Review: assessment of cell proliferation in histological material.
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
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TL;DR: The Annexin V assay offers the possibility of detecting early phases of apoptosis before the loss of cell membrane integrity and permits measurements of the kinetics of apoptotic death in relation to the cell cycle.
5,291 citations
TL;DR: Data suggest that in normal tissues and lymphoid neoplasms, PCNA immunolocalization can be used as an index of cell proliferation, however, in some forms of neoplasia, including breast and gastric cancer and in vitro cell lines, the simple relation between PCNA expression and cell proliferation is lost.
Abstract: Proliferating cell nuclear antigen (PCNA) is a 36 kD nuclear protein associated with the cell cycle A monoclonal antibody, PC10, that recognizes a fixation and processing resistant epitope has been used to investigate its tissue distribution Nuclear PCNA immunoreactivity is found in the proliferative compartment of normal tissues PCNA immunoreactivity is induced in lectin stimulated peripheral blood mononuclear cells in parallel with bromodeoxyuridine incorporation and the number of cells with PCNA immunoreactivity is reduced by induction of differentiation in HL60 cells In non-Hodgkin's lymphomas a linear relation between Ki67 and PCNA staining was demonstrated These data suggest that in normal tissues and lymphoid neoplasms, PCNA immunolocalization can be used as an index of cell proliferation However, in some forms of neoplasia, including breast and gastric cancer and in vitro cell lines, the simple relation between PCNA expression and cell proliferation is lost In some breast and pancreatic tumours there is apparent deregulation of PCNA with increased expression in tissues adjacent to the tumours The over-expression in some tumours and in adjacent morphologically normal tissue may represent autocrine or paracrine growth factor influence on PCNA gene expression
1,441 citations
TL;DR: The results challenge the dogma that the adult heart is a postmitotic organ and indicate that the regeneration of myocytes may be a critical component of the increase in muscle mass of the myocardium.
Abstract: Background The scarring of the heart that results from myocardial infarction has been interpreted as evidence that the heart is composed of myocytes that are unable to divide. However, recent observations have provided evidence of proliferation of myocytes in the adult heart. Therefore, we studied the extent of mitosis among myocytes after myocardial infarction in humans. Methods Samples from the border of the infarct and from areas of the myocardium distant from the infarct were obtained from 13 patients who had died 4 to 12 days after infarction. Ten normal hearts were used as controls. Myocytes that had entered the cell cycle in preparation for cell division were measured by labeling of the nuclear antigen Ki-67, which is associated with cell division. The fraction of myocyte nuclei that were undergoing mitosis was determined, and the mitotic index (the ratio of the number of nuclei undergoing mitosis to the number not undergoing mitosis) was calculated. The presence of mitotic spindles, contractile ri...
1,389 citations
TL;DR: Monoclonal antibody Ki‐67 is of use in research, providing a means of measuring proliferative activity in a variety of conditions besides malignancy, and may prove of value in monitoring tumour response to established and trial therapies.
Abstract: Monoclonal antibody Ki-67 is a reliable and easy means of accurately assessing the growth fraction of human neoplasms. Although the number of long-term follow-up studies is limited, it does appear to provide valuable prognostic information particularly in lymphoproliferative disease. Since the estimation of growth fraction is only one factor influencing tumour behaviour it would be naive to believe that measurement of this parameter alone, no matter how accurately, would provide the clinician with definitive prognostic information for all tumours. The antibody is also of use in research, providing a means of measuring proliferative activity in a variety of conditions besides malignancy, and may prove of value in monitoring tumour response to established and trial therapies.
637 citations
TL;DR: In situ end‐labelling stains cells with the morphological characteristics of apoptosis, and greatly simplifies their identification, and in two model systems, the number of labelled cells parallels thenumber of cells undergoing apoptosis as measured by alternative techniques.
Abstract: We have investigated the use of a novel technique, in situ end-labelling, as a means of the specific identification of apoptotic cells in formalin-fixed, paraffin-processed tissue sections. The technique relies on the presence of DNA strand breaks in apoptotic cells, caused by activation of endogenous nuclease activity during the process of cell death. These strands are labelled with a non-isotopic reporter molecule in the presence of a DNA polymerase, and labelled DNA is identified immunohistochemically. We show that in situ end-labelling stains cells with the morphological characteristics of apoptosis, and greatly simplifies their identification. Furthermore, in two model systems, the number of labelled cells parallels the number of cells undergoing apoptosis as measured by alternative techniques. The ability of the Klenow fragment of DNA polymerase to label apoptotic nuclei suggests that the characteristic DNA fragmentation seen during this process involves the formation of DNA breaks with a 5' overhang. In situ end-labelling will be valuable for the identification and quantitation of apoptosis in a range of normal tissues and in a variety of pathological states. However, the technique is not specific for programmed cell death, and results must be interpreted with caution and correlated with morphological criteria of apoptosis.
