Thymidine kinase

Thymidine kinase TK, is an enzyme, a phosphotransferase (a kinase): 2'-deoxythymidine kinase, ATP-thymidine 5'-phosphotransferase,. 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.

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 found to be more abundant in adult tissue, and initially they were termed fetal and adult thymidine kinase. Soon it was shown that TKI is present in the cytoplasm only in anticipation of cell division (cell cycle-dependent), whereas TKII is located in mitochondria and is cell cycle-independent. The genes of the two types were localized in the mid-1970s. 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.

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 molecule, a reaction catalysed by a DNA polymerase (enzyme) and a complementary DNA molecule (or an RNA molecule 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, whereas thymidine kinase II is not cell cycle-dependent.

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.

Thymidine kinase for drug design
Some drugs are specifically directed against dividing cells. They can be used against tumours and viral diseases, as the diseased cells replicate 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, such as acyclovir and ganciclovir, make use of the specificity for viral thymidine kinase, as opposed to human thymidine kinases. These drugs act as prodrugs, which in themselves are not toxic, but are converted to toxic drugs by phosphorylation by viral thymidine kinase. Cells infected with the virus therefore produce highly-toxic triphosphates that lead to cell death. Human thymidine kinase, in contrast, with its more narrow specificity, is unable to phosphorylate and activate the prodrug. In this way, only cells infected by the virus are susceptible to the drug. Such drugs are effective only against viruses from the herpes group with their specific thymidine kinase.

The herpesvirus thymidine kinase gene has also been used as a “suicide gene” as a safety system in gene therapy experiments, allowing cells expressing the gene to be killed using ganciclovir. This is desirable in case the recombinant gene causes a mutation leading to uncontrolled cell growth (insertional mutagenesis). The thymidine kinase produced by these modified cells may diffuse to neighboring cells, rendering them similarly susceptible to gancliclovir, a phenomenon known as the "bystander effect." This approach has been used to treat cancer in animal models, and is advantageous in that the tumor may be killed with as few as 10% of malignant cells expressing the gene.

A similar use of the thymidine kinase makes use of the presence in some tumor cells of substances not present in normal cells (tumor markers). Such tumor markers are, for instance, CEA (carcinoembryonic antigen) and AFP (alpha fetoprotein). The genes for these tumor markers may be used as promoter genes for thymidine kinase. Thymidine kinase can then be activated in cells expressing the tumor marker but not in normal cells, such that treatment with ganciclovir kills only the tumor cells. Such gene therapy-based approaches are still experimental, however, as problems associated with gene transfer have not yet been completely solved.

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 because they have no source of thymidine monophosphate. The lymphocytes eventually die because 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.

Thymidine kinase in clinical chemistry
Thymidine kinase is a salvage enzyme, and therefore only present in anticipation of cell division. Therefore it will 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-growing 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, whereas 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. Whereas PSA is considered to give an indication of the tumour mass, thymidine kinase indicates the rate of proliferation.

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 wherein 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.

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.