Ki-67 expression and BrdUrd incorporation as markers of proliferative activity in human prostate tumour models

Abstract. The validity of the use of the monoclonal antibodies Ki-67 and anti-BrdUrd to evaluate proliferative activity of human prostate tumour models was studied. Growth of the transplantable PC-82 and PC-EW prostate tumours, as assessed by tumour volume measurements, was significantly correlated with the proliferative activity as reflected by BrdUrd incorporation into DNA ( r = 0.64 and r = 0.78, respectively). The proliferative activity of PC-82 tumours detected by Ki-67 antigen expression paralleled the pattern observed with BrdUrd ( r = 0.51) and a significant correlation ( r = 0.60) between the results obtained with both markers was found. In growing PC-82 and PC-EW tumours only small variations in the Ki-67 and BrdUrd indices were observed. In contrast, Ki-67 expression in regressing PC-82 tumours varied considerably (2.7 ± 2.2%). The BrdUrd index in regressing PC-32 tumours showed less variation (1.3 ± 0.2%), but part of the BrdUrd-positive cells were found in the stromal (murine) part of the regressing tissue. It is concluded that the Ki-67 and BrdUrd proliferation markers are reliable parameters to monitor changes in growth of prostate tumour lines, but that in slow growing or regressing tumours Ki-67 and BrdUrd data should be interpreted with caution.     

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.     

PCNA independence of Ki-67 expression in HPV infection.

12 Infectious Wart lesions were stained using the streptavidin-biotin immunoperoxidase method for PCNA10, MIB-1 (Ki-67 equivalent antigen) and Human Papilloma Virus antigen to study the effect of HPV presence on epidermal proliferation. Using strict methods to avoid observer bias, Ki-67 antigen was found in a high proportion of nuclei in the suprabasal layers together with HPV antigens, in the absence of PCNA staining. This finding indicates that DNA synthesis related, Ki-67 antigen bearing structures can be raised in the human nucleus in the absence of induction of PCNA bearing structures, suggesting also structural independence between the antigen bearing molecules.     

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.     

Comparison of monoclonal antibodies PC 10 and MIB 1 on microwave-processed paraffin sections

Abstract. Antibodies against the proliferation-associated nuclear antigen (PCNA) and against the Ki-67 protein are widely used as operational proliferation markers in human tumour diagnostics. The original Ki-67 antibody had the inherent drawback in that it could only be used when fresh-frozen material was available. The antibody PClO was supposed to offer the advantage that it could be applied on formalin-fixed and paraffin-embedded tissues. However, in cases in which the formalin fixation exceeded 4 h, PC10 staining proved to be inconsistent and often failed.
The aim of this study was to compare a recently prepared Ki-67 equivalent monoclonal antibody (MIB 1) and PC10 in routinely fixed histopathological material using antigen retrieval by microwave processing.
Antibody MIB 1 stained the nuclei of cells known to belong to the proliferative compartments in microwave-processing paraffin sections of formalin-fixed tissues. Quiescent cells were consistently negative for MIB 1 staining. In contrast, PC10 was positive in almost all nuclei of different tissues in microwave-treated paraffin sections. Thus, antigen retrieval by microwave processing is beneficial for the detection of the Ki-67 protein in paraffin sections, whereas it is not needed for the detection of the PCNA.     

An improved method for the immunocytochemical detection of bromodeoxyuridine labeled nuclei using flow cytometry

A new staining protocol is described for the immunocytochemical detection of BrdUrd labeled nuclei. Pepsin treatment of ethanol fixed cells or tissue, followed by DNA denaturation at low pH, resulted in increased sensitivity of BrdUrd staining comparable to the thermal denaturation protocol, and decreased background binding. This technique is applicable to cell suspensions, including cultured cells and bone marrow cells. Furthermore, pepsin digestion of ethanol fixed tissue fragments resulted in a high recovery of nuclei in which incorporated BrdUrd could be detected. This possibility, together with the high sensitivity, make this method especially suitable for cell kinetic studies of human solid tumors in vivo.     

Simultaneous immunohistochemical demonstration of antigen expression and 5-bromodeoxyuridine incorporation in plastic embedded sections

Summary In this study a double immunohistochemical staining procedure is described for the simultaneous demonstration of antigen expressing cells and replicating cells in rat thymus. As markers for cell surface antigen expression a monoclonal antibody against Ia-expressing cells (His 19) and a monoclonal antibody against cells of the monocyte-macrophage lineage (ED2) were used. Replicating cells were demonstrated by the incorporation of 5-bromodeoxyuridine (BrdUrd). Tissue pieces were fixed in a periodate-lysine-paraformaldehyde fixative and embedded in glycol methacrylate. To demonstrate Ia-expressing cells or ED2-positive macrophages in plastic embedded sections a digestion with trypsin is necessary. The staining procedure was applied sequentially and was performed with a peroxidase and an alkaline phosphatase labeled reagent yielding respectively a brown and a blue reaction product. Results with this staining procedure on plastic embedded sections of rat thymus, an organ with a high DNA synthesizing capacity, showed incorporation of BrdUrd predominantly in the cortex. ED2-positive macrophages were only found in the cortex. The la-positive epithelial reticular cells demonstrated extremely well their stellate form.     

