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PubMedDate not supplied

Cell proliferation in rat colon measured with bromodeoxyuridine, proliferating cell nuclear antigen, and [3H]thymidine.

Epithelial cell proliferation was studied in the normal colonic mucosa of 5-week-old Sprague-Dawley rats, comparing [3H]thymidine incorporation (group 1) with two newer proliferation markers, bromodeoxyuridine (group 2) and proliferating cell nuclear antigen (group 3). Microautoradiography (group 1) or immunoperoxidase assays (groups 2 and 3) were carried out. Cells were counted for positive reaction and position along 50 colonic crypt columns/animal. No significant differences were found in number or distribution of labeled epithelial cells in proliferative compartments in crypt columns of normal colonic mucosa; labeled cells were mainly in the lower 60% of colonic crypts. Thus, in this model, bromodeoxyuridine and proliferating cell nuclear antigens were comparable to [3H]thymidine as reliable markers of proliferating epithelial cells in rat colon.

Animals↗

PubMed1990

Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms.

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.

Adult↗

PubMed1992

Proliferating cell nuclear antigen. A marker for cell proliferation in autopsy tissues.

Antibodies to the proliferating cell nuclear antigen allow identification of proliferating cells in fresh tissue specimens using routine immunocytochemical methods. However, the use of such proliferation markers has not been verified for autopsy-derived tissue specimens, in which there is often a significant delay between the time of death and tissue specimen fixation. To assess the reliability of anti-proliferating cell nuclear antigen antibodies to identify proliferating cells in autopsy tissue specimens, an autopsy simulation was performed using fresh monkey and rat tissue specimens. These tissue specimens were kept at room temperature for predetermined numbers of hours before fixation. The proliferation specific staining was most reliable for tissue specimens obtained within 6 hours of death. There was reliable staining of proliferating regions up to 12 hours, although sensitivity was decreased. The only exception was skin, which was able to withstand much longer periods. Quantitative data from monkey spleen white-pulp regions showed 63% of the cells to stain for proliferating cell nuclear antigen when fixed immediately; this decreased to 29% of the cells after 12 hours and only 19% by 18 hours of postmortem simulation. Representative tissue specimens obtained from human autopsy material revealed similar postmortem staining patterns. Rapid procurement and fixation of tissue specimens and the use of control tissue specimens derived from the same autopsy material (eg, lymph node tissue) are recommended. These studies do suggest that anti-proliferating cell nuclear antigen antibodies can be used to identify proliferating cells in human autopsy tissue specimens obtained within approximately 12 hours of death, with some compromise in overall sensitivity.

Animals↗

PubMed1989

Cell proliferation, cell death and aging.

An integrated view of the processes which most likely play a critical role in the aging process at the cellular level is proposed. Cells are continuously exposed to a variety of internal and external stressors, potentially dangerous for the maintenance of the functional integrity of the cell (UV and gamma radiation, heat, oxygen free radicals, glucose, bacteria, viruses). In the course of evolution a number of mechanisms [DNA repair, production of heat shock and other stress proteins, enzymatic and non-enzymatic antioxidant defence systems, poly(ADP-ribose) polymerase activation] have emerged which allow the cell to cope with such a variety of potentially harmful agents. These mechanisms are in fact interconnected and constitute a network of cellular defence systems. It is suggested that they play a physiological role, being involved in the control of gene expression. A failure of these mechanisms does not allow the cell to maintain homeostasis and has profound consequences as far as two of the major programs of the cell are concerned, i.e. cell proliferation and cell death. Recent data suggesting that these are two physiologically active phenomena tightly linked and regulated are examined. Thus, activation of cell cycle related genes and active inhibition of suicide genes appear to be a part of an integrated process. Conversely, deprivation of growth factors seems able to induce an active process of programmed cell death characterized by Ca++,Mg+(+)-dependent endonuclease activity and DNA fragmentation (apoptosis). Similar phenomena have been shown to accompany the terminal differentiation process in several cellular systems. The understanding of the factors which favour or prevent cell death (a phenomenon which has been recognized as one of the most important in fetal development and morphogenesis) will help to unravel and eventually to manipulate the aging process. In an evolutionary perspective, cell senescence appears to be the price paid to avoid unlimited capability of proliferation, i.e. cell transformation and cancer.

Aging↗

PubMed1992

Cell proliferation in childhood acute leukemia. Comparison of Ki-67 and proliferating cell nuclear antigen immunocytochemical and DNA flow cytometric analysis.

