Investigation on cell proliferation with a new antibody against thymidine kinase 1.
01 Jan 2001-Analytical Cellular Pathology (Hindawi Publishing Corporation)-Vol. 23, Iss: 1, pp 11-19
TL;DR: A polyclonal anti‐TK1 antibody against a synthetic peptide from the C‐terminus of human TK1 is developed and demonstrated, demonstrating the exclusive location of TK 1 in the cytoplasm of cells.
Abstract: 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.
Citations
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TL;DR: The potential clinical applications of proliferative indices are discussed, including their use as prognostic indicators and predictors of response to systemic therapy.
Abstract: Various methods are available for the measurement of proliferation rates in tumours, including mitotic counts, estimation of the fraction of cells in S-phase of the cell cycle and immunohistochemistry of proliferation-associated antigens. The evidence, advantages and disadvantages for each of these methods along with other novel approaches is reviewed in relation to breast cancer. The potential clinical applications of proliferative indices are discussed, including their use as prognostic indicators and predictors of response to systemic therapy.
193 citations
Cites background from "Investigation on cell proliferation..."
...Wang and colleagues [78] have developed a polyclonal anti-TK1 antibody and demonstrated cell cycle-dependent expression of the enzyme....
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TL;DR: Chicken egg yolk immunoglobulins generated to a synthetic 31-amino acid peptide from the C-terminal of human HeLa thymidine kinase 1 (TK1) enzyme are suggested to be potentially useful for serological and immunohistochemical detection of TK1 as an early prognosis and for monitoring patients undergoing treatment.
74 citations
Journal Article•
TL;DR: The results indicate that the S-TK1 concentration is higher in patients developing distant and/or loco-regional recurrence 3 months post-surgery.
Abstract: Background The prognostic value of the concentration of serum thymidine kinase 1 (S-TK1) with regard to recurrence in low risk breast cancer patients, 3 months after surgery was evaluated. Patients and methods The concentration of S-TK1 in serum was determined in 120 breast cancer patients at the time of surgery and in 67 patients 3 months after surgery, by anti-TK1 chicken IgY antibody, using a dot-blot immuno-assay. The S-TK1 concentration was compared with the serological activity of thymidine kinase (STK) and of carbohydrate antigen (CA 15-3). Results A statistically significant trend (unadjusted) was found for recurrence (distant or loco-regional) in patients with a higher S-TK1 concentration, as compared with patients with a lower S-TK1 concentration. A multivariate analysis gave the same results. The hazard rate ratio for developing distant and/or loco-regional recurrence in patients with a higher S-TK1 concentration was about six to seven times higher than in patients with a lower S-TK1 concentration. Conclusion Our results indicate that the S-TK1 concentration is higher in patients developing distant and/or loco-regional recurrence 3 months post-surgery.
72 citations
TL;DR: The status of TK1 for cancer monitoring and its use as a proliferation marker are summarized and a comprehensive overview about the association of Tk-1 with various entities is given.
70 citations
Cites background from "Investigation on cell proliferation..."
...The presence of TK1 positive cells could be demonstrated in both, normal and malignant tissue [11]....
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TL;DR: Determination of thymidine kinase helps to monitor the follow-up of solid tumours and haematological malignancies as well as indicating the efficacy of adjuvant and palliative chemotherapy.
Abstract: Thymidine kinase 1 (TK 1-fetal) is a cell cycle-dependent marker that increases dramatically during the S-phase of the cell cycle. In this review, the authors discuss serum levels of thymidine kinase in a variety of neoplasias. Determination of thymidine kinase helps to monitor the follow-up of solid tumours and haematological malignancies as well as indicating the efficacy of adjuvant and palliative chemotherapy. Elevated levels of thymidine kinase must always be interpreted together with a detailed knowledge of the patient's condition because nonspecific elevations of serum levels (inflammatory and autoimmune diseases) must be excluded.
70 citations
Cites methods from "Investigation on cell proliferation..."
...Although immunochemical methods have been reported, a highly sensitive but convenient approach for routine measurement of serum TK1 in haematological malignancies has not been forthcoming [16-19] ....