Simultaneous immunohistochemical demonstration of antigen expression and 5-bromodeoxyuridine incorporation in plastic embedded sections

In this study a double immunohistochemical staining procedure is described for the simultaneous demonstration of antigen expressing cells and replicating cells in rat thymus. As markers for cell surface antigen expression a monoclonal antibody against Ia-expressing cells (His 19) and a monoclonal antibody against cells of the monocyte-macrophage lineage (ED2) were used. Replicating cells were demonstrated by the incorporation of 5-bromodeoxyuridine (BrdUrd). Tissue pieces were fixed in a periodate-lysine-paraformaldehyde fixative and embedded in glycol methacrylate. To demonstrate Ia-expressing cells or ED2-positive macrophages in plastic embedded sections a digestion with trypsin is necessary. The staining procedure was applied sequentially and was performed with a peroxidase and an alkaline phosphatase labeled reagent yielding respectively a brown and a blue reaction product. Results with this staining procedure on plastic embedded sections of rat thymus, an organ with a high DNA synthesizing capacity, showed incorporation of BrdUrd predominantly in the cortex. ED2-positive macrophages were only found in the cortex. The Ia-positive epithelial reticular cells demonstrated extremely well their stellate form.     

Analytical methods for the study of liver cell proliferation.

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase-isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size-dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two-step analysis. A non-immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.     

The Labeling of Proliferating Cells by Ki67 and MIB-1 Antibodies Depends on the Binding of a Nuclear Protein to the DNA

The antigen Ki-67 Ag, regarded as a marker for proliferating cells, was identified as a protein(s) (pKi-67) which can exist free or associated with DNA as evidenced by DNA digestion of cells before or after immunolabeling with Ki-67. The dual nature of this antigen was also supported by reconstitution of Ki-67 Ag from purified DNA and nuclear proteins extracted from the K562 cell line. The immunoreactivity of the resulting complexes was examined in solution using Ki-67 and MIB-1 antibodies. The interaction between Ki-67 or MIB-1 antibodies and pKi-67 was enhanced in the presence of undegraded ds DNA, indicating that ds DNA modulates the conformation of pKi-67 and that the altered conformation of pKi-67 is more reactive than the pure protein to both Ki-67 and MIB-1 antibodies.     

The use of bromodeoxyuridine labeling in the human lymphocyte HGPRT somatic mutation assay

The autoradiographic assay developed by Strauss and Albertini (1979) to quantitate human in vivo somatic mutation at the hypoxanthine guanine phosphoribosyl-transferase locus uses tritiated thymidine to identify mutant cells by their ability to pass through 'S' phase in the presence of 6-thioguanine. An alternative method, based on the incorporation of bromodeoxyuridine (BrdUrd) into the DNA of proliferative cells, followed by differential staining with the fluorescence-plus-Giemsa method, was used to identify 3 classes of lymphocyte nuclei: (a) small darkly stained nuclei, (b) large, reddish-colored nuclei with an apparent nucleolus, and (c) large, bluish-colored nuclei. By double labeling with BrdUrd and tritiated thymidine, it was determined that only the nuclei of the third class had incorporated BrdUrd. These results demonstrate that the technique used for sister-chromatid differentiation can be used to detect putative HGPRT mutants and to determine variant frequencies at the HGPRT locus.     

DNA flow cytometry of breast carcinoma after acetic-acid fixation

Aqueous acetic acid was used to fix and store specimens of tissue prior to dissociation into nuclear suspensions for flow cytometric quantitation of DNA. The optimum concentration was 20 volumes of glacial acetic acid in 80 volumes of distilled water. Both neoplastic and benign nuclei were easily released from the fixed tissue blocks by slicing and shaking. Residual undissociated tissue was suitable for microscopic examination to confirm its identity. The nuclei fluoresced brightly after staining with propidium iodide, and yielded histograms similar to those from unfixed samples. Acetic-acid fixation resulted in slightly broader G1 and G0 peaks in the DNA histograms in comparison to unfixed cells, but fluorescent debris was less and correlation between the flow cytometric S-phase fraction (SPF) and in vitro thymidine labelling index (TLI) was better than with unfixed cells. Twenty-one of thirty-nine acetic-acid-fixed breast carcinomas had DNA indices in excess of 1.0 (increased nuclear DNA content in comparison to benign cells), and eighteen had DNA indices of 1.0 (normal or near-normal). The SPF was usually in excess of the TLI, but the two were significantly correlated (r = 0.72, P less than 0.0001). However, a significant correlation of SPF with TLI held only for the group with DNA index greater than 1.0. DNA indices greater than 1.0 were associated with high SPF and TLI, and high SPF and TLI each associated with low content of estrogen receptor.     

Computerized image analysis of Ki-67 in ductal breast carcinoma in situ.

