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Journal ArticleDOI

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.

AbstractThe 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.

Topics: Bromodeoxyuridine (61%), Thymidine kinase 1 (60%), Cell cycle (60%), Cell growth (59%), Mitosis (57%)

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Citations
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Journal ArticleDOI
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.

183 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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Journal ArticleDOI
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.
Abstract: Egg yolk is a good source of highly specific antibodies against mammalian antigens because of the phylogenetic distance between birds and mammals. Chicken egg yolk immunoglobulins (IgY) were generated to a synthetic 31-amino acid peptide from the C-terminal of human HeLa thymidine kinase 1 (TK1) enzyme. The anti-TK1 IgY antibody was purified using affinity chromatography against the 31-amino acid peptide. The purified antibody inhibited the catalytic activity of the TK1 enzyme in the CEM TK1(+) cells and recognized the 25-kDa subunit and tetrameric form of TK1, which has a pI value of 8.3. No immunoreaction was observed in CEM TK1(-) cells. Western blot of the serum TK1 (S-TK1) also showed that only a single band was found in the serum of patients with malignancies. No band was seen in healthy serum. Furthermore, dot blots and enhanced chemiluminescence (ECL) detection of S-TK1 performed on sera of preoperative patients with gastric cancer (GC) (n=31) and healthy controls (n=62) showed that the levels of S-TK1 in the sera of cancer patients were significantly different (P<0.01). Using ECL dot blots, 0.1 pg of TK1 in 3 microl sera could be detected. Immunohistostaining of tissues in the 11 advanced-stage cancer patients (four breast carcinomas, three hepatocarcinomas and four thyroid carcinomas) indicated that a strong staining of TK1 enzyme was found in the cytoplasm of malignant cells. No staining or weak staining was seen in normal tissues. We suggest that screening for TK1 using anti-TK1 IgY may be potentially useful for serological and immunohistochemical detection of TK1 as an early prognosis and for monitoring patients undergoing treatment.

67 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.

65 citations


Journal ArticleDOI
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.

62 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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Journal ArticleDOI
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.
Abstract: Thymidine kinases (TK) have a key function in the synthesis of DNA. Two isoenzymes have been characterized: TK1 is cell cycle-dependent and present in the cytoplasm whereas TK2--located in mitochondria--is cell cycle-independent. The diagnostic and prognostic role of TK1 has recently been investigated. TK1 might be helpful for screening and monitoring of human malignancies. TK1 may also serve as a prognostic factor for progression. Herein, we summarize the status of TK1 for cancer monitoring and point out its use as a proliferation marker. A comprehensive overview about the association of TK-1 with various entities is given.

60 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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References
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Journal ArticleDOI
05 Jul 1991-Science
Abstract: Mutations in the evolutionarily conserved codons of the p53 tumor suppressor gene are common in diverse types of human cancer. The p53 mutational spectrum differs among cancers of the colon, lung, esophagus, breast, liver, brain, reticuloendothelial tissues, and hemopoietic tissues. Analysis of these mutations can provide clues to the etiology of these diverse tumors and to the function of specific regions of p53. Transitions predominate in colon, brain, and lymphoid malignancies, whereas G:C to T:A transversions are the most frequent substitutions observed in cancers of the lung and liver. Mutations at A:T base pairs are seen more frequently in esophageal carcinomas than in other solid tumors. Most transitions in colorectal carcinomas, brain tumors, leukemias, and lymphomas are at CpG dinucleotide mutational hot spots. G to T transversions in lung, breast, and esophageal carcinomas are dispersed among numerous codons. In liver tumors in persons from geographic areas in which both aflatoxin B1 and hepatitis B virus are cancer risk factors, most mutations are at one nucleotide pair of codon 249. These differences may reflect the etiological contributions of both exogenous and endogenous factors to human carcinogenesis.

7,906 citations


Journal ArticleDOI
TL;DR: A first series of immunostainings of tumour biopsies indicated that Ki‐67 may be a potent tool for easy and quick evaluation of the proportion of proliferating cells in a tumour.
Abstract: The production of a mouse monoclonal antibody, Ki-67, is described. The Ki-67 antibody recognized a nuclear antigen present in proliferating cells, but absent in resting cells. Immunostainings with Ki-67 revealed nuclear reactivity in cells of germinal centres of cortical follicles, cortical thymocytes, neck cells of gastrointestinal mucosa, undifferentiated spermatogonia and cells of a number of human cell lines. The Ki-67 antibody did not react with cells known to be in a resting stage, such as lymphocytes, monocytes, parietal cells and Paneth's cells of gastrointestinal mucosa, hepatocytes, renal cells, mature sperm cells, brain cells, etc. Expression of the antigen recognized by Ki-67 could be induced in peripheral blood lymphocytes after stimulation with phytohaemagglutinin, whereas it disappeared from HL-60 cells stimulated with phorbol esters to differentiate into mature macrophages in a resting stage. These findings suggest that Ki-67 is directed against a nuclear antigen associated with cell proliferation. A first series of immunostainings of tumour biopsies indicated that Ki-67 may be a potent tool for easy and quick evaluation of the proportion of proliferating cells in a tumour.

