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.     

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.     

The proteasome controls the expression of a proliferation-associated nuclear antigen Ki-67

The proteasome is a protease complex responsible for rapid, selective, and irreversible removal of regulatory proteins, as well as many other cellular proteins. In this study, we have demonstrated that a proliferation-associated nuclear protein Ki-67 depended on the proteasome for its rapid degradation. A proteasome-specific inhibitor lactacystin augmented Ki-67 protein levels in pancreatic cancer BxPC-3 cells while repressed the level of steady-state Ki-67 mRNA. Inhibition of the proteasome also led to accumulation of two CDK inhibitors p27(kip1) and p21(cip1) in the BxPC-3 cells. Failed reduction of Ki-67 protein and enhanced levels of the two CDK inhibitors are likely contributing factors for the suppressed BxPC-3 proliferation after proteasome inhibition.     

Further characterization of the human inducer T cell subset defined by monoclonal antibody.

Evidence is presented that the OKTA+ T cell subset in man, defined by a monoclonal hybridoma antibody, provides help for B lymphocyte differentiation in a PWM driven system. Both B cell proliferation and intracytoplasmic immunoglobulin synthesis are facilitated by OKT4+ and not by OKT4- T cells. Given earlier studies demonstrating that OKT4+ T cells were necessary for generation of T cytotoxic cells and the present study that OKT+ T cells are necessary for the differentiation of B cells, it would appear that the OKT+ population is the major human T helper (inducer) subset.     

Immunobiochemical characterization of the antigen detected by monoclonal antibody IND.64. Evidence that IND.64 reacts with the cell proliferation associated nuclear antigen previously defined by Ki-67.

The immunohistochemical characteristics of the monoclonal antibody IND.64 are very similar to those of the monoclonal antibody Ki-67. The aim of this study was to further characterize this new antibody and to compare it with Ki-67 using immunobiochemical methods. Our results demonstrate that the similarity between the antibodies holds true even at the molecular level. Immunoblot analysis of IM-9-cell lysates with both antibodies showed a double band with apparent molecular weights of 395 kD and 345 kD, respectively. Competition ELISAs using a synthetic peptide derived from the thus far determined Ki-67 cDNA sequence as competitor, indicate that IND.64 may recognize the same epitope as Ki-67. The IND.64 epitope resides at least within a 20 amino acid sequence which also contains the Ki-67 epitope. Since IND.64 is of the IgG2b subclass, while Ki-67 is of the IgG1 subclass, the two antibodies may be useful for double immunostaining. In addition, IND.64 may help in determining the still unknown function of the antigen it recognizes.     

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.     

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.     

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.     

The proliferating cell marker monoclonal antibody Ki-67 recognizes specific antigens associated with the nuclear envelope of the early Drosophila embryo

Summary— Immunofluorescence and immunoelectron microscopy indicated that the antibody raised against the nuclear antigen Ki-67 of mammalian cells recognized antigenic determinants of early Drosophila embryos, localized on the outside of the nuclear envelope. Hence, the nuclear envelope of Drosophila appears to share a similar epitope with the chromosome scaffold of mitotic mammalian cells. With the progression of mitosis the antigen persisted around the mitotic spindle region and was also found in the pole regions at metaphase and anaphase. The antibody also stained the equatorial regions of the spindles from anaphase to late telophase. The antibody may therefore be used as a biochemical marker of the nuclear envelope for studying nuclear membrane biogenesis and behavior during the mitotic divisions of the Drosophila embryo.     

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.     

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.     

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.     

A major heat-shock protein defined by a monoclonal antibody.

A monoclonal antibody reacts with a polypeptide of 68 000 mol. wt. (p68) that accumulates to high levels during heat shock. The intracellular distribution of this antigen in normal and heat-shocked cells has been studied. It is a major component of non-stressed cells, where it is located predominantly in the cytoplasm, but also occurs in the nucleus. The nuclear accumulation is growth regulated, in that exponentially growing cells have strong nuclear immunofluorescence and confluent cells little. It is concentrated at the leading edge of motile fibroblasts and co-distributes with actin-containing microfilaments. Heat shock causes cytoplasmic and nuclear accumulation and there is new deposition in the periphery of cells. In normal cells the antigen in the nucleus is located in the nuclear lamina and matrix which increases during heat shock. The distribution of this molecule and the structures with which it interacts suggests that it is important in mediating the effects of heat shock.     

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.     

Characterization of the human trophoblast-leukocyte antigenic molecules defined by a monoclonal antibody

The murine monoclonal antibody H316 recognizes cell surface glycoproteins shared in expression by human trophoblast and peripheral blood leukocytes. It is also strongly expressed by many human tumor cell types, including choriocarcinoma and teratocarcinoma. The H316 antigenic determinant from these different cell types is carried on two wheat germ agglutinin-reactive glycoproteins of approximately 65,000 and 55,000 m.w. The antigenicity is not dependent on N-linked glycosylation. Biochemical fractionation procedures suggest that the molecules are related to each other, are not associated through intermolecular disulfide bonds, but do exhibit intramolecular disulfide interactions. These glycoproteins demonstrate m.w. heterogeneity between different cell lines and individual trophoblast membrane preparations. Such properties suggest that H316 molecules may be representative of TLX antigens (trophoblast-leukocyte common antigens), which have been suggested to influence maternofetal immunogenetic interactions.     

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.     

Immunohistochemical studies on a human thymic epithelial cell subset defined by the anti-cytokeratin 18 monoclonal antibody

Summary Two monoclonal antibodies respectively recognizing cytokeratins (CK) 18 and 19 were applied to the human thymic epithelium (in vivo and in vitro) in normal and pathological conditions, including 12 thymomas. We observed that in both normal and hyperplastic thymuses (from patients with myasthenia gravis) virtually the entire epithelial network was CK19-positive as were the majority of cells growing in culture. In four thymomas, however, the expression of cytokeratin 19 was not detected by immunofluorescence. On the other hand, CK18 was expressed by a discrete subset of medullary thymic epithelial cells in normal and in hyperplastic thymuses. Among the thymomas a large majority was either negative or contained few isolated CK18-positive cells scattered within the tumour. Conversely, in the two undifferentiated epithelial thymomas, virtually all the tumoral network was strongly labeled with the anti-CK18 monoclonal antibody. The present investigation thus not only defines the human thymic epithelial cell subset on the basis of differential cytokeratin expression but also indicates that anti-CK antibodies with single cytokeratin specificities can be regarded as useful tools to study the heterogeneity of thymomas.     

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.     

A novel heteromorphic human cell surface alloantigen, gp60, defined by a human monoclonal antibody

A human mAb (DSM1) generated from a patient immunized with irradiated allogeneic melanoma cells detects a new cell surface alloantigen of restricted cell type distribution. The Ag is a 60,000-Da glycoprotein (gp60) that displays considerable heteromorphism in its cytosolic and cytoskeletal (52 to 62 kDa) and membrane forms (60 to 64 kDa). The gp60 Ag has been purified using lectin affinity, ion exchange, and Mono P fast performance liquid chromatography. Rabbit antiserum against purified gp60 recognizes a homologous gp60 molecule on DSM1-nonreactive cells. Molecular properties of gp60 and a partial amino acid sequence of a tryptic gp60-derived peptide distinguish it from other known human alloantigens. This is the first report of a human alloantigenic system whose definition required a cell type other than those of bone marrow derivation.