The proliferative activity of bone marrow leukemia cells was determined by DNA flow cytometric (FCM) analysis and labeling index (LI) of Ki-67 monoclonal antibodies and proliferating cell nuclear antigen (PCNA) autoantibodies in 73 children with acute leukemia. LI of Ki-67 varied greatly from patient to patient (range, 0.4% to 42.2%; mean, 18.8%) and differed significantly between acute lymphoblastic leukemia (ALL) and acute nonlymphoblastic leukemia (ANLL). In ALL, the Ki-67 LI showed a positive correlation with the S-phase fraction (SPF) determined by DNA FCM analysis, whereas, in ANLL, there was a discrepancy between the Ki-67 LI and SPF. In contrast, LI of PCNA varied less among the patients (range, 57.2% to 100%; mean, 90.3%), and the value was always higher than that of the Ki-67 LI in individual patients. A significant relationship between PCNA LI and the percentage of blast cells was found in peripheral blood leukocytes from patients with leukemia. These results suggest that the Ki-67 LI reflects differences in the proliferative activity depending on the subtype of the disease and that the PCNA LI is useful as a marker of proliferating cells.

Blast Crisis↗

PubMed1992

Inhibition of smooth muscle cell proliferation by an antisense oligodeoxynucleotide targeting the messenger RNA encoding proliferating cell nuclear antigen.

BACKGROUND: The process by which normally quiescent vascular smooth muscle cells (SMCs) change into proliferating cells, which express and respond to multiple growth factors, plays a major role in restenosis after coronary angioplasty. We are attempting to inhibit SMC proliferation by interventions that inhibit specific factors involved in signal transduction pathways leading to cell division. To date, all studies taking this approach have attempted to block the effects of mitogens acting on the cell surface. In contrast, we have focused on a strategy that bypasses cell surface-mediated events by directly inhibiting the expression of proliferating cell nuclear antigen (PCNA), an intranuclear protein that functions in a final common pathway shared by diverse mitogen-induced signals. In the present investigation, we determined whether antisense oligodeoxynucleotides (ODNs) complementary to the messenger RNA of PCNA will inhibit PCNA expression and thereby reduce SMC proliferation. METHODS AND RESULTS: When antisense ODNs (15- or 18-mer), modified to inhibit their degradation, are introduced into the medium of rat aortic SMCs in concentrations ranging from 10 to 100 microM, the 18-mer ODN, in a concentration-related manner, decreases SMC growth (as assessed by cell counting) by more than 50%. This effect persists for at least 9 days. An ODN with the same nucleotides but a scrambled sequence has little effect. Western blots and immunocytochemistry indicate that the antisense ODN reduces expression of PCNA protein. CONCLUSIONS: Our results demonstrate that an antisense ODN directed at the messenger RNA of PCNA decreases expression of the PCNA gene product and reduces SMC proliferation. In addition, these results provide an important impetus to initiating in vivo studies to determine the feasibility of antisense strategies in the prevention of coronary restenosis.

Angioplasty, Balloon, Coronary↗

PubMed1992

A study of cell proliferation in formalin-fixed, wax-embedded bone marrow trephine biopsies using the monoclonal antibody PC10, reactive with proliferating cell nuclear antigen (PCNA).

We have investigated proliferation in bone marrow trephine biopsies from 32 patients with normal or abnormal haemopoiesis, using the monoclonal antibody PC10, which detects proliferating cell nuclear antigen (PCNA), together with immunohistochemical markers of haemopoietic cell lineage. PCNA immunostaining revealed the pattern of proliferation within individual haemopoietic lineages in normal marrow. Two unexpected observations were made: of erythroid cells, only pro-erythroblasts and occasional early normoblasts reacted, and positivity of megakaryocytes was unrelated to nuclear lobulation or CD61 expression. The pathological cases represented conditions in which haemopoiesis is increased (reactive hyperplasia, chronic granulocytic leukaemia, myeloproliferative and myelodysplastic syndromes, megaloblastic anaemia). Increases in the number, and disturbances of the spatial organization, of PCNA-expressing cells were present to a variable extent in all cases. Sheets of PCNA-positive megaloblastoid erythrocytes were frequently found in myelodysplastic and myeloproliferative tissue, associated with marked disturbances in the spatial organization of all haemopoietic lineages. Cases of megaloblastic anaemia due to vitamin B12/folate deficiency also demonstrated greatly increased erythroid PCNA expression, with positivity in some giant metamyelocytes. In addition to reflecting increased proliferation, elevated PCNA expression in some bone marrow pathologies may be due to altered kinetics of the protein induced by disturbances in growth factor production.

