Thymidine Kinase 1 Expression Research Articles

Thymidylate Synthase (TS) Expression In Diffuse Large B-Cell Lymphoma (DLBCL): Identification of a Potential Therapeutic Target

AbstractAbstract 5107 Purpose:TS is critical for the de novo synthesis of thymidine needed for tumor cell proliferation and DNA damage repair, whereas thymidine kinase 1 (TK1) is key to the salvage pathway providing thymidine recovered from plasma. While TK1 expression has been previously shown to correlate with proliferation in DLBCL, little is known about TS levels in these neoplasms. We hypothesized that, similarly to TK1, TS expression levels associate with increased proliferation and thus may represent a novel therapeutic target in large cell lymphoma. Methods:To address this hypothesis, we determined the expression of TS in lymph node biopsies from 43 DLBCL patients sequentially enrolled in the study with no pre-selection criteria other than the primary histological diagnosis of lymphoma, and then compared it to TK1 expression. In addition, Ki-67 (MIB1) was included as an established marker of cell proliferation. Pearson correlation along with the correlation coefficient and two-tailed P values were calculated to measure the extent of covariation. Results:TS expression and both MIB1 and TK1 were significantly correlated with correlation coefficients of 0.43 and 0.65, respectively (95% CI 0.13 to 0.65 and 0.44 to 0.8; P values = 0.006 and <0.0001, respectively). The correlation between TS and MIB1 expression was in fact stronger than between MIB1 and TK1 (P values 0.006 and 0.04, respectively). Mean percent positive cells and standard deviations for MIB1, TS and TK1 were 44.5% (SD=35), 51.8% (SD=33.4), and 44% (SD=28.5), respectively. Despite the general good correlation, however, six patients had relatively high TS levels (defined as % cell staining above the mean for TS) and relatively low TK1 levels (% cell staining below the mean for TK1), while the reverse was true in seven patients. Eight patients had high levels of TK1 and TS with ≥ 70% cells positive for both; seven patients had low levels of both proteins (≤30% of cells stained). Conclusion and Significance:This is the first report of TS expression in DLBCL. TS is frequently expressed in DLBCL and there is a significant correlation between TS and TK1 or MIB1 expression. Further studies are warranted to determine the correlation between TS or TK1 expression and patient prognosis in comparison to MIB1. Importantly, the common expression of TS in patients with DLBCL indicates that TS may be a suitable target for TS inhibitors (TSIs) in these neoplasms. Preclinical studies are underway to test this hypothesis. Disclosures:No relevant conflicts of interest to declare.

Thymidine Kinase 1 is a Potential Marker for Prognosis and Monitoring the Response to Treatment of Patients with Breast, Lung, and Esophageal Cancer and Non-Hodgkin's Lymphoma

Thymidine kinase 1 (TK1) is converting thymidine to thymidine monophosphate, and is related to DNA replication and cell proliferation. The use of the TK1 protein levels as a proliferation marker in malignancies is here summarized. TK1 protein in serum (STK1p) and TK1 expression in tissues were determined by a chemoluminescent dot blot assay and by immunohistochemistry staining, respectively. The expression of TK1 in tumor tissues correlated to pathological stages and clinical grades of carcinomas (ca) of esophagus, lung and in premalignancy of breast ductal ca. STK1p could monitor the out-come of tumor therapy by being correlated to remission [breast ca, non-Hodgkin's lymphoma], relapse [breast ca] and to survival [non-Hodgkin's lymphoma] of patients. In a health screening study of 12,641 persons, STK1p seemed to predict the risk of development of neoplasia related diseases at early stage.

Abstract 3654: Clofarabine: factors influencing cytotoxicity and resistance in hematologic and solid malignancies

