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.     

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.     

Cell cycle dependent distribution of the proliferation-associated Ki-67 antigen in human embryonic lung cells

The cell cycle-dependent distribution of the proliferation-associated Ki-67 antigen has been evaluated immunocytochemically in L-132 human fetal lung cells. The cells were synchronized and cell cycle phases were determined: G1 = 6.7 h, S = 5.4 h, G2 = 8.5 h and mitosis = 1.3 h. The Ki-67 patterns were strictly correlated with the cell cycle phases. In late G1-phase, Ki-67 antigen was present only in the perinucleolar region. In the S-phase, Ki-67 staining was found homogeneously in the karyoplasm and in the perinucleolar region. G2-phase cells contained a finely granular Ki-67 staining in the karyoplasm with Ki-67-positive specks and perinucleolar staining. In early mitotic cells (pro- and metaphase) an intense perichromosomal Ki-67 staining was observed in addition to a homogeneously stained karyoplasm in prophase, and cytoplasm in metaphase. During ana- and telophase the Ki-67 antigen disappeared rapidly. In resting cells there was no Ki-67 staining.     

The Ki-67 protein: from the known and the unknown

The expression of the human Ki-67 protein is strictly associated with cell proliferation. During interphase, the antigen can be exclusively detected within the nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes. The fact that the Ki-67 protein is present during all active phases of the cell cycle (G(1), S, G(2), and mitosis), but is absent from resting cells (G(0)), makes it an excellent marker for determining the so-called growth fraction of a given cell population. In the first part of this study, the term proliferation marker is discussed and examples of the applications of anti-Ki-67 protein antibodies in diagnostics of human tumors are given. The fraction of Ki-67-positive tumor cells (the Ki-67 labeling index) is often correlated with the clinical course of the disease. The best-studied examples in this context are carcinomas of the prostate and the breast. For these types of tumors, the prognostic value for survival and tumor recurrence has repeatedly been proven in uni- and multivariate analysis. The preparation of new monoclonal antibodies that react with the Ki-67 equivalent protein from rodents now extends the use of the Ki-67 protein as a proliferation marker to laboratory animals that are routinely used in basic research. The second part of this review focuses on the biology of the Ki-67 protein. Our current knowledge of the Ki-67 gene and protein structure, mRNA splicing, expression, and cellular localization during the cell-division cycle is summarized and discussed. Although the Ki-67 protein is well characterized on the molecular level and extensively used as a proliferation marker, the functional significance still remains unclear. There are indications, however, that Ki-67 protein expression is an absolute requirement for progression through the cell-division cycle.     

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.     

Additive effects of radiation and docetaxel on murine SCCVII tumors in vivo: special reference to changes in the cell cycle

The purpose of the present study was to investigate the effects of a combination of docetaxel and irradiation in vivo with special reference to docetaxel-arrested G(2)/M-phase cells. At 24 and 48 h after intraperitoneal administration of docetaxel (90 mg/kg), tumor-bearing mice were irradiated with (60)Co gamma rays. Cell cycle distribution was analyzed by a DNA-Ki-67 double staining method using flow cytometry. An accumulation of cells in the G(2)/M phase of up to approximately 40% was observed 24 h after administration of docetaxel. Between 24 and 72 h, the percentage of cells arrested in G(2)/M phase that expressed Ki-67 decreased from 37.2% to 13.8%, in accordance with the increase in the Ki-67-negative G(2)/M-phase fraction. More than half of the cells arrested in G(2)/M phase lost their expression of Ki-67 protein between 24 and 72 h. The G(1)-phase fraction decreased from 28.4% to 8.6% at 24 h after docetaxel treatment; this remained unchanged at 72 h. These flow cytometry data suggested that docetaxel-arrested G(2)/M-phase cells did not enter the next cell cycle and were killed by docetaxel alone. Our data showed that arrest of cells in G(2)/M phase does not contribute to the synergism that has been reported for combinations of docetaxel and radiation in in vivo tumor models.     

Cell cycle-dependent reactivity with the monoclonal antibody Ki-67 during myeloid cell differentiation

The specificity and sensitivity of the monoclonal antibody Ki-67 in identifying proliferating cell compartments was tested with the human promyelocytic leukemia cell line HL-60 using multi-parameter flow cytometry. While correlated measurements of DNA content and Ki-67 immunofluorescence indicated that the antigen was present in all phases of the cell cycle, reactivity with the antibody was highest in proliferating S and G2+M cells. The analysis of the BrdU content of cells sorted on the basis of reactivity with Ki-67 showed a correlation between Ki-67 reactivity and BrdU uptake. In HL-60 cells induced to differentiate with dimethyl sulfoxide (DMSO), the loss of reactivity with Ki-67 paralleled the exit of cells from the cell cycle. This was not observed in DMSO-resistant HL-60 cells. These results validate the usefulness of the Ki-67 antibody for determining the proliferative stage of mammalian cells in culture.     

Ki-67 labeling in postmitotic cells defines different Ki-67 pathways within the 2c compartment

Simultaneous quantification of DNA and Ki-67 proliferation-associated antigen was performed using fluorescence image cytometry. In the MCF-7 cell line, the Ki-67 antigen content increases during the cell cycle, and its intranuclear distribution pattern varies. Quantitative evolution of Ki-67 content as a function of nuclear area makes it possible to define several pathways followed by cells going through the 2c compartment. 1) In some cells, the amount of Ki-67 antigen remains constant during G1 (Ki-67 stable pathway), and a characteristic speckled pattern can be observed. 2) In the larger fraction of cells analyzed, there is a postmitotic decrease in the Ki-67 (Ki-67 decrease pathway) content. In this pathway, labeling is located in the nucleoplasm in small nuclei, is located in nucleoli in intermediate-sized nuclei, and is absent from larger nuclei (G0). A progressive increase in Ki-67 content (Ki-67 increase pathway) was observed from intermediate-sized nuclei to S phase nuclei. From these results, we hypothesize that the Ki-67 stable pathway is the G1 phase of newly formed cells going directly to S phase in local optimal conditions of growth and that Ki-67 decrease pathway and Ki-67 increase pathway correspond to cells whose progression to S phase is regulated by extracellular factors.     

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.     

Correlation of Ki-67 and MCM-2 proliferative marker expression with grade of histological malignancy (G) in ductal breast cancers

The study aimed at examining a relationship between expression of Ki-67 antigen and minichromosome maintenance 2 protein (MCM-2) and a grade of histological malignancy G in ductal breast cancers. The function of widely used marker of proliferation Ki-67 is still not clear. In contrast, the MCM-2 protein is well known to play an important role in controlling the cell cycle. Both proteins represent small protein molecules, which manifest nuclear expression only during cell division of normal and neoplastic cells. Their expression is noted in several malignant tumours. These studies were conducted on 56 archival paraffin blocks of ductal breast cancers. Immunohistochemical reactions were performed using monoclonal Ki-67- and MCM-2-specific antibodies. Statistical analysis demonstrated a positive correlation between expressions of two proteins (r=0.6; p<0.05). The most intense expression of these two markers was demonstrated in G3 grade cancers. Statistical analysis showed more pronounced expression of Ki-67 antigen in G3 grade cancers as compared to cancers of G1 and G2 grades (p<0.001) and, in the case of MCM-2 protein, a more pronounced expression in G3 grade cancers, as compared to those of G1 (p<0.05) or G2 grade (p<0.01). The results obtained in our study suggest that MCM-2 could be used as a marker of proliferation in breast carcinomas.     

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.     

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.     

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.     

The effect of pre-eclamptic umbilical cord serum on fibroblast division in culture

Edema, proteinuria, hypertension (EPH-gestosis), most commonly termed as pre-eclampsia, is the most common pregnancy-associated pathological syndrome. It is accompanied by a thorough remodelling of extracellular matrix in the umbilical cord tissues. It is commonly known that the presence of serum in culture medium strongly stimulates many functions of cells cultured in vitro. It was decided to check how the pre-eclamptic serum affects the fibroblast division in culture. Ki-67 is a protein present in proliferating cells and can be detected during all phases of the cell cycle (G1, S, G2/M) but not in resting (G0) cells. PCNA (proliferating cell nuclear antigen) is an intranuclear polypeptide whose synthesis rate is at its maximum during the S-phase of the cell cycle. The expression of Ki-67 and PCNA was measured by immunocytochemical methods and biosynthesis of DNA was evaluated by [14C]-thymidine incorporation. The activity of pre-eclamptic umbilical cord serum (UC-serum) was found to be distinctly lower in comparison to control one. The expression of Ki and PCNA in fibroblast cultures treated with pre-eclamptic serum was also distinctly lower. Also the incorporation of [14C]-thymidine to DNA was lower than in the cultures treated with control UC-serum. It may by concluded that pre-eclampsia reduces the mitogenic activity of the umbilical cord serum.     

Cutting edge: polycomb gene expression patterns reflect distinct B cell differentiation stages in human germinal centers

Polycomb group (Pc-G) proteins regulate homeotic gene expression in Drosophila, mouse, and humans. Mouse Pc-G proteins are also essential for adult hematopoietic development and contribute to cell cycle regulation. We show that human Pc-G expression patterns correlate with different B cell differentiation stages and that they reflect germinal center (GC) architecture. The transition of resting mantle B cells to rapidly dividing Mib-1(Ki-67)+ follicular centroblasts coincides with loss of BMI-1 and RING1 Pc-G protein detection and appearance of ENX and EED Pc-G protein expression. By contrast, differentiation of centroblasts into centrocytes correlates with reappearance of BMI-1/RING1 and loss of ENX/EED and Mib-1 expression. The mutually exclusive expression of ENX/EED and BMI-1/RING1 reflects the differential composition of two distinct Pc-G complexes. The Pc-G expression profiles in various GC B cell differentiation stages suggest a role for Pc-G proteins in GC development.     

The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins

The antigen defined by mAb Ki-67 is a human nuclear protein the expression of which is strictly associated with cell proliferation and which is widely used in routine pathology as a "proliferation marker" to measure the growth fraction of cells in human tumors. Ki-67 detects a double band with apparent molecular weights of 395 and 345 kD in immunoblots of proteins from proliferating cells. We cloned and sequenced the full length cDNA, identified two differentially spliced isoforms of mRNA with open reading frames of 9,768 and 8,688 bp encoding for this cell proliferation-associated protein with calculated molecular weights of 358,761 D and 319,508 D, respectively. New mAbs against a bacterially expressed part and a synthetic polypeptide deduced from the isolated cDNA react with the native Ki-67 antigen, thus providing a circle of evidence that we have cloned the authentic Ki-67 antigen cDNA. The central part of the Ki-67 antigen cDNA contains a large 6,845-bp exon with 16 tandemly repeated 366-bp elements, the "Ki-67 repeats", each including a highly conserved new motif of 66 bp, the "Ki-67 motif", which encodes for the epitope detected by Ki-67. Computer analysis of the nucleic acid and the deduced amino acid sequence of the Ki-67 antigen confirmed that the cDNA encodes for a nuclear and short-lived protein without any significant homology to known sequences. Ki-67 antigen-specific antisense oligonucleotides inhibit the proliferation of IM-9 cell line cells, indicating that the Ki-67 antigen may be an absolute requirement for maintaining cell proliferation. We conclude that the Ki-67 antigen defines a new category of cell cycle-associated nuclear nonhistone proteins.     

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.