Characterization of a Monoclonal Antibody, HTA28, Recognizing a Histone H3 Phosphorylation Site as a Useful Marker of M-phase Cells
Division of Oncological Pathology, Aichi Cancer Center Research Institute, Nagoya, Japan (AH,KI,TT,HS,TM,MT); Department of Veterinary Pathology, Gifu University, Gifu, Japan (AH,HS,TY,TM); and Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Japan (HG,MI)
Correspondence to: Masae Tatematsu, Div. of Oncological Pathology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan. E-mail: mtatemat{at}aichi-cc.jp
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Summary |
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Key Words: M-phase cell cell proliferation immunohistochemistry histone phosphorylation rat liver partial hepatectomy
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Introduction |
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Histones, the major protein constituents of chromatin in the nuclei of eukaryotic cells, are divided into two main groups, core and linker. Core histones are wrapped around DNA as octamers, consisting of two H2A-H2B dimers and one H3-H4 tetramer (D'Anna and Isenberg 1974; Moss et al. 1976
; Luger et al. 1997
). The N-terminal tails of core histones are subjected to multiple modifications such as acetylation, phosphorylation, methylation, and ubiquitination to maintain several chromatin functions and structures (Cheung et al., 2000
; Strahl and Allis, 2000
). Histone H3 (H3) phosphorylation is closely linked to mitotic chromosomal condensation, occurring at serine 10 in diverse organisms (Hans and Dimitrov 2001
). Recently we reported that H3 phosphorylation occurs not only at serine 10 but also at serine 28 and demonstrated that this coincides with mitotic chromosomal condensation in several types of cultured cells using immunocytochemical and biochemical approaches (Goto et al. 1999
). A site- and phosphorylation state-specific antibody was generated to recognize the region around phosphoserine 28 of H3 and named HTA28. The HTA28 antibody could be shown to specifically bind to mitotic but never to interphase cells by immunofluorescence of in vitro cultured cells and by immunoblotting analysis.
Cell proliferation can be induced by various methods in rodents. One well-known and highly reproducible animal model that includes all cell-cycle events is regenerating rat liver after partial hepatectomy (PH), because the liver cells are initially in G0 and then they traverse the cell cycle one to three times before finally decycling back in G0 (Wright and Alison 1984). This animal model has been widely used in studies of methods for measurement of cell proliferation (Alison et al. 1994
; Gerlach et al. 1997
).
In this study we therefore investigated the utility of the HTA28 antibody for detecting M-phase cells by IHC in regenerating rat liver tissue after PH. The temporal patterns and spatial distributions of HTA28-positive cells were compared with those of BrdU- and Ki-67-positive cells, as well as mitotic figures, in hematoxylin and eosin (H and E)-stained serial sections. Because some proliferating markers, such as PCNA (Hall et al. 1990; Leong et al. 1993
), can be readily destroyed with the prolonged fixation, we also investigated the stability of HTA28 antigenicity with prolonged fixation or delay before fixation to provide information about optimal preparation of clinical samples.
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Materials and Methods |
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Antibodies
Rat monoclonal antibody (MAb) HTA28 was produced using a synthetic peptide PH28 (CKKAARKpSAPATGGV; pS represents phosphoserine), corresponding to the region around the H3 phosphorylation site at serine 28, as an antigen. The specificity of HTA28 has been characterized by immunocytochemistry and immunoblotting analyses as described earlier (Goto et al. 1999). A mouse (MAb) against BrdU (clone Bu20a) and an anti-rat Ki-67 antibody (MIB-5) were purchased from Dako Japan (Kyoto, Japan).
Experimental Protocol
In experiment I (Figure 1A)
, all rats underwent PH under diethyl ether anesthesia and groups of three to five rats were sacrificed at 0, 12, 18, 24, 30, 36, 48, 60, or 72 hr thereafter. They received an IP injection of BrdU (Sigma Chemical; St Louis, MO) at a dose of 100 mg/kg body weight 1 hr before sacrifice. Liver slices of the right lobe were fixed in 10% neutral buffered formalin immediately after excision for 24 hr at room temperature. Tissues were then processed routinely, embedded in paraffin and sectioned serially at 2 µm for H and E staining and immunostaining for HTA28, BrdU, and Ki-67.
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Immunohistochemistry
After removal of paraffin, endogenous peroxidase was blocked with incubation in 0.3% H2O2 in methanol for 20 min at RT. Sections for HTA28 and Ki-67 staining were then pretreated in a microwave oven (type MI-77; Azumaya, Tokyo, Japan) in 0.01 M citrate buffer at pH 6.0 for 10 min at 98C with continuous monitoring of solution temperature using a temperature probe. After rinsing in PBS, nonspecific binding sites were blocked by incubation with diluted normal horse serum for 20 min at RT. Primary antibodies diluted in PBS were applied (diluted at 1:500, 1:50, and 1:100 for HTA28, BrdU, and Ki-67, respectively) and incubated overnight at 4C. Influence of the staining procedure itself was assessed in negative controls by omitting the primary antibodies using regenerating rat liver tissue 24 hr after PH. After incubation, sections were washed with PBS and processed for avidinbiotinperoxidase staining (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. Sections were then incubated in 3,3'-diaminobenzidine and H2O2 to provide the chromogen, washed in running tapwater, and lightly counterstained in Meyer's hematoxylin.