Autoantigens↗

PubMed1992

Comparison of BrdUrd and [3H]TdR incorporation to estimate cell proliferation, cell loss, and potential doubling time in tumor xenografts.

In this study, two different methods of estimating cell proliferation were compared: cell loss and potential growth rate of xenografted head and neck cancer grown in nude mice based on the detection of DNA incorporation of bromodeoxyuridine (BrdUrd) in one method, and [3H]thymidine ([3H]TdR) in the other. The 21-d-old xenografts were labelled in vivo, either with BrdUrd or [3H]TdR and excised at intervals during 65.5 h. In tumors containing BrdUrd, the percent labelling was measured in mid-S and mid-G1 phase windows of cytograms from bivariate DNA flow cytometry (FCM). In [3H]TdR-labelled tumors, the percent labelled mitoses (PLM) was determined by light microscopy evaluation of autoradiographs. With a computer program based on a theoretical model, the percent labelling versus time after injection was used to analyze cell cycle time, cell loss, tumor growth fraction, and potential doubling time. The values calculated from DNA incorporation with BrdUrd agreed well with those obtained from labelling with [3H]TdR, i.e., cell cycle time 2.3 vs. 2.4 d, and growth fraction 67 vs. 70%. The estimated potential doubling time was 3.1 d and cell loss factor 40% by both methods. Flow cytometry analysis of BrdUrd-labelling is considerably faster than the evaluation of [3H]TdR-labelling, and the present results provide further support for the BrdUrd labelling method as a promising alternative to the PLM method in cell cycle studies designed to evaluate the relevance of cell proliferative properties in relation to biological behavior in xenografted head and neck cancer.

Animals↗

PubMed1992

Assessment of rejection in orthotopic human heart transplantation using proliferating cell nuclear antigen (PCNA) as an index of cell proliferation.

Myocardial biopsies taken during the management of cardiac transplantation were stained for proliferating cell nuclear antigen (PCNA). Counts of PCNA-positive interstitial cells were compared, in retrospect, with the reported histological grade of rejection. Biopsies without rejection had negligible numbers of PCNA-positive cells. Ascending grades of rejection were paralleled by an increase in the number of PCNA-positive cells [grade 1, 13 +/- 35 (mean +/- SD); grade 2a, 38 +/- 40; grade 2b, 91 +/- 75; grade 3, 170 +/- 78]. While highly significant, in statistical terms, the overlap in the counts between different grades means that prediction of rejection from the PCNA count alone is not feasible. Biopsies graded as 0 or 1 and which immediately preceded more severe rejection episodes showed no increase in PCNA-positive cells. The majority of PCNA-positive cells are fibroblasts, although in grade 2b and 3 rejection a small population of PCNA-positive T lymphocytes occurs. PCNA staining is also seen in cardiac myocytes immediately after transplantation, during rejection episodes, and late after transplantation in the absence of rejection. The positive PCNA staining of cardiac myocytes probably reflects DNA synthesis that occurs with the shift toward polyploidy in hypertrophy.

Autoantigens↗

PubMedDate not supplied

Prognostic value of cell proliferation in breast cancer as determined by proliferating cell nuclear antigen (PCNA) immunostaining.

A series of 175 biopsies from primary tumours were subjected to immunostaining for proliferation cell nuclear antigen (PCNA) and histopathological analysis for prognostic factors in 175 women with breast cancer followed up for nine years. In normal breast epithelium, only occasional nuclei were positive for PCNA. In breast carcinomas, the fraction of nuclei positive for PCNA showed considerable intratumoural variation, usually being highest at the invasive tumour margins. The fraction of positive nuclei was significantly related to histological grade (p less than 0.001), histological type (p = 0.049) and tumor recurrence (p = 0.01). The fraction of positive nuclei predicted recurrence-free survival (p = 0.007) and overall survival (p = 0.0158) in univariate analysis. In the multivariate analysis including also the standard prognostic factors, the PCNA positivity predicted recurrence-free survival (p = 0.004) and overall survival (p = 0.030) independently. The results show that the light microscopic assessment of the growth fraction as determined by PCNA immunolabeling has independent prognostic value in breast carcinomas like some other (e.g. S phase fraction, mitotic index, Ki-67) previously characterized proliferation indices.

