Thymidine kinase

Thymidine kinase 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, TK1 and TK2. 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.

Classification
Two different classes of thymidine kinases have been identified and are included in this super family:


 * one family groups together thymidine kinase from herpesvirus as well as cellular thymidylate kinases,


 * the second family groups TK from various sources that include, vertebrates, bacteria, the Bacteriophage T4, poxviruses, African swine fever virus (ASFV) and Fish lymphocystis disease virus (FLDV). The major capsid protein of insect iridescent viruses also belongs to this family.

The Prosite pattern recognises only the cellular type of thymidine kinases.

Biochemistry
Higher organisms have two isoenzymes, that are chemically very different, TK1 and TK2. 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 TK1 is present in the cytoplasm only in anticipation of cell division (cell cycle-dependent), whereas TK2 is located in mitochondria and is cell cycle-independent. The genes of the two types were localized in the mid-1970s. The gene for TK1 was cloned and sequenced. The corresponding protein has a molecular weight of about 25 kD. Normally, it occurs in tissue as a dimer. It can be activated by ATP. After activation, it has been converted to a tetramer. The recombinant TK1 cannot be activated and converted to a tetramer in this way, showing that the enzyme occurring in cells has been modified after synthesis. TK1 is synthesized by the cell during the S phase of cell division. After cell division is completed, TK1 is degraded intracelluarly, so that it does not pass to body fluids after normal cell division. There is a feed-back regulation of the action of thymidine kinase in the cell: thymidine triphosphate (TTP), the product of the further phosphorylation of thymidine, acts as an inhibitor to thymidine kinase. This serves to maintain a balanced amount of TTP available for nucleic acid synthesis, not oversaturating the system. 5'-Aminothymidine, a non-toxic analogue of thymidine, interferes with this regulatory mechanism and thereby increases the cytotoxicity of thymidine analogues used as antineoplastic drugs.

Genes for virus specific thymidine kinases have been identified in Herpes simplex virus, Varicella zoster virus and Epstein-Barr virus.

+ ATP ---> + ADP

Deoxythymidine reacts with ATP to give deoxythymidine monophosphate and ADP.

Physiological context
Deoxythymidine monophosphate, the product of the reaction catalysed by thymidine kinase, is in turn phosphorylated to deoxythymidine diphosphate by the enzyme thymidylate kinase and further to deoxythymidine triphosphate by the enzyme nucleoside diphosphate kinase. The triphosphate is included in a DNA molecule, a reaction catalysed by a DNA polymerase 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, a product of other metabolic pathways unrelated to thymidine, by the enzyme thymidylate synthethase. 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 1 is such a salvage enzyme, whereas thymidine kinase 2 is not cell cycle-dependent.

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.

PET scan of active tumours
3'-Deoxy-3'-[(18)F]fluorothymidine is a thymidine analogue. Its uptake is regulated by thymidine kinase 1, and it is therefore taken up preferentially by rapidly proliferating tumour tissue. The fluorine isotope 18 is a positron emitter that is used in positron emission tomography (PET). This marker is therefore useful for PET imaging of active tumour proliferation, and compares favourably with the more commonly used marker 2-[(18)F]fluoro-2-deoxy-D-glucose.

Selection of hybridomas
Hybridomas are cells obtained by fusing tumour cells (which can divide infinitely) and immunoglobulin-producing lymphocytes (plasma cells). 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 (TK-) tumour cell lines for the fusion. The thymidine kinase negative cells are obtained by growing the tumour cell line in the presence of thymidine analogues, that kill the thymidine kinase positive (TK+) cells. The negative cells can then be expanded and used for the fusion with TK+ plasma cells. After fusion, the cells are grown in a medium with methotrexate or aminopterin that inhibit the enzyme dihydrofolate reductase thus blocking the de novo synthesis of thymidine monophosphate. One such medium that is commonly used is HAT medium, which contains hypoxanthine, aminopterin and thymidine. 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 plasma cell survive. Those that produce the desired antibody are then selected and cultured to produce the monoclonal antibody.

However, instead of focussing on thymidine kinase, the hybridoma cells can be isolated using the same principle as described with respect to another gene the HGPRT, which synthesises IMP necessary for GMP nucleotide synthesis in the salvage pathway.

Clinical chemistry
Thymidine kinase is a salvage enzyme that is only present in anticipation of cell 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. It is interesting to note that the form of enzyme present in the circulation does not correspond to the protein as encoded by the gene: the gene corresponds to a protein with molecular weight around 25 kD. It is a dimer with a molecular weight of around 50 kD, if activated by ATP a tetramer with molecular weight around 100 kD. The main fraction of the active enzyme in the circulation has a molecular weight of 730 kD and is probably bound in a complex to other proteins.

The most dramatic increases are seen in hematologic malignancies. 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.

Also in dogs, lymphomas cause elevations of serum TK levels, indicative of the disease activity and useful for management of the disease.

Similar patterns can be seen in other hematological malignancies (leukemia,  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. There are also reports of the utility of thymidine kinase measurements in serum in small cell lung cancer, in breast cancer and in kidney cancer.

