indirectly as a measure of the aggressivity of the tumour. The main use of thymidine kinase assay now is in Non-Hodgkin lymphoma. This disease has a wide range of aggressivity, from slow growing indolent disease that hardly requires treatment to highly aggressive rapidly growing forms that should be treated urgently. This is reflected in the values of serum thymidine kinase, that range from close to the normal range for slow growing tumours to very high levels for rapidly rowing forms. Similar patterns can be seen in other hematological malignancies (leukemia, lymphoma, myeloma, myelodysplastic syndrome). A very interesting case is the myelodysplastic syndrome: some of them rapidly change to acute leukemia, while others remain indolent for very long time. Identification of those tending to change to overt leukemia is important for the treatment. Also solid tumours give increased values of thymidine kinase. Reports on this have been published for prostatic carcinoma, where thymidine kinase has been suggested as a supplement to PSA (Prostate Specific Antigen), the tumor marker now most frequently used in prostate cancer. While PSA is considered to give an indication of the tumour mass, thymidine kinase indicates the rate of proliferation.
[edit] Measurement technique for thymidine kinase in serum
The level of thymidine kinase in serum or plasma is so low that the measurement is best based on the enzymatic activity. In commercial assays, this is done by incubation of a serum sample with a substrate analogue. The oldest commercially available technique uses iodo-deoxyuridine where a methyl group in thymidine has been replaced with radioactive iodine. This substrate is well accepted by the enzyme. The monophosphate of iododeoxyuridine is adsorbed on aluminium oxide that is suspended in the incubation medium. After decantation and washing the radioactivity of the aluminium oxide gives a measure of the amount of thymidine kinase in the sample. A newly developed technique uses another thymidine analogue, bromo-deoxyuridine, as substrate to the enzyme. The product of the reaction (in microtiter plates) binds to the bottom of the wells in the plate. There it is detected with ELISA technique: the wells are filled with a solution of a monoclonal antibody to bromo-deoxyuridine. The monoclonal antibody has been bound (conjugated) to alkaline phosphatase (an enzyme). After the unbound antibody with attached alkaline phosphatase has been washed away, a solution of a substrate to the alkaline phosphatase, 4-nitrophenyl phosphate, is added. The product of the reaction, 4-nitrophenol, is yellow and can be measured by photometry.
[edit] Measurement of thymidine kinase in tissue
Thymidine kinase has been determined in tissue samples after extraction of the tissue. No standard method for the extraction or for the assay has been developed. The results indicate that there is a relationship between tissue thymidine kinase in tumour tissue and malignant character of the tumour, but no practical use for the determination has been found. Antibodies against thymidine kinase are available for immunohistochemical detection. The frequency of positivity in a tumour tissue sample reflects the rate of proliferation, but this is not a standard procedure to assess the malignancy of tumours.
[edit] Reference
- Nanda D, Vogels R, Havenga M, Avezaat CJ, Bout A, Smitt PS. Treatment of malignant gliomas with a replicating adenoviral vector expressing herpes simplex virus-thymidine kinase. Cancer Res 2001;61:8743-50. Fulltext. PMID 11751394.
