Cell cycle-related proteins: a flow cytofluorometric study in human tumors

We used 2-parameter flow cytometry (FCM) to investigate the relationship between the cell cycle phases and 3 proteins whose expression is known to increase in proliferating cells: the surface antigen transferrin receptor (Trf-r), the "cyclin" (a proliferating cell nuclear antigen, PCNA), and the nuclear antigen recognized by the monoclonal antibody (MoAb) Ki-67. FITC-labeled antibodies against Trf-r, PCNA, and the Ki-67-reactive antigen, as well as propidium iodide-DNA distribution, were simultaneously measured on human leukemia HL-60 and K562, and breast carcinoma MCF-7 cell lines and on fresh human leukemic and glioblastoma cells. The 70% ethanol fixation for Trf-r and PCNA and the 95% acetone fixation for Ki-67 plus permeabilization (with 0.1% and 1% Triton X100, respectively, for the surface and the nuclear antigens) produced cell suspensions with negligible cell clumping, high-quality DNA profiles, and bright specific immunofluorescent staining. The investigated proteins have different relationships with the proliferative state of the cell. Trf-r is expressed mainly at the transition from G0/G1 to S-phase. PCNA expression is prominent in late G1 and through S-phase and decreases in G2-M. The Ki-67-reactive antigen is widely distributed in G1, S, and G2-M phases. Knowledge regarding the relationships between proliferation-associated antigens and cell cycle phase in normal and neoplastic cells could improve our understanding of the mechanisms underlying growth regulation and neoplastic transformation. Bivariate FCM is an easy method for obtaining these data from large numbers of cells.     

Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL-60 cells

The expression and stability of the proliferation-associated nuclear antigen detected by Ki-67 antibody have been investigated in human promyelocytic leukaemic HL-60 cells in relation to their progression through the cell cycle. Expression of this antigen was minimal in late G1 and early S phase cells. The antigen accumulated in the cells predominantly during S phase, and its rate of increase per cell accelerated during the second half of this phase. The accumulation of Ki-67 antigen during S exceeded the increase in DNA content, and thus the Ki-67/DNA ratio rose 80% from late G1 to G2 + M. This antigen rapidly disappeared from post-mitotic cells. The half-life of this protein estimated in post-mitotic cells during stathmokinesis induced by vinblastine appeared to be shorter than 1 h. This rapid turnover should be compared with the relatively long (6-8 h) duration of G1 of the studied cells. In cells in which de novo protein synthesis was inhibited by 0.1 microgram/ml cycloheximide, the half-life of the Ki-67 antigen was also found to be about 1 h regardless of the cell position in the cell cycle. Thus, the data suggest that variations in the level of this protein during the cell cycle are a consequence of its different synthesis rate rather than phase-specific changes in the rate of its degradation. Because the late G1 and very early S phase cells express the antigen at levels only slightly above background, it is possible that, when using Ki-67 antibody as a marker of the cell growth fraction, some late G1 cells can be erroneously classified as non-cycling cells.     

Regional differences of proliferation activity in the spinal cord ependyma of adult rats

Increased proliferation activity in the central canal ependyma of adult rodent spinal cord was described after injury and is thought to participate in recovery processes. Proliferation activity is scarce under physiological conditions, but still could be of importance, as in vitro studies showed that the spinal cord ependyma is an internal source of neural stem cells. Data from these studies indicate that there are regional differences in the distribution of proliferation activity along the rostro-caudal axis. We analyzed the proliferation activities in the ependyma within the entire extent of intact adult rat spinal cord. To identify proliferating cells we performed immunohistochemistry either for cell cycle S-phase marker BrdU or for the nuclear protein Ki-67. BrdU and Ki-67 positive cells were counted on sections selected from four spinal cord regions — cervical, thoracic, lumbar and sacral/coccygeal. Analysis showed that the number of BrdU positive cells within the ependyma was very low in all subdivisions of the spinal cord. Both BrdU and Ki-67 labeling revealed a significantly higher number of proliferating cells in the ependyma of sacrococcygeal part in comparison to all other spinal cord regions, suggesting that the caudal spinal cord might have potentially higher regeneration capacity compared to more rostral parts.     