OBJECTIVE: To develop and determine the staining protocols and computerized image analysis methods that are the most effective combination for performing quantitative analysis of Ki-67. STUDY DESIGN: We compared conventional bright-field light microscopy and refractive optical enhancement methods in combination with various immunodetection and filter enhancement methods, including immunogold in combination with epipolarization refractive optics and enzymatic conversion of chromogenic substrates in combination with optical filter enhancement. Initial Ki-67 tests were performed on lymph node tissues and cultured human breast cells and then applied to 200 ductal carcinoma in situ (DCIS) samples. DCIS acini were digitally acquired, and a region of interest was manually outlined in each one with a digital stylus to include only the cellular component; then the Ki-67 staining index was quantified by segmentation analysis. RESULTS: Although combining epipolarization analysis with immunohistogold staining was the most sensitive detection method, nonspecific binding was too high. The streptavidin-horseradish-peroxidase enzymatic conversion of 3,3'-diaminobenzidine (DAB) in combination with optical enhancement filters was the most effective method tested. Ki-67 stain was associated with dense fibrillar structures of the nucleoli in the less intensely staining nuclei and was most intense in paired nuclei. CONCLUSION: The method of measuring Ki-67 expression by DAB staining combined with optical enhancement filters and quantification via computer-assisted image analysis techniques produced objective and reproducible results. As such, this method can offer (1) less intraobserver and interobserver variability, (2) a digital archival record, and (3) a baseline for digital exchange of information between studies.     

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.     

Direct comparison of bromodeoxyuridine and Ki-67 labelling indices in human tumours

Direct comparison of bromodeoxyuridine (BrdUrd) and Ki-67 labelling indices was achieved by selecting similar areas from serial sections of human tumours. Fifteen patients were selected who had been administered BrdUrd in vivo and both proliferation markers were assessed by immunohistochemistry. The data show a good correlation between both BrdUrd LI and MIB-1 LI and T_pot (calculated using the flow cytometry derived duration of S phase) and MIB-1 LI. The contribution of BrdUrd LI to growth fraction varied as a function of proliferation characteristics. In tumours with a high LI, the number of DNA synthesizing cells represented half the growth fraction, whilst in tumours with lower LI's (<10%) the ratio of DNA precursor labelled cells as a function of growth fraction fell to between 10% and 20%. T_pot showed a linear correlation with MIB-1/BrdUrd ratio with a slope approaching unity. It was apparent that both intra- and interpatient variation in proliferation index was greater for BrdUrd labelling than for MIB-1 expression.     

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.     

Cell proliferation in human tumour xenografts: measurement using antibody labelling against bromodeoxyuridine and Ki-67

Cell proliferation was investigated in human tumour xenografts using bromodeoxyuridine (BrdUrd) labelling, evaluated either by flow cytometry or in tissue sections, and also using the proliferation marker Ki-67. BrdUrd labelling was found to increase when cryostat tumour sections were digested with an enzymic solution. This yielded a labelling index up to four times higher than that obtained using the flow cytometer. Ki-67 indices were found to be higher than those reported for human tumour biopsies, as may be expected due to the enhanced growth rate of the xenografts. Significant heterogeneity was observed in the results for cervix, breast and bladder tumours, and the results of the three methods were poorly correlated. However, three of the four tumour types showed that the tumour with the lowest Ki-67 index also had the longest potential doubling time. Since the measurement of Ki-67 index was found technically easier to perform, and also adequately reflects relative tumour cell proliferation, it is preferred over the other techniques.     

Rapid in vitro bromodeoxyuridine labeling method for monitoring of therapy response in solid human tumors

In vitro bromodeoxyuridine (BrdUrd) incubated single-cell suspensions obtained from solid tumors were fixed on slides for subsequent sample processing. As dispersal of nuclei largely was avoided, only small amounts of cells were needed for examination. The sensitivity of detecting even low BrdUrd incorporation rates could be improved by treatment with intense DNA denaturation conditions. This technique was applied to monitor cytokinetic response to chemotherapy and radiation in oral carcinomas by analysing biopsies taken consecutively in the course of treatment. By combining BrdUrd labeling and DNA flow cytometry, cells arrested in S phase easily could be distinguished from cells showing continuous proliferation.     

Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part I: Historical perspectives, histochemical methods and cell kinetics

Summary Bromodeoxyuridine (BrdUrd), a thymidine analogue incorporated into DNA, can be quantified by fluorescent or chromophoric quenching of dyes bound to DNA or with antibodies to BrdUrd. These technologies have been used since the 1970s as tools for measuring DNA synthesis in isolated chromosomes and in cells and tissues. This paper is Part I of a three-part comprehensive review of the literature over the last 20 years (to the end of 1993) describing the histochemical methods for measuring BrdUrd in cells and tissues. Fixation, denaturation and staining procedures are compared for quantifying BrdUrd for microscopy and flow cytometry. Non-immunochemical methods related to the quenching of fluorescent DNA stains by BrdUrd are also described. Methods are described for the comparative assay of cell kinetic parameters by tritiated thymidine and bromodeoxyuridine. The multivariate BrdUrd/DNA assay of T_s, and T_c, and a comparison of recent methods based on the single biopsy bivariate analysis of T_pot, is presented. Recent developments in the use of double halopyrimidine label to determine kinetic parameters are also reviewed.