Non-malignant causes for elevation of thymidine kinase in serum are vitamin B12 deficiency, leading to pernicious anemia, viral infections (particularly by virus from the herpes group)  and wound healing after trauma and operation.

Therapeutic
Some drugs are specifically directed against dividing cells. They can be used against tumours and viral diseases (both against retrovirus and against other virus), as the diseased cells replicate much more frequently than normal cells and also against some non-malignant diseases related to too fast cell replication (for instance psoriasis). There are different classes of drugs to control too fast cell division that are directed against thymidine metabolism and thereby involving thymidine kinase   :

Chain terminators are thymidine analogues that are included in the growing DNA chain, but modified so that the chain cannot be further elongated. As analogues of thymidine, they are readily phosphorylated to 5'-monophophates. The monophosphate is further phosphorylated to the corresponding triphosphate and incorporated in the growing DNA chain. The analogue has been modified so that it does not have the hydorxyl group in the 3'-position that is required for continued chain growth. In zidovudine (AZT; ATC: ) the 3'-hydroxyl group has been replaced by an azido group, in Stavudine (ATC: ) it has been removed without replacement. AZT is used as substrate in one of the methods for determination of thymidine kinase in serum. This implies that AZT interferes with this method and may be a limitation: AZT is a standard component of HAART therapy in HIV infection. One common consequence of AIDS is lymphoma and the most important diagnostic application of thymidine kinase determination is for monitoring of lymphoma.

Other thymidine analogues, for instance Idoxuridine (ATC: ) act by blocking base pairing during subsequent replication cycles, thereby making the  resulting DNA chain defective. This may also be combined with radioactivity to achieve apoptosis of malignant cells.

Some antiviral drugs, such as acyclovir (ATC: ) and ganciclovir (ATC: ) as well as other recently developed nucleoside analogs 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.

After smallpox was declared eradicated by WHO in December 1979, vaccination programs were terminated. A re-emergence of the disease either by accident or as a result of biological warfare would meet an unprotected population and could result in an epidemic that could be difficult to control. Mass vaccination would be unethical, as the only efficient vaccines against smallpox include live vaccinia virus with severe adverse effects on rare occasions. As one protective measure, large amounts of vaccine are kept in stock, but an efficient drug against smallpox has high priority. One possible approach would be to use the specificity of the thymidine kinase of poxvirus for the purpose, in a similar way that it is used for drugs against herpesvirus. One difficulty is that the poxvirus thymidine kinase belongs to the same family of thymidine kinases as the human thymidine kinases and thereby is more similar chemically. The structure of poxvirus thymidine kinases has therefore been determined to find potential antiviral drugs. The search has however not yet resulted in a usable antiviral drug against poxviruses.

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 ganciclovir, 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.

Incorporation of a thymidine analogue with boron has been suggested and tried in animal models for boron neutron capture therapy of brain tumours.

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. Kits using this principle are commercially available from the companies Immunotech/Beckman and DiaSorin.

A non-radioactive assay method has been developed by the company Dia-Sorin. In this technique 3'-azido-2',3'-deoxythymidine (AZT)is first phosphorylated to AZT 5'-monophosphate (AZTMP) by TK1 in the sample. AZTMP is measured in a immunoassay with anti-AZTMP antibodies and AZTMP-labeled peroxidase. The assay runs in a closed system on the laboratory robot from DiaSorin

Another newly-developed technique uses a 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 at alkaline pH and can be measured by photometry. This assay gives a considerably more sensitive determination. It is commercially available from the company Biovica.

Direct determination of the thymidine kinase protein by immunoassay has also been used. The amounts of thymidine kinase found by this method did not correlate well with the activities and found to have less clinical significance, and the method has been withdrawn from the market.

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 and TK determination in extracts from cells and tissues have not been validated in relation to any specific clinical question, see however Romain et al. and Arnér et al. A method has been developed for specific determination of TK2 in cell extracts using the substrate analogue 5-Bromovinyl 2'-deoxyuridine. In the studies referred to below the methods used and the way the results are reported are so different that comparisons between different studies are not possible.

The TK1 levels in fetal tissues during development are higher than those of the corresponding tissues later.

Certain non-malignant diseases also give rise to dramatic elevation of TK values in cells and tissue: in peripheric lymphocytes during monocytosis and in bone marrow during pernicious anemia.

As TK1 is present in cells during cell division, it is reasonable to assume that the TK activity in malignant tissue should be higher than in corresponding normal tissue. This is also confirmed in most studies. A higher TK activity is found in neoplastic than in normal tissue,  in brain tumours, in hematological malignancies, in cancer and polyps in colon,      in breast cancer,      in lung cancer,   in gastric cancers, in ovarian cancer, in mesotheliomas, in melanomas and in thyroid tumours.

In leukemia and in breast cancer  therapy that influences the rate of cell proliferation influences the TK values correspondingly.

Immunohistochemical staining for thymidine kinase
Antibodies against thymidine kinase are available for immunohistochemical detection. Staining for thymidine kinase was a reliable technique for identification of patients with stage 2 breast carcinoma. The highest number of patients identified was obtained by combination of thymidine kinase and Ki-67 staining.

The technique has also been validated for lung cancer, for colorectal carcinima, for lung cancer and for renal cell carcinoma.