Evaluation of cell proliferation in rat tissues with BrdU, PCNA, Ki-67(MIB-5) immunohistochemistry and in situ hybridization for histone mRNA.

The standard method for assessment of cell proliferation in paraffin-embedded tissue sections is 5-bromodeoxyuridine (BrdU) immunohistochemistry (IHC). BrdU can be administered to laboratory animals via IP injections, is readily incorporated into nuclei during the DNA synthetic phase of the cell cycle, and is detected with an anti-BrdU antibody. This method has several disadvantages, and an accurate method for evaluation of proliferative activity that can substitute for BrdU IHC, when necessary, is of great interest to investigators. Alternative methods for detection of proliferating cells in tissue sections are proliferating cell nuclear antigen (PCNA) IHC, Ki-67 IHC, and in situ hybridization (ISH) for histone mRNA. To determine the optimal choice, we analyzed the correlation of anti-PCNA, anti-Ki-67(MIB-5), and histone mRNA labeling indices (LIs) with anti-BrdU LI in rat highly replicative (renewing) tissues. The correlation between anti-BrdU and histone mRNA LIs, as well as the correlation between anti-BrdU and anti-Ki-67 LIs, was statistically significant. There was no significant correlation between anti-BrdU and anti-PCNA LIs. These results suggest that both ISH for histone mRNA and IHC with MIB-5 are preferable techniques for assessment of cell proliferation in rat paraffin-embedded renewing tissues compared to PCNA IHC. They can substitute for BrdU IHC when necessary.     

Ki-67 monoclonal antibody (MAb) reacts with a proliferation associated nuclear antigen in the rabbit Oryctolagus cuniculus

The Ki-67 monoclonal antibody which recognizes a human nuclear antigen expressed by cycling cells but not by resting cells was found to react immunohistochemically with tissues from the rabbit Oryctolagus cuniculus. Ki-67 immunoreactivity was restricted to the nucleus. A comparative study with bromodeoxyuridine labelling patterns was carried out to study the association with proliferating cells. In lingual, jejunal and appendix mucosa, skin, adrenal gland, thymus, spleen, bone marrow, testis, growth cartilage, periosteum and periochondrium of long bones the distribution of Ki-67 positive and bromodeoxyuridine labelled cells was similar and consistent with the distribution of proliferating cells in these tissues. In tissue from the brain, kidney, skeletal or cardiac muscle and liver no Ki-67 positive or bromodeoxyuridine labelled cells were seen. In cartilage labelled in vivo with tritiated thymidine, all thymidine labelled cells were also Ki-67 positive. These results suggest that the Ki-67 antibody recognizes a nuclear antigen in the rabbit that is associated with cell proliferation and is expressed by cells in S-phase as well as in other phases of the cell cycle.     

Ki-67 monoclonal antibody (MAb) reacts with a proliferation associated nuclear antigen in the rabbitOryctolagus cuniculus

Summary The Ki-67 monoclonal antibody which recognizes a human nuclear antigen expressed by cycling cells but not by resting cells was found to react immunohistochemically with tissues from the rabbitOryctolagus cuniculus. Ki-67 immunoreactivity was restricted to the nucleus. A comparative study with bromodeoxyuridine labelling patterns was carried out to study the association with proliferating cells. In lingual, jejunal and appendix mucosa, skin, adrenal gland, thymus, spleen, bone marrow, testis, growth cartilage, periosteum and perichondrium of long bones the distribution of Ki-67 positive and bromodeoxyuridine labelled cells was similar and consistent with the distribution of proliferating cells in these tissues. In tissue from the brain, kidney, skeletal or cardiac muscle and liver no Ki-67 positive or bromodeoxyuridine labelled cells were seen. In cartilage labelled in vivo with tritiated thymidine, all thymidine labelled cells were also Ki-67 positive. These results suggest that the Ki-67 antibody recognizes a nuclear antigen in the rabbit that is associated with cell proliferation and is expressed by cells in S-phase as well as in other phases of the cell cycle.     

Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: a new view of the cell cycle

Flow cytometric multiparameter analysis of two proliferation-associated nuclear antigens (proliferating cell nuclear antigen (PCNA)/cyclin and Ki-67) was performed on seven human hematopoietic cell lines. PCNA/cyclin, an S phase-related antigen, was detected using an autoantibody and a fluorescein isothiocyanate-labeled anti-human antibody. The Ki-67 antigen, which in cycling cells is expressed with increasing levels during the S phase with a maximum in the M phase, was detected using a monoclonal antibody and a phycoerythrin-conjugated anti-mouse antibody. In some experiments the PCNA/Ki-67 staining was combined with a DNA stain, 7-amino actinomycin D, and simultaneous detection of the three stains was performed by a single laser flow cytometer. Using this technique four distinct cell populations, representing G1, S, G2, and M, respectively, could be demonstrated in cycling cells on the basis of their PCNA/cyclin and Ki-67 levels. The cell cycle phase specificity could be verified using metaphase (vinblastine, colcemide) and G2 phase (mitoxantrone) blocking agents, as well as by stainings with a mitosis-specific antibody (MPM-2). Also, G0 cells could be discriminated from G1 cells in analysis of a mixture of resting peripheral mononuclear blood cells and a proliferating cell line. This technique can be valuable in detailed cell cycle analysis, since all cell cycle phases can be visualized and calculated using a simple double staining procedure.     

Cell cycle analysis using the monoclonal antibody Ki-67

We determined by flow cytometry the proportion of cells in cycle with the monoclonal antibody Ki-67 and also in S-phase after incorporation of bromodeoxyuridine (BrdUrd) with monoclonal antibody to BrdUrd. The monoclonal antibody Ki-67 was useful to detect a nuclear antigen present only in proliferating cells but not expressed in resting (Go) cells. Cell preparation to measure BrdUrd amount incorporated into cellular DNA was difficult but this anti-BrdUrd antibody was useful for measuring the rate of DNA synthesis and for the analysis of precious cell kinetics. These antibodies may provide useful information of cell kinetics.     

Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67

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.     

The cell cycle associated change of the Ki-67 reactive nuclear antigen expression

The change of human nuclear antigen expression in proliferating cells recognized by a monoclonal antibody, Ki-67, during the cell cycle was investigated in HeLa S3 cells using a bivariate-flow-cytometric analysis. The antigen was immunocytochemically stained with FITC, and DNA was stained with propidium iodide (Pl). The expression of the antigen increased with cell-cycle progression, especially in the latter half of S-phase and reached a maximum at G2M-phase, although its content varied greatly from cell to cell. The cells in which DNA synthesis was inhibited by treatment with hydroxyurea increased markedly in the antigen expression (as compared to untreated cells). Treatment with adriamycin also elevated the antigen content. After digestion with DNase I, but not after RNase treatment, FITC fluorescence from the antigen disappeared. These results suggest that the Ki-67 antigen is bound to DNA and its expression does not depend on DNA replication. Although the biological implications of the antigen remain unresolved, the antigen may be considered to be essential for maintaining the proliferating state of cells.     

Effect of ionic strength in immunocytochemical detection of the proliferation associated nuclear antigens p120, PCNA, and the protein reacting with Ki-67 antibody.

This study was aimed at revealing whether or not ionic interactions between the epitope of the antigen detected by Ki-67 antibody, or the proliferation-associated proteins PCNA or p120, and neighboring cellular constituents impede detectability of these antigens in HL-60 cells by indirect immunofluorescence assay. To this end, the ionic strength (NaCl concentration) of the solutions in which cells were suspended during their fixation with 0.5% paraformaldehyde was increased, to up to 1.65 M NaCl, to weaken the intra- and/or intermolecular ionic interactions during the process of crosslinking, and the cells were then immunostained. Fluorescence of cells reacting with Ki-67 antibody was maximally increased after their treatment with 1.15 M NaCl; the average increase was nearly 110% above the level seen with the standard methodology utilizing 0.15 M NaCl. The increase was greater for cells in the G1 phase of the cell cycle compared to cells in S or G2. Fluorescence of cells stained with the PCNA antibody was maximally enhanced after cell treatment with 0.65 M NaCl. The enhancement, however, varied depending on the source of the antibody; it was nearly 200% in the case of the antibody provided by Boehringer and over 100% by DAKO. Detection of the nucleolar antigen p120 was not significantly affected by 0.65-1.65 M NaCl. The data indicate that ionic interactions between cellular constituents indeed play a role in masking the epitope of PCNA and the antigen detected by Ki-67.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous flow cytometric analysis of surface markers and nuclear Ki-67 antigen in leukemia and lymphoma

The monoclonal antibody Ki-67 identifies an antigen present during the late G1, S, G2, and M phases of the cell cycle, whereas resting cells do not express this antigen. Immunostaining with Ki-67 provides a simple method with which to determine the growth fraction of a malignant cell population without requiring a laborious procedure or use of radioactive materials. Thus far, detection of Ki-67-positive cells by flow cytometry was limited because of nuclear location of the antigen. In this study, periodate-lysine-paraformaldehyde (PLP) fixation of cells in suspension, labeling with Ki-67, and the subsequent flow cytometric analysis of the tumor growth fraction is described. Fixation with PLP at -10 degrees C for 15 min rendered the plasma membrane permeable without destroying cell surface antigens. Thus double immunofluorescence studies using both a surface marker and Ki-67 could be performed. This offers the additional advantage of being able to define the phenotype of proliferating cells. This method was applied to determine the growth fraction in peripheral blood and bone marrow samples of patients with leukemia and non-Hodgkin's lymphoma. The results of Ki-67 studies in 91 patients are shown. A wide variability of individual Ki-67 values was observed within each entity. Use of this flow cytometric procedure substantially facilitates the quantification of proliferating cells in pathological blood and bone marrow samples.     

Evidence for quiescent S- and G2-phase cells in human colorectal carcinomas: a flow cytometric study with the Ki-67 antibody

The expression of certain antigens specific for proliferating cells can be determined simultaneously with cell cycle distribution by means of two-dimensional flow cytometry. In this way, a tumour's growth potential is characterized more precisely than with any one parameter alone. Here we describe such simultaneous measurements of DNA content and labelling with the Ki-67 antibody that distinguishes between cycling and non-cycling cells. Having overcome a number of technical problems we were able to analyse material from 29 biopsies of human colorectal tumours. In a number of cases, Ki-67 negative cells were found with a DNA-content of G_0/1 only, whereas all cells with an S- or G₂-phase DNA-content were Ki-67 positive. There were other cases in which cells with an S- and G₂-phase DNA-content had obviously become quiescent (Ki-67 negative), sometimes even outnumbering the proliferating (Ki-67 positive) cells in the respective compartments of the cycle. Generally, however, when Ki-67 negative and positive subpopulations were analysed separately it was found that the former had a significantly lower (S + G₂)-phase fraction than the latter. There was evidence for a correlation between Ki-67 index and (S + G₂)-phase fraction at least in the subgroup of aneuploid tumours. Neither of the two parameters was correlated with stage according to Duke's classification or tumour size. However, a positive correlation was found between the fraction of unlabelled S- and G₂-phase cells and tumour size as reflected in the T category.     

Simultaneous staining of exponentially growing versus plateau phase cells with the proliferation-associated antibody Ki-67 and propidium iodide: analysis by flow cytometry

The antibody Ki-67, which detects proliferating cells, was used in combination with propidium iodide, a DNA-specific dye. The double-staining method allowed discrimination of cells in the phases of the cell cycle as well as the recognition of Ki-67 staining characteristics. Suspension cultures of U937 cells were measured in exponential growth and plateau phase in nutritional deprivation. The fraction of Ki-67 positive cells was nearly 100% 2 days after dilution and 46% 7 days after dilution of the cultures. Stathmokinetic measurements with colchicine and flow cytometry measurements with the BrdU-Hoechst technique yielded close to 100% proliferation at day 2 but only 18% and 6%, respectively, at day 7. The discrepancy between Ki-67 results and the results of the two other methods is considered to be a characteristic of nutritionally deprived cells.     

Simultaneous staining of exponentially growing versus plateau phase cells with the proliferation-associated antibody Ki-67 and propidium iodide: analysis by flow cytometry

Abstract. The antibody Ki-67, which detects proliferating cells, was used in combination with propidium iodide, a DNA-specific dye. The double-staining method allowed discrimination of cells in the phases of the cell cycle as well as the recognition of Ki-67 staining characteristics. Suspension cultures of U937 cells were measured in exponential growth and plateau phase in nutritional deprivation. The fraction of Ki-67 positive cells was nearly 100% 2 days after dilution and 46% 7 days after dilution of the cultures. Stathmokinetic measurements with colchicine and flow cytometry measurements with the BrdU-Hoechst technique yielded close to 100% proliferation at day 2 but only 18% and 6%, respectively, at day 7. The discrepancy between Ki-67 results and the results of the two other methods is considered to be a characteristic of nutritionally deprived cells.     

Assignment of the gene(s) involved in the expression of the proliferation-related Ki-67 antigen to human chromosome 10

Summary The antigen recognized by the monoclonal antibody Ki-67 is a proliferation-related nucleolus-associated constituent used as a marker for cycling cells in tumor diagnosis. Antibody Ki-67 reacts with human proliferating cells, but not with hamster and mouse cells. Expression of the Ki-67 antigen was studied in a panel of human-rodent somatic cell hybrids. The results indicate that a gene involved in the expression of the antigen is located on chromosome 10.     

Proliferating cell nuclear antigen and Ki-67 antigen expression in human haematopoietic cells during growth stimulation and differentiation

Abstract. By flow cytometric dual parameter analysis of proliferating cell nuclear antigen (PCNA) and the Ki-67 antigen a detailed cell cycle analysis can be performed. In this study the co-ordinated expression of these two growth-related antigens was investigated in human haematopoietic cells at entrance into the cell cycle as well as at exit from the cycle. In mitogen-stimulated peripheral blood lymphocytes entering the first cell cycle, the Ki-67 antigen was found to be expressed in S phase cells and not in G₁ cells. Thus, the Ki-67 antigen expression in PCNA-positive S phase cells differed between continuously cycling cells and cells entering the cell cycle. Based on this difference, it was possible to visualize and evaluate the recruitment of cells into the first cell cycle from a resting stage. This new cell cycle parameter can give additional information concerning tumour growth. The Ki-67 antigen was also studied during different stages of G₁ and was found to be expressed at high levels in early G₁ cells compared with other parts of G₁.     

Evolutionary conservation in various mammalian species of the human proliferation-associated epitope recognized by the Ki-67 monoclonal antibody

The human proliferation-associated epitope recognized by the Ki-67 monoclonal antibody (MAb) was detected in proliferating normal and neoplastic cells of many mammalian species (lamb, calf, dog, rabbit, rat) besides human. In contrast, Ki-67 stained proliferating cells from other species weakly (mouse) or not at all (swine, cat, chicken, pigeon). The immunostaining pattern of Ki-67 in animal tissues was identical to that previously described in human: Ki-67 reacted only with cells known to proliferate (e.g., germinal center cells, cortical thymocytes) but not with resting cells (e.g., hepatocytes, brain cells, renal cells); this MAb produced a characteristic nuclear staining pattern (e.g., stronger labeling of nucleoli than of the rest of the nuclei and staining of chromosomes in mitotic figures); and Ki-67 crossreacted with the squamous epithelium in both animal and human tissues. In vitro studies showed that when quiescent (Ki-67-negative) NIH 3T3 fibroblasts or bovine peripheral blood lymphocytes were induced to proliferate, the appearance of Ki-67-positive cells paralleled the induction of cell proliferation caused by addition of fetal calf serum or PHA, respectively, to the cultures, and in both human and rat proliferating cells the Ki-67 expression closely paralleled the incorporation of [3H]-thymidine. These findings indicate that the epitope recognized by the Ki-67 MAb in human and animal species is the same. The widespread evolutionary conservation of the human proliferation-associated epitope recognized by the Ki-67 MAb suggests that it and/or its carrier molecule may play an important role in regulation of cell proliferation.