Abstract Clofarabine is a new-generation nucleoside analog that was synthesized to combine the most favorable pharmacological properties of its congener fludarabine and cladribine. It acts as a strong inhibitor of DNA polymerase, DNA synthesis and ribonucletide reductase (R1/R2). Like other nucleoside analogues, clofarabine must be activated within cells to the 5′-triphodphate form initially by deoxycytidine kinase (dCK). In this study, the anticancer effect of clofarabine was investigated in three solid tumor cell lines, HCT116 (a colon carcinoma cell line), U1810 (a human non-small cell lung carcinoma cell line), and MCF7 (a mammary carcinoma cell line) and compared to four leukemic cell lines, CCRF-CEM, HL60, K562 and MOLT4. Clofarabine was investigated to enter into HCT116, U1810, and MCF7 in a clearly detectable manner by LigandTracer® White. At 10 µM a high signal is achieved and approaches equilibrium after 1∼2 hrs. Pre-incubation with 10 µM clofarabine resulted in rapid inhibition of thymidine incorporation and DNA synthesis. Induction of apoptosis by clofarabine was cell line dependent, but the average apoptotic level was higher in leukemic cell lines than solid tumor cell lines; however it seemed the sensitivity for apoptosis of cell line is correlated with the activity of dCK and thymidine kinase 1 (TK1) in the solid tumor cell lines. Upon treatment with 10 μM clofarabine for 24 hrs, apoptosis induction increased compared to untreated cells 3.1-, 2.9- and 2.1-fold; the activity of dCK 7.3-, 3.7- and 3.1-fold and the activity of TK1 4.2-, 1.4- and 1.2-fold in U1810, HCT116 and MCF7 cells, respectively. In general, the expression of R1, dCK and TK1 in leukemic cells was higher than in solid tumor cells, but the expression of R2, p53R2 and p53 varied among the cell lines. However, there was an inverse correlation between induction of apoptosis at 100nM clofarabine (upon 24 hours incubation) and the expression of p53R2 (spearman rank test −0.67, p=0.1). In addition, clofarabine treatment causes retardation in S-phase, which seems to be related to the activity of TK1 and the protein expression of R2 in U1810, HCT116 and MCF7 cells. This study is a first step to extend the application of clofarabine toward the solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3654.

Immunohistochemical characterization of pyrimidine synthetic enzymes, thymidine kinase-1 and thymidylate synthase, in various types of cancer

Thymidine kinase-1 (TK-1) and thymidylate synthase (TS) are key enzymes for salvage and de novo pyrimidine synthesis, respectively. Numerous studies have suggested that increased TS levels are associated closely with resistance to fluoropyrimidine-based chemotherapy. TAS-102 is a novel drug containing trifluorothymidine, which is phosphorylated by TK-1 to its active monophosphated form, that in turn can inhibit TS. TAS-102 has been shown to exhibit antitumor activity in fluoropyrimidine-resistant human cancer cells. TAS-102 is currently undergoing clinical trials for use in gastrointestinal cancers. In the present study, we used immunohistochemistry to investigate the expression of TK-1 and TS in various types of cancer. TK-1 and TS expression was markedly different between cancer types. High TK-1 expression was detected prominently in gastrointestinal adenocarcinomas and esophageal and uterine squamous cell carcinomas. Gastrointestinal adenocarcinomas and squamous cell uterine carcinomas were often accompanied by high TS expression, indicating activation of pyrimidine synthesis through both the salvage and de novo pathways. These results led us to consider that TAS-102 may also be effective for esophageal and uterine squamous cell carcinomas, as well as for gastrointestinal adenocarcinomas, even in fluoropyrimidine-resistant cases with high TS expression. In contrast, thyroid papillary carcinomas, lung adenocarcinomas, hepatocellular carcinomas, pancreatic ductal carcinomas, and renal cell carcinomas, which exhibit low TK-1 expression, may be resistant to TAS-102. In non-small cell lung cancers, high TK-1 expression was demonstrated in squamous cell carcinomas, but not in adenocarcinomas. This result suggests that TAS-102 efficacy and the pyrimidine synthetic pathway may differ depending on histological type. Our results indicate that administration of TAS-102 could be selected on the basis of the immunohistochemical evaluation of TK-1 and TS.

The Proliferation Marker Thymidine Kinase 1 Level is High in Normal Kidney Tubule Cells Compared to other Normal and Malignant Renal Cells

The activity of the proliferation related enzyme thymidine kinase 1 (TK1) was reported to be 3-fold higher in extracts from normal kidney tissue as compare to renal carcinoma extracts [3]. To verify these unexpected results, determinations of the protein levels of TK1 in normal kidney and in samples from different types of renal cell carcinoma (RCC) were done with immunohistochemistry and Western blot analysis. Two anti-TK1 peptide antibodies reacting with different TK1 epitops were used. TK1 levels were high in tubule cells as compared to glomerulus cells and connective tissue cells, while an intermediary TK1 was observed in renal cell carcinoma (RCC) cells. Western blot analysis demonstrated high levels of TK1 in extract from normal kidney, and lower levels of TK1 in the RCC extracts. The specificity of TK1 staining was demonstrated in competition experiments with excess TK1 antigen. The high TK1 levels in normal kidney tubule cells suggest that they are in a form of activated G1-state. The relatively low TK1 level in RCC, representing TK1 expression in S-phase cells, is in accordance with the low overall proliferation rate of these tumors. These results suggest that cell cycle regulation of TK1 in normal tubule cells differ from that in other type of normal and malignant renal cells.

