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.
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TL;DR: It is concluded that TK1 might be a more accurate marker than PCNA for estimation of cell proliferation and malignant potentials in breast carcinomas.
Abstract: 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 c...
36 citations
Cites background or methods from "Investigation on cell proliferation..."
...The new anti-TK1 antibody works in archival specimens and is a specific marker of tumor cell proliferation.([19,20]) In the present study, we estimated the proliferation index of TK1 in breast cancer patients by using anti-TK1 antibody (pAb1)....
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...Typically, the positive staining was observed by spots of granules and concentrated mostly near the nuclei as previously described by Wang et al.[19] The staining for PCNA was found in the nuclei with a diffuse or granular pattern or a mixture of both....
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...Typically, the positive staining was observed by spots of granules and concentrated mostly near the nuclei as previously described by Wang et al.([19]) The staining for PCNA was found in the nuclei with a diffuse or granular pattern or a mixture of both....
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...Here we present a new tumor marker, thymidine kinase 1, an enzyme recently shown to correlate with tumor proliferative rate.([17,19,20]) The expression of TK activity has been studied thoroughly in human breast cancer cell lines([24]) and in human serum of breast cancer patients, – 28] but corresponding studies in malignant breast tissues and normal breast tissues are still few....
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TL;DR: The value of the half-life of STK1 measured by ECL dot blot can be used as a potential marker for monitoring the response to surgery in patients with gastric or other cancers one month after surgery.
Abstract: Thymidine kinase 1 in serum (STK1) of patients with gastric cancer was determined by two methods: ECL dot blot and radioactivity assay. Both measurements showed significantly different values for preoperative STK1 and healthy STK1 (p=0.012 for ECL dot blot and p=0.003 for the radioactivity assay). The preliminary results of ECL dot blot STK1 measurement showed that in tumor-free subjects the level of the enzyme was significantly reduced to 52.7% 35 days after surgery (n=8, p=0.0106). The decrease in STK1 levels in the tumor-free subjects paralleled the decline of the half-life of the STK1 enzyme. In patients with distant metastases (n=6) the enzyme level had increased to 173% 35 days postoperatively. By contrast, with the radioactivity assay no significant differences in thymidine kinase activity for 0-day-postoperative patients and 35-day-postoperative tumor-free patients was found (p=0.329). The activity decreased to 80% in 35-day-postoperative patients with metastatic disease. We suggest that the value of the half-life of STK1 measured by ECL dot blot can be used as a potential marker for monitoring the response to surgery in patients with gastric or other cancers one month after surgery.
36 citations
TL;DR: 18F‐FLT, a nucleoside analog, could monitor effects of molecularly targeted therapeutics on tumor proliferation as well as investigate the role of EMTs in tumor proliferation.
Abstract: Defining response to therapy by traditional anatomic imaging of tumor size requires serial observations over many months. In contrast, nucleoside analog radiopharmaceuticals that are being developed for noninvasive imaging of tumor cell proliferation have significant potential for showing tumor response within days to weeks of initiating therapy. One investigational radiopharmaceutical that is seeing increased use in cancer research is the proliferation-specific positron emission tomography (PET) ligand 3′-18F-fluoro-3′-deoxy-fluorothymidine (18F-FLT). Several clinical trials have demonstrated that 18F-FLT can be used to image a variety of tumor types, and there is a strong correlation between the extent of FLT uptake and the proliferation index in individual tumors.1–4
FLT enters cells through active and passive nucleoside transport mechanisms and is subsequently phosphorylated by thymidine kinase-1 (TK1).5,6 The resulting negative charge of FLT-monophosphate prevents efflux out of the cell, and this TK1-mediated phosphorylation reaction is the rate-limiting step involved in FLT cellular uptake/retention.7,8 Consistent with this, in vitro studies have correlated TK1 expression levels with the extent of cellular FLT retention.9,10 Although TK1 is not a specific proliferation marker, TK1 is regulated within the cell cycle,11 and the extent of FLT uptake within a tumor usually reflects the fraction of tumor cells in S-phase where TK1 expression is highest. Because TK1 expression and FLT tumor uptake should be suppressed following pharmacologic inhibition of tumor proliferation, nucleoside analogs which act as reporters of TK1 activity may be suitable for assessing the antiproliferative effects of a variety of molecularly targeted therapeutics.
Several epidermal growth factor receptor (EGFR) inhibitors have demonstrated activity, either alone or in combination with conventional cytotoxic agents, in several tumor types, including colorectal, lung, and head and neck cancer. Unfortunately, only a minority of patients benefit from these molecularly targeted agents, and development of techniques to identify those patients most likely to benefit from a specific molecular therapy could facilitate the individualization of cancer therapy. In the current study, we have applied 18F-FLT PET imaging to monitor the effects of 2 clinically approved EGFR inhibitors: erlotinib, a small molecule EGFR kinase inhibitor, and cetuximab, an anti-EGFR therapeutic monoclonal antibody. The current study demonstrates that EGFR inhibitor therapy suppresses FLT uptake as early as 3 days after initiating drug treatment and suggests that this technique could be used to identify patients who are responding to EGFR inhibitor therapy early during their course of therapy.
36 citations
TL;DR: FMAU is preferably phosphorylated by TK2 and can track TK1 activity and mitochondrial mass in cellular stress and may provide an early marker of treatment effects.
Abstract: Purpose
Fluoropyrimidines like 1-(2′-deoxy-2′-fluoro-β-d-arabinofuranosyl)-thymine (FMAU) and 3′-deoxy-3′-fluorothymidine (FLT) accumulate in tumors and are being used as positron emission tomography tumor-imaging tracers. Proliferating tissues with high thymidine kinase 1 (TK1) activity retain FLT; however, the mechanism of selective accumulation of FMAU in tumors and certain other tissues requires further study.
35 citations
TL;DR: It is demonstrated that the UV-induced activation of Erbb2 regulates the response of the skin to UV, and a role for ErBB2 is suggested inUV-induced pathologies such as skin cancer.
Abstract: Exposure to ultraviolet (UV) irradiation is the major cause of nonmelanoma skin cancer, the most common form of cancer in the United States. UV irradiation has a variety of effects on the skin associated with carcinogenesis, including DNA damage and effects on signal transduction. The alterations in signaling caused by UV regulate inflammation, cell proliferation, and apoptosis. UV also activates the orphan receptor tyrosine kinase and proto-oncogene Erbb2 (HER2/neu). In this study, we demonstrate that the UV-induced activation of Erbb2 regulates the response of the skin to UV. Inhibition or knockdown of Erbb2 before UV irradiation suppressed cell proliferation, cell survival, and inflammation after UV. In addition, Erbb2 was necessary for the UV-induced expression of numerous proinflammatory genes that are regulated by the transcription factors nuclear factor-κB and Comp1, including interleukin-1β, prostaglandin-endoperoxidase synthase 2 (Cyclooxygenase-2), and multiple chemokines. These results reveal the influence of Erbb2 on the UV response and suggest a role for Erbb2 in UV-induced pathologies such as skin cancer.
35 citations
References
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TL;DR: The p53 mutational spectrum differs among cancers of the colon, lung, esophagus, breast, liver, brain, reticuloendothelial tissues, and hemopoietic tissues as mentioned in this paper.
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.
8,063 citations
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.
2,655 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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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.
1,024 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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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
453 citations
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.
440 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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