Adult↗

PubMed1990

Thrombospondin gene expression by endothelial cells in culture is modulated by cell proliferation, cell shape and the substratum.

Endothelial cells plated on the surface of a two-dimensional substratum (gelatin-coated dishes, dishes coated with native type I collagen or collagen gels) form a cobblestone monolayer at confluence, whereas cells plated within a three-dimensional gel matrix elongate into a sprouting morphology and self-associate into tube-like structures. In this study, we have compared the synthesis of thrombospondin by quiescent endothelial cells displaying (a) the same morphological phenotype (cobblestone) on different substrata (gelatin and collagen) and (b) different morphological phenotypes (cobblestone and sprouting) on the same substratum (collagen). We demonstrate that thrombospondin is a major biosynthetic product of confluent, quiescent cells cultured on dishes coated with either gelatin or collagen, and that the synthesis of this protein is markedly decreased when cells are plated on or in three-dimensional collagen gels. Moreover, we demonstrate that cells plated in gel (sprouting) secrete less thrombospondin than do cells plated on the gel surface (cobblestone). The regulation of thrombospondin synthesis is reversible and occurs at the level of transcription, as steady-state mRNA levels for thrombospondin decrease in a manner comparable with the levels of protein secreted by these cells. We also show that mRNA levels for laminin B2 chains are increased when cells are cultured on and in collagen gels compared with on gelatin-coated dishes, suggesting that the syntheses of thrombospondin and laminin are regulated by different mechanisms. When cells are cultured on gelatin- or collagen-coated dishes, thrombospondin gene expression is directly proportional to the proliferative state of the cultures. By contrast, the synthesis of thrombospondin by cells cultured on collagen gels remains at equally low levels whether they are labelled when they are sparse and rapidly proliferating or when they are confluent and quiescent. Fibronectin synthesis was found to increase with increasing confluency of the cells plated on all three substrata. These results demonstrate that thrombospondin gene expression is modulated by cell shape, cell proliferation and the nature of the substratum used for cell culture.

Animals↗

PubMed1978

Cell proliferation and cell density of mesenchyme in the maxillary process and adjacent regions during facial development in the chick embryo.

Cell proliferation, as measured by DNA labeling indices was analyzed during the early development of the maxillary process. Chick embryos were labeled with [3H]thymidine for 1 h and processed for autoradiography. The percentage of labeled mesenchymal cells was determined within delineated areas in the maxillary processes and in adjacent regions. Analysis of labeling indices in each of the areas at successive stages of development demonstrated a pattern of declining rates of cell proliferation with advancing developmental age. Cell proliferation in adjacent regions declined earlier and, in some instances, faster than it did in the maxillary process. Cell density was measured in the maxillary process and the roof of the stomodeum and was found to be higher in the maxillary process throughout the period studied. Cell density and cell proliferation data were analyzed with reference to the operation of 'density-dependent inhibition' of growth as a regulatory mechanism for the observed changes. 'Density-dependent inhibition' of growth was not a satisfactory explanation for the observed differences between the maxillary process and adjacent regions.

Animals↗

PubMed1989

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.

Animals↗

PubMed1989

G1 events and regulation of cell proliferation.

Cells prepare for S phase during the G1 phase of the cell cycle. Cell biological methods have provided knowledge of cycle kinetics and of substages of G1 that are determined by extracellular signals. Through the use of biochemical and molecular biological techniques to study effects of growth factors, oncogenes, and inhibitors, intracellular events during G1 that lead to DNA synthesis are rapidly being discovered. Many cells in vivo are in a quiescent state (G0), with unduplicated DNA. Cells can be activated to reenter the cycle during G1. Similarly, cells in culture can be shifted between G0 and G1. These switches in and out of G1 are the main determinants of post-embryonic cell proliferation rate and are defectively controlled in cancer cells.

Animals↗

PubMed1975

Kinetics of cell proliferation and cell loss in the peripheral and central parts of Walker tumours growing in rats and nude mice.