Thymidine kinase 1 expression in atypical ductal hyperplasia significantly differs from usual ductal hyperplasia and ductal carcinoma in situ: A useful tool in tumor therapy management

Thymidine kinase 1 (TK1) is an enzyme involved in the synthesis of DNA precursors. In studies using immunohistochemistry, it was reported to be a more useful proliferation marker than Ki-67 and PCNA in breast, lung and colorectal carcinoma. In this study, we extend the work of prior breast carcinoma studies by investigating the expression of TK1 in 132 patients with usual ductal hyperplasia (UDH), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). TK1 and Ki-67 expression were determined with monoclonal antibodies using the SP technique. The expression of TK1 was found to be significantly increased in the breast ductal carcinomas in the following manner: UDH<ADH<DCIS<IDC. TK1 expression was correlated with histological grade in DCIS and IDC patients and with pathological stage in IDC patients. The degree of TK1-positive staining in the tumors of patients with ADH, DCIS and IDC was 80-90%, while the corresponding value for UDH patients was less than 5%. TK1 and Ki-67 expression showed very high correlation in the four tumor groups investigated. Since the expression of TK1 varied significantly between the breast ductal subgroups, we concluded that TK1 is a reliable proliferation marker for the determination of breast tumor proliferation, particularly of pre-cancerous lesions (ADH). This may enable timely treatment in the early stages of tumor development using minimal access surgery, thus avoiding extensive radical breast surgery and improving survival rates and the quality of life.

Thymidine kinase 1 expression defines an activated G1 state of the cell cycle as revealed with site-specific antibodies and ArrayScan™ assays

Thymidine kinase 1 (TK1) is a DNA salvage enzyme involved in the synthesis of thymidine triphosphate needed during S phase. Although TK1 has been utilized as a cell proliferation marker for many years no well-characterized antibodies are available. The preparation and properties of two types of poly- and monoclonal anti-TK1 peptide antibodies are described and they are used to determine the levels of TK1 in intact cells. Expression of TK1, c-fos, cyclin B1, Ki67, phosphorylated histone H3, phosphorylated ribosomal protein S6, as well as bromodeoxyuridine (BrdU) incorporation in human normal dermal fibroblast cultures were studied with high-content ArrayScan™ fluorescence microscopy. The levels of TK1 increased 6–7 h after serum re-addition to starved cells as they passed through G1, S and G2/M phases, which was earlier than the increase in Ki67 protein levels and before BrdU incorporation was detected. Thus, a population of activated G1 cells with high TK1 and low Ki67 expression could be identified and their role in cell proliferation can now be clarified.

Thymidine Kinase 1 and Thymidine Phosphorylase Expression in Non-Small-cell Lung Carcinoma in Relation to Angiogenesis and Proliferation

The thymidine salvage pathway enzymes thymidine kinase 1 (TK1) and thymidine phosphorylase (TP) compete for thymidine as a substrate and catalyze opposing synthetic and catabolic reactions that have been implicated in the control of proliferation and angiogenesis, respectively. We investigated the relationship between the expression of TK1 and TP as they relate to proliferation (Ki-67 labeling index) and angiogenesis (Chalkley count of CD31-stained blood vessels) in a series of 110 non-small-cell lung cancer (NSCLC) tumors from patients prospectively enrolled in an imaging trial. TK1 and TP exhibited similar patterns of immunohistochemical distribution, in that each was found in both the nucleus and the cytoplasm of tumor cells. Each enzyme exhibited a significant positive correlation between its levels of nuclear and cytoplasmic expression. A significant positive correlation between TK1 expression and the Ki-67 labeling index (r = 0.53, p<0.001) was observed. TP was significantly positively correlated with Chalkley scoring of CD31 staining in high vs low Chalkley scoring samples (mean TP staining of 115.8 vs 79.9 scoring units, p<0.001), respectively. We did not observe a substantial inverse correlation between the TP and TK1 expression levels in the nuclear compartment (r = -0.17, p=0.08). Tumor size was not found to be associated with TK1, TP, Ki-67, or Chalkley score. These findings provide additional evidence for the role of thymidine metabolism in the complex interaction of proliferation and angiogenesis in NSCLC.