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References
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TL;DR: The promoter of the murine thymidine kinase gene contains a binding site for transcription factor E2F that is the target for trans activation by the viral protein.
Abstract: The promoter of the murine thymidine kinase gene contains a binding site for transcription factor E2F. Using cell lines (3T3-LT) conditionally expressing polyomavirus large T antigen from a hormone-responsive promoter and reporter gene constructs carrying the thymidine kinase promoter with intact or mutated E2F sites, we show that this E2F site is the target for trans activation by the viral protein. Transcription of the growth-regulated endogenous thymidine kinase gene can be activated in serum-starved, quiescent 3T3-LT cells by induction of T antigen. Activation of transcription from the thymidine kinase promoter requires an intact binding site for the retinoblastoma protein in the T antigen. The same promoter region was furthermore shown to play a major role in growth regulation of the gene. As several other DNA synthesis enzymes also carry E2F binding sites in their promoters, our observations suggest a common mechanism of growth regulation of these genes and that they all might be targets for trans activation by DNA tumor virus proteins.
92 citations
"Investigation on cell proliferation..." refers background in this paper
...Overexpression of the E2F gene products induces quiescent cells to enter S-phase combined with overexpression of TK1 [27]....
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TL;DR: The results indicate that the previously reported increase in TK enzyme levels seen after either treatment is paralleled by an equivalent increase in the steady-state levels of TK mRNA.
Abstract: The thymidine kinase (TK) gene has been isolated from human genomic DNA. The gene was passaged twice by transfection of LTK- cells with human chromosomal DNA, and genomic libraries were made in lambda Charon 30 from a second-round TK+ transformant. When the library was screened with a human Alu probe, seven overlapping lambda clones from the human TK locus were obtained. None of the seven contained a functional TK gene as judged by transfection analysis, but several combinations of clones gave rise to TK+ colonies when cotransfected into TK- cells. A functional cDNA clone encoding the human TK gene has also been isolated. Using this cDNA clone as a probe in restriction enzyme/blot hybridization analyses, we have mapped the coding sequences and direction of transcription of the gene. We have also used a single-copy subclone from within the coding region to monitor steady-state levels of TK mRNA in serum-stimulated and simian virus 40-infected simian CV1 tissue culture cells. Our results indicate that the previously reported increase in TK enzyme levels seen after either treatment is paralleled by an equivalent increase in the steady-state levels of TK mRNA. In the case of simian virus 40-infected cells, the induction was delayed by 8 to 12 h, which is the length of time after infection required for early viral protein synthesis. In both cases, induction of TK mRNA coincides with the onset of DNA synthesis, but virally infected cells ultimately accumulate more TK mRNA than do serum-stimulated cells.
83 citations
"Investigation on cell proliferation..." refers background in this paper
...chemical studies that the cell cycle related increase in TK1 activity is combined with increased expression of mRNA as well as of the TK1 protein [2, 14,19,25,32,35,38]....
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TL;DR: Reconstitution of a deoxycytidine kinase-deficient cell line with the wild-type nuclear or the mutant cytosolic enzymes both restored sensitivity toward anticancer nucleoside analogs.
Abstract: Human deoxyribonucleoside kinases are required for the pharmacological activity of several clinically important anticancer and antiviral nucleoside analogs. Human deoxycytidine kinase and thymidine kinase 1 are described as cytosolic enzymes in the literature, whereas human deoxyguanosine kinase and thymidine kinase 2 are believed to be located in the mitochondria. We expressed the four human deoxyribonucleoside kinases as fusion proteins with the green fluorescent protein to study their intracellular locations in vivo. Our data showed that the human deoxycytidine kinase is located in the cell nucleus and the human deoxyguanosine kinase is located in the mitochondria. The fusion proteins between green fluorescent protein and thymidine kinases 1 and 2 were both predominantly located in the cytosol. Site-directed mutagenesis of a putative nuclear targeting signal, identified in the primary structure of deoxycytidine kinase, completely abolished nuclear import of the protein. Reconstitution of a deoxycytidine kinase-deficient cell line with the wild-type nuclear or the mutant cytosolic enzymes both restored sensitivity toward anticancer nucleoside analogs. This paper reports that a deoxyribonucleoside kinase is located in the cell nucleus and we discuss the implications for deoxyribonucleotide synthesis and phosphorylation of nucleoside analogs.