In previous experiments it was shown that, in the submucosal part of Walker tumours transplanted to the gastric wall of rats, a lower rate of cell proliferation was seen in the peripheral zone, defined as the outer 100-120 mu of the tumours, than in the main tumour mass. The purpose of the present experiments was to investigate whether such differences are independent of the location of the Walker tumour, or were caused by local factors specific for the gastric mucosa, and whether specific cellular immunity cell proliferation at the periphery of a transplanted tumour. Cells from Walker 256 tumour were injected into the subcutaneous space in rats and in mutant nude mice, which lack T lymphocytes. In one series, the rats and mice were injected with 3H-TDR at different time intervals before sacrifice. In a second series vinblastine sulfate was injected 3 hours before sacrifice. Although all the animals were given the same tumour dose, the tumours in mice increased in size more slowly than those in rats. In the first-mentioned series, the mitotic counts, the labelled cells and the percentage labelled mitoses (PLM) in the main tumour mass and at the tumour perphery were counted. In the second series the mitotic rate in the same two regions was determined. A significantly lower rate of cell proliferation was demonstrated at the periphery compared to the main tumour mass in both rats and mice. Differences between the PLM curves in the two regions were also found. Possible explanations of these findings are discussed. It is concluded that the described growth pattern is probably a general characteristic of the Walker tumour, and that the low rate of proliferation at the periphery is not caused by specific immunological mechanisms mediated through T lymphocytes. If the growth rates were calculated on the assumtion that the actual tumour growth followed a Gompertz function, then the rate of cell loss in the tumour in mice was higher than that in the tumour in rats.

Animals↗

PubMed1991

Studies on the relationship between cell proliferation and cell death: opposite patterns of SGP-2 and ornithine decarboxylase mRNA accumulation in PHA-stimulated human lymphocytes.

To assess the relationship between cell proliferation and cell death, the mRNA accumulation of ornithine decarboxylase (ODC) and sulfated glycoprotein 2 (SGP-2) were measured in human peripheral blood lymphocytes (HPBL) 2-6 hours after stimulation with phytohemagglutinin (PHA). ODC is the rate limiting enzyme of polyamines biosynthesis and its early induction in mitogen-stimulated lymphocytes has been reported. On the other hand, SGP-2, a glycoprotein present in most mammalian tissues, is induced in classical models of apoptosis, such as dexamethasone-treated thymocytes. Indeed, a consistent amount of SGP-2 mRNA in quiescent HPBL, an early and progressive decrease of SGP-2 mRNA and a parallel increase of ODC mRNA accumulation, were observed, in PHA-stimulated HPBL, suggesting that concomitant repression of SGP-2 and induction of ODC genes contribute for the cell entering the cell cycle.

Blotting, Northern↗

PubMed1992

Studies of the relationship between cell proliferation and cell death. II. Early gene expression during concanavalin A-induced proliferation or dexamethasone-induced apoptosis of rat thymocytes.

Several data in the literature suggest that an intriguing relationship exists between cell proliferation and cell death. Accordingly, we studied the early expression of different genes in the same cells, i.e. rat thymocytes, undergoing cell proliferation upon stimulation with Concanavalin A or cell death following dexamethasone treatment. We showed that an early accumulation of c-fos, c-jun and c-myc mRNA occurred in both phenomena but with different kinetics. It can be speculated that the early induction of nuclear oncogenes is necessary to allow the later induction of other genes probably regulated at the transcriptional level by the AP-1 complex and/or by Myc protein. The accumulation of the transcript for another gene, i.e. poly(ADP-ribose)polymerase, an enzyme responsible for posttranslational modifications of several nuclear proteins, could instead be related to chromatin modifications occurring in both processes.

Animals↗

PubMed1991

The MTT-assay as a rapid test for cell proliferation and cell killing: application to human peripheral blood lymphocytes (PBL).

The possibility to use the colorimetric MTT assay for measuring proliferation and cell death of human peripheral blood lymphocytes (PBL) was studied. In a range from 100,000-800,000 cells/well a linear correlation between the optical signal (OD signal at 570 nm) and the cell number was found. It is necessary to incubate the cells with the MTT at least 2 hours. After stimulation by different PHA concentrations a very good correlation between [3H] thymidine incorporation and MTT assay was found. A comparison of daunomycin cytotoxicity, measurement by trypan blue exclusion and MTT assay, gave also a good correlation between both methods. It can be pronounced that the MTT assay is a suitable method to measure cell proliferation and cell death of human PBL. The assay is easy to handle, a large number of probes can be assayed in a relatively short time and no radioactivity is necessary. For the measurement of the colored product a common ELISA reader can be used.

Cell Death↗