Thymidine kinase activity in serum of renal cell carcinoma patients is a useful prognostic marker

It is known that the concentration and activity of the DNA precursor enzyme thymidine kinase 1 (TK1) in serum is significantly elevated in patients with malignancies, as compared with levels in patients with benign tumours and those in healthy individuals. For the first time, the use of serum TK1 as a prognostic marker for patients with renal cell carcinoma (RCC) was examined. Serum TK1 protein (STK1p) concentration and serum TK1 activity (STK1a) were determined by a dot blot chemoluminescence assay and a radio enzyme assay, respectively. There was no correlation between STK1p and STK1a in the same sera from 27 RCC patients. Only one STK1p value as compared with 15 STK1a values was clearly above the cut-off values (2 pmol/l and 6 U/l, respectively) for healthy individuals. STK1a values did not correlate with the level of TK1 expression in tumour sections from the RCC patients, estimated by immunohistochemistry staining. However, there was a significant correlation between STK1a levels and the grade, stage and size of the RCC tumours. The discrepancy between the STK1p and the STK1a results is likely to be because of reduced ability of the TK1 antibody to recognize the STK1 in sera from RCC patients. We conclude that the activity of STK1 is a useful tool for evaluating the prognosis of patients with RCC.

Activation of thymidine salvage pathway measured with FLT PET, as a potential indicator of response to 5FU in colorectal cancer

3572 Background: The commonly used positron emission tomography (PET) tracer is [18F]-Fluoro-2-deoxy-D-glucose (FDG). [18F]-3-fluoro-3-deoxy-L-thymidine (FLT) is a novel tracer, with cellular uptake mechanisms linked to the pyrimidine salvage enzyme, thymidine kinase-1 (TK-1). The colorectal cancer chemotherapeutic agent, 5-Fluorouracil (5FU), inhibits de novo pyrimidine synthesis. Therefore, we investigated changes in FLT and FDG uptake after 5FU treatment, to determine their potential in detecting early evidence of response to 5FU in vitro. Methods: Human colorectal cancer cell cultures (SW480, SW620) were treated with 5FU (50μg/ml, 2h). Cells were washed, incubated with FLT or [18F]-Fluoro-2-deoxy-D-glucose (FDG) for comparison (100kBq, 90 min). Cells were subsequently harvested and uptake was determined using a gamma counter. TK-1 expression was detected by Western blotting. Changes in S-phase fraction (SPF) of treated cell were examined by flow cytometry. Results: Cytotoxic dose of 5FU chosen showed minimal cell killing immediately after treatment (0–10% (p=NS). However, 50% cell killing was achieved at 24 hours. A mean 2.96 fold increase (2.82 ± 0.2, SW480; 3.10± 0.3 SW620) in total FLT uptake (per 10 5 cells) was observed in treated cells vs controls (p<0.01). However, no difference was observed in FDG uptake. The increase in FLT uptake post 5FU treatment correlated with increased TK-1 expression in both cell lines. No significant change in the SPF of 5FU treated cells was observed. Conclusions: An early biological response to 5FU treatment was measured with FLT and not FDG. This effect correlated with an increased expression of thymidine salvage enzyme TK-1, before changes in the cell cycle were observed. FLT PET may potentially be used as an indicator of response to 5FU based chemotherapeutic agents before changes in morphology become apparent. No significant financial relationships to disclose.

Expression of cell proliferating genes in patients with non-small cell lung cancer by immunohistochemistry and cDNA profiling

Thymidine kinase 1 (TK1) is a key enzyme involved in the synthesis of DNA precursors and thus, cell proliferation-dependent. Antibodies against TK1 have provided attractive tools for cancer diagnosis. Expression of TK1 in 158 non-small cell lung cancer (NSCLC) patients with 59 adenocarcinoma (AC) and 99 squamous cell carcinoma (SCC) was determined by anti-TK1 monoclonal antibody (mAb) 1E3 (AC, n=50; SCC, n=70). Parallel tumor sections were stained for Ki-67 (MIB-1), and TK1 expression was also investigated with anti-TK1 chicken IgY Ab (AC, n=9; SCC, n=29; normal lung tissues, n=10). In one AC and one SCC patient, gene profiling was done by cDNA array. Using the mAb 1E3, a significantly higher TK1 labeling index (LI) of AC patients was found (68%) compared to the LI of Ki-67 (36%). This difference was due to a significantly higher TK1 LI of tumor stage II and grade 2. Although no difference in the LI of TK1 and Ki-67 of SCC patients was found (54 vs. 53%), significantly higher TK1 LI of SCC patients of tumor grade 1 was found. Using the anti-TK1 IgY Ab, a higher TK1 LI of AC patients (78%) and SCC patients (66%) was found compared to staining with mAb 1E3 (68 vs. 54%), but it was not significantly different. Samples stained only for TK1 represented mostly tumor stages I and II and grades 1 and 2 of both AC and SCC. AC patients whose samples stained only for Ki-67 were found to be in stage I and grade 1. cDNA profiling showed that the expression of BRCA1, cyclin B1 and cdc2p34 was higher in AC compared to SCC, while the expression of IGFBP-3 and EGFR was higher in SCC. TK1 is apparently a more reliable marker in AC patients than Ki-67. However, a combination of the two markers may help identify patients of different stages and grades more efficiently, and cyclin/kinase complexes and growth factors/receptors may be useful markers in distinguishing AC from SCC.