80 citations
"Investigation on cell proliferation..." refers background in this paper
...The intracellular localizations of various viral TKs and as well the other deoxnucleoside kinases were recently reinvestigated and nuclear forms of these enzymes were reported [6,13,18]....
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...In studies with fluorescent protein fused with human or viral TK1, cytoplasmic fluorescence but also low level of fluorescence in the cell nuclei was found [18]....
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TL;DR: The formalin-subtilisin Carlsberg technique resulted in good agreement of ploidy measurements compared to the standard method, a higher number of evaluable histograms, an improved detectability of aneuploid cell populations, and an improved accuracy of the S- and G2-phase analysis, particularly in samples with low proliferation.
Abstract: An easy method for preparation of bare cell nuclei from fresh solid tissues for DNA flow cytometry is described. Pieces of up to 2 × 2 × 2 mm3 size from fresh tissues were fixed in formalin. After removal of formalin by washing with ethanol and rehydration with tap water, the tissue pieces were incubated with subtilisin Carlsberg (pronase, Sigma protease XXIV) and then stained directly with DAPI. Staining with ethidium bromide gave unsatisfactory results. Neither mechanical disaggregation nor centrifugation were used. The resulting cell nucleus suspensions had extremely low frequencies of debris particles and of clumped cell nuclei. A good yield, a minimized loss, and a good stainability of cell nuclei were obtained.
The applicability of the method was exemplified by the analysis of biopsies from the colon-rectum in patients with ulcerative colitis and of biopsies from the bladder in patients with bladder cancer and compared to the standard method of this laboratory, which uses mechanical disaggregation, ethanol fixation, pepsin treatment, and staining with ethidium bromide. The formalin-nubtilisin Carlsberg technique resulted in good agreement of ploidy measurements compared to the standard method, a higher number of evaluable histograms, an improved detectability of aneuploid cell populations, and an improved accuracy of the S- and G2-phase analysis, particularly in samples with low proliferation. The method also makes it possible to use long-term storage and to transport samples by post. © 1993 Wiley-Liss, Inc.
71 citations
"Investigation on cell proliferation..." refers methods in this paper
...The cell cycle composition was determined by DNA flow cytometry as described previously [3]....
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01 Jan 1990
TL;DR: The respective pretreatment prognostic impacts of the following markers were evaluated in 125 patients with small cell lung cancer: lactic dehydrogenase, serum thymidine kinase, S‐TK, carcinoembryonic antigen, neuronspecific enolase, and tissue polypeptide antigen.
Abstract: The respective pretreatment prognostic impacts of the following markers were evaluated in 125 patients with small cell lung cancer (SCLC): lactic dehydrogenase (LDH), serum thymidine kinase (S‐TK), carcinoembryonic antigen (CEA), neuronspecific enolase (NSE), and tissue polypeptide antigen (TPA). More traditional clinical and serologic markers were also evaluated. Univariate analysis showed that all of the biochemical markers mentioned above, the Karnofsky index (KI) and the patient's sex were related to both the stage of disease (limited/extensive disease: LD/ED) and to survival. The strongest marker for the clinical stage was S‐TK, whereas TPA showed the strongest relationship with survival. Multivariate analyses produced a model consisting of S‐TK, CEA, NSE, and the patient's sex for determining the clinical stage. To compare the prognostic capacity of easily determined biochemical and simple clinical variables to the more resource‐demanding variable of the clinical stage, three multivariate analyses in relation to survival were performed: (1) biochemical markers and simple clinical variables; (2) LD/ED and simple clinical variables; and (3) all available variables. The model obtained from the first analysis included TPA, KI, age, and the patient's sex; the model from the second analyses included LD/ED, patient's age, and KI; and the model from the third analysis, TPA, KI, age, sex, and LD/ED. Indices based on these three multivariate models were calculated for each patient and the prognostic capacity of these indices was compared. Pretreatment serum marker levels also had the capacity to predict both the grade and the duration of the response to therapy.
69 citations