586 citations
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TL;DR: The data suggest that the early stages of mitogen stimulation represent initial sequences of proliferation and not parts of the cell cycle, and immunostaining with monoclonal antibody Ki-67 provides a reliable means of rapidly evaluating the growth fraction of normal and neoplastic human cell populations.
Abstract: The monoclonal antibody Ki-67 detects a nuclear antigen that is present only in proliferating cells. The aim of the present investigation was to clarify whether the Ki-67 nuclear antigen is restricted in its expression to certain phases of the cell cycle. All experiments consistently showed that the Ki-67 nuclear antigen is present in S, G2, and M phase, but is absent in G0. However, the results concerning Ki-67 antigen expression in G1 phase varied: cells passing the early events of mitogen triggered transition from G0 to G1, i.e., G1T and first G1A, lacked the Ki-67 nuclear antigen, whereas G1 cells after mitosis were constantly Ki-67-positive. This result suggests that after mitosis cells might not follow the same metabolic pathways as G0 cells do when entering G1 for the first time. Therefore, we suggest that the early stages of mitogen stimulation represent initial sequences of proliferation and not parts of the cell cycle. Because our data show that the Ki-67 nuclear antigen is present throughout the cell cycle, immunostaining with monoclonal antibody Ki-67 provides a reliable means of rapidly evaluating the growth fraction of normal and neoplastic human cell populations.
4,093 citations
TL;DR: Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication on a cell-by-cell basis in vitro and do not cross-react with thymidine.
Abstract: Monoclonal antibodies specific for 5-bromodeoxyuridine have been produced and applied in detecting low levels of DNA replication on a cell-by-cell basis in vitro. The immunoglobulin-producing hybridomas were derived from spleen cells of mice immunized with a conjugate of iodouridine and ovalbumin. The cells were fused with the plasmacytoma line SP2/0Ag14. The antibodies produced are highly specific for bromodeoxyuridine and iododeoxyuridine and do not cross-react with thymidine. DNA synthesis in cultured cells exposed to bromodeoxyuridine for as short a time as 6 minutes can be detected easily and rapidly by an immunofluorescent staining method and quantitated by flow cytometry.
2,722 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
TL;DR: There was a good correlation between the DNA histograms produced using this method and those obtained using unfixed tissue from the same tumor stained with ethidium bromide plus mithramycin.
Abstract: A method has been developed that allows flow cytometry to be used for measuring the cellular DNA content of paraffin-embedded human tumors. Thick (i.e., 30 micron) sections were cut from tissue blocks using a microtome and dewaxed in xylene. The sections were then rehydrated by sequentially immersing them in 100, 95, 70, and 50% ethanol before finally washing in distilled water. Single cell suspensions were then prepared by incubation in 0.5% pepsin, pH 1.5, at 37 degrees C for 30 min. The cells were counted, washed, and stained with 1 microgram/ml 4',6'-diamidino-2-phenylindole for 30 min, and DNA content was measured using an ICP 22 flow cytometer. There was a good correlation between the DNA histograms produced using this method and those obtained using unfixed tissue from the same tumor stained with ethidium bromide plus mithramycin. This method allows the retrospective study of archival material where the clinical outcome is already known, and it should, therefore, be particularly useful for determin...
2,005 citations
TL;DR: Cyclin and the auxiliary protein of DNA polymerase-δ are identical, and it is reported here that these two are identical.
Abstract: Identification of the cellular proteins whose expression is regulated during the cell cycle in normal cells is essential for understanding the mechanisms involved in the control of cell proliferation. A nuclear protein called cyclin of relative molecular mass 36,000 (Mr 36K), whose synthesis correlates with the proliferative state of the cell, has been identified in several cell types of human, mouse, hamster and avian origin. The rate of cyclin synthesis is very low in quiescent cells and increases several fold after serum stimulation shortly before DNA synthesis. Immunofluorescence and autoradiography studies have shown that the nuclear staining patterns of cyclin during S phase have a sequential order of appearance and a clear correlation can be found between DNA synthesis and cyclin positive nuclei. The proliferating cell nuclear antigen (PCNA) and cyclin have many common properties and it has been shown that these two are identical. Recently a protein which is required by DNA polymerase-delta for its catalytic activity with templates having low primer/template ratios has been isolated from calf thymus. We report here that cyclin and the auxiliary protein of DNA polymerase-delta are identical.
1,747 citations