Actinomycin D induces high-level resistance to thymidine analogs in replication of human immunodeficiency virus type 1 by interfering with host cell thymidine kinase expression.

Actinomycin D (ActD) is a transcription inhibitor and has been used in the treatment of certain forms of cancer. ActD has been reported to be a potential inhibitor of human immunodeficiency virus type 1 (HIV-1) replication due to its ability to inhibit reverse transcription. In contrast to what was expected, low concentrations of ActD (1 to 10 nM) upregulated HIV-1 replication 8- to 10-fold in MT-2 cells and had no effect on HIV-2 replication or on HIV-1 replication in MT-4, Jurkat, or peripheral blood mononuclear cells. The upregulation of HIV-1 replication was associated with an increase in HIV-1 transcription and a decrease in CD4 and CXCR4 expression. To further evaluate the effects of ActD on emergence of drug resistance in HIV-1 replication, a series of drug resistance assays were performed. Of interest, treatment of MT-2 cells with ActD also led to a high level of resistance to thymidine analogs (>1,000-fold increase in resistance to zidovudine and >250-fold to stavudine) but not to other nucleoside reverse transcriptases (RT), nonnucleoside RT, or protease inhibitors. This resistance appeared to be due to a suppression of host cell thymidine kinase-1 (TK-1) expression. These results indicate that ActD leads to a novel form of thymidine analog resistance by suppressing host cell TK-1 expression. These results suggest that administration of combination drugs to HIV-1-infected patients may induce resistance to antiretroviral compounds via a modification of cellular factors.

A Comparative Study: Immunohistochemical Detection of Cytosolic Thymidine Kinase and Proliferating Cell Nuclear Antigen in Breast Cancer

To explore the expression of cytosolic thymidine kinase 1 (TK1) as a cell proliferative marker in human breast cancers, immunohistochemistry was used to detect the expression of TK1 in 52 malignant breast lesions, 20 benign breast lesions, and 16 normal breast tissues. The results were compared to the expression of proliferating cell nuclear antigen (PCNA) in the same specimens. The TK1-labelling index (TK1-LI) and PCNA-labeling index (PCNA-LI) were significantly higher in malignant lesions than in nonmalignant lesions (p<0.0001 and p<0.0013, respectively). The TK1-LI (78.9%) in malignant lesions was higher compared to PCNA-LI (64.5%). No significant difference was found for TK1-LI and PCNA-LI between benign lesions and normal tissues. Concerning the tumor stages and the tumor grades, TK1-LI showed a significant correlation with the increased tumor stages (p=0.023) and tumor grades (p=0.009). However, PCNA-LI was neither significantly different in tumor stages (p=0.062) nor in tumor grades (p=0.073). We conclude that TK1 might be a more accurate marker than PCNA for estimation of cell proliferation and malignant potentials in breast carcinomas.

Investigation on cell proliferation with a new antibody against thymidine kinase 1.

The cytosolic thymidine kinase 1 (TK1) is one of the enzymes involved in DNA replication. Based on biochemical studies, TK1 is activated at late G1 of cell cycle, and its activity correlates with the cell proliferation. We have developed a polyclonal anti‐TK1 antibody against a synthetic peptide from the C‐terminus of human TK1. Using this antibody, here we demonstrate the exclusive location of TK1 in the cytoplasm of cells. Cell cycle dependent TK1 expression was studied by simultaneous fluorescence staining for TK1 and bromodeoxyuridine, by using elutriated cells, and by quantitation of the amount TK1 in relation to the cellular DNA content. TK1, which was strongly expressed in the cells in S+G2 period, raised at late G1 and decreased during mitosis. The amount of TK1 increased three folds from late G1 to G2. TK1 positive cells were demonstrated in areas of proliferation activity of various normal and malignant tissues. The new anti‐TK1 antibody works in archival specimens and is a specific marker of cell proliferation.