Analysis of Sections
In experiment I, the numbers of HTA28-, BrdU-, and Ki-67-positive hepatocytes, as well as mitotic figures in H and E sections, were counted at x400 magnification and the percentages of positive cells to a total of 1000 or more hepatocytes were calculated for each time point. Here we counted metaphase and anaphase cells in H and E sections as mitotic figures to calculate mitotic indices (MIs).
In experiment II, only mitotic figures (metaphase and anaphase cells) were counted at x400 magnification. One hundred or all mitotic figures (sometimes mitotic figures were less than 100 in one section) were counted and the percentages of HTA28-positive cells were calculated.
Statistical Analysis
In experiment I, the significance of differences was statistically evaluated using the Mann-Whitney U-test for comparisons between the HTA28 staining index (SI) and MI at each time point after PH. Linear regression analysis was used to determine the relationship between the HTA28 SI and MI. In experiment II, the significance of differences was statistically evaluated using the Mann-Whitney U-test for comparisons among groups and the Kruskal-Wallis test for comparisons within the same time group (within groups 1, 2, and 3).
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Results |
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Experiment II
Table 1 shows the results of experiment II. The HTA28 SI significantly decreased with time in PBS before fixation (group 1-1 vs 2-1 and 3-1; p<0.01) and with the fixation period (among groups 1-1, 1-2, and 1-3; p<0.01).
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Discussion |
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The temporal pattern of HTA28 SI showed the expected similarity with that for BrdU LI with a time lag of 6 hr, although the magnitudes of the labeling indices were quite different, presumably reflecting the respective phase duration (Wright and Alison 1984). Because the S-phase is much longer than the M-phase, it is reasonable that the BrdU LI was much higher. There have been many reports of a time lag of 46 hr between S- and M-phase peaks (Grisham 1962
; Fabrikant 1968
; Whitfield et al. 1980
; Wright and Alison 1984
).
The spatial distribution of HTA28-positive cells in the liver after PH also corresponded with that of BrdU-positive cells in the present study. It is widely accepted that DNA synthesis in the regenerating liver shows a periportal preponderance in the first 36 hr after PH (Wright and Alison 1984). The subsequent shift towards the midzonal area has also been previously reported (Harkness 1952
; Grisham 1962
; Fabrikant 1967
,1968
).
The temporal pattern and distribution of Ki-67-positive cells found here were in line with previous reports (Gerlach et al. 1997). Ki-67 SI was the highest at every time point among indices of other proliferative markers because this antigen is expressed throughout all active phases of the cell cycle. Therefore, the MIB-5 antibody would be expected to have the greatest sensitivity to detect proliferating cells.
In the present IHC study, reactivity for HTA28 was maintained through all stages of M-phase from prophase to telophase, although telophase cells were stained relatively weakly, indicating primary expression of the antigen epitope in the early stages of M-phase. Immunoreactivity of HTA28 was found to decrease at the beginning of anaphase and to completely disappear during late anaphase in cultured cells with an indirect immunofluorescence technique (Goto et al. 1999). This difference in findings may be caused merely by differences of fixatives, labeling methods, or conditions of cells between in vivo and in vitro. Indeed, we have established that it is possible to readily detect cells in telophase by increasing the amount of the antibody and/or extending the development time with chromogens (unpublished results).
In experiment II, antigenicity was severely lost with introduction of a period before fixation, with 30% reduction of an HTA28 SI by soaking tissues in PBS for only 2 hr, even when they were appropriately fixed (for 24 hr in formalin) afterward. Moreover, antigenicity was also lost after elongated fixation times, with statistical significance in group 1. Actually, a few cells (3.4%) were already not positive although they showed mitotic figures (metaphase and anaphase) even after only 24 hr fixation. Loss of antigenicity might be caused by dephosphorylation at serine 28 of H3 because obvious changes such as degeneration were not apparent on H and E staining. It has been confirmed that HTA28 reacts with the synthetic peptide PH28 (CKKAARKpSAPATGGV; pS represents phosphoserine) but not with H28 (CKKAARKSAPATGGV) (Goto et al. 1999
). Therefore, surgeons and pathologists are recommended to immediately and precisely fix samples if clinical specimens are to be examined.
In the present study, we clearly demonstrated that HTA28 is a useful IHC marker of M-phase cells. HTA28 might find application to human and other animal tissues because H3 phosphorylation at serine 28, as well as at serine 10, may be conserved across species (Wei et al. 1998). Assessment of proliferative activity of tumor cells is clinically very important for the evaluation of patients' prognosis. Miscellaneous parameters have been proposed as prognostic markers for different human neoplasms (Hall and Levison 1990
; Linden et al.1992
; Sallinen et al. 1994
), and HTA28 might be particularly useful because it can detect actual mitoses more sensitively than can morphological assessment. Moreover