2,552 citations


"Investigation on cell proliferation..." refers methods in this paper

  • ...In clinical investigations, mitotic count, 3H-thymidine labeling index, bromodeoxyuridine (BrdU) incorporation, expression of Ki-67, proliferating cell nuclear antigen, and cyclins, as well as measurement of S-phase by means of DNA flow cytometry have been used in the assessment of cell proliferation [5,8,17,22,33,36,37]....

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Journal ArticleDOI
TL;DR: It is concluded that overexpression of p53 is synonymous with mutation, but some mutations would not be detected by a simple immunohistochemical analysis.
Abstract: Immunohistological staining of primary colorectal carcinomas with antibodies specific to p53 demonstrated gross overexpression of the protein in approximately 50% of the malignant tumors examined. Benign adenomas were all negative for p53 overexpression. To determine the molecular basis for this overexpression we examined p53 protein expression in 10 colorectal cancer cell lines. Six of the cell lines expressed high levels of p53 in ELISA, cell-staining, and immunoprecipitation studies. Direct sequencing and chemical-mismatch-cleavage analysis of p53 cDNA by using the polymerase chain reaction in these cell lines showed that all cell lines that expressed high levels of p53 were synthesizing mRNAs that encoded mutant p53 proteins. In two of those four cell lines where p53 expression was lower, point mutations were still detected. Thus, we conclude that overexpression of p53 is synonymous with mutation, but some mutations would not be detected by a simple immunohistochemical analysis. Mutation of the p53 gene is one of the commonest genetic changes in the development of human colorectal cancer.

1,010 citations


"Investigation on cell proliferation..." refers background in this paper

  • ...Recently, also cell cycle related gene expression such as the expression of the p53 tumor suppressor gene has been the subject of studies relating to the prognosis of malignancies [4,16,30]....

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Journal ArticleDOI
TL;DR: This brief overview will illustrate current ideas about cellular proliferation and its regulation and the advantages and disadvantages of the better known methods for assessing cellular proliferation in histopathological material.
Abstract: Introduction There can be little dispute that cellular proliferation is one of the most fundamental of biological processes.' Nor can there be disagreement over the importance of assessing cellular proliferation in the study of many biological processes: indeed, Leblond2 used such assessment for identifying the three major functional types of cellular populationnamely, static, conditional renewal, and continually renewing. The practice of histopathology involves direct or, more usually, indirect assessment of cellular proliferation (and related phenomena such as differentiation) in many situations.3 It is intended that this brief overview will illustrate current ideas about cellular proliferation and its regulation and the advantages and disadvantages of the better known methods for assessing cellular proliferation in histopathological material

451 citations


Journal ArticleDOI
TL;DR: Two different post-transcriptional mechanisms largely account for the periodic behavior of the enzyme activity during the cell cycle, indicating that the efficiency of translation of thymidine kinase mRNA increases as cells begin DNA replication.
Abstract: As an approach to defining the molecular basis for the periodic expression of thymidine kinase activity during the cell cycle, we have examined properties of the cytosolic enzyme in cycling HeLa cells synchronized by centrifugal elutriation and mitotic selection. By immunoblot analyses with a specific antiserum raised against the purified HeLa enzyme, we have demonstrated that changes in the levels of thymidine kinase activity reflect similar changes in the levels of thymidine kinase polypeptide. In contrast, the steady state levels of thymidine kinase mRNA show relatively small changes during the cell cycle. Using pulse labeling methods, we have shown that the synthetic rate of thymidine kinase protein is about 10-fold greater in S phase than in G1 phase, indicating that the efficiency of translation of thymidine kinase mRNA increases as cells begin DNA replication. In addition, the stability of thymidine kinase protein dramatically decreases upon cell division, resulting in the rapid clearance of the enzyme from newly divided G1 cells. Thus, two different post-transcriptional mechanisms largely account for the periodic behavior of the enzyme activity during the cell cycle.

425 citations


"Investigation on cell proliferation..." refers background or result in this paper

  • ...Some mitotic cells had the same level of TK1 as G2- cells while others had much lower levels, which indicates a rapid degradation of the TK1 protein during mitosis, as was also described by Sherley and Kelly [32]....

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  • ...cells while others had much lower levels, which indicates a rapid degradation of the TK1 protein during mitosis, as was also described by Sherley and Kelly [32]....

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  • ...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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  • ...There is a good correlation between TK activity and the amount of TK1 protein during the cell cycle [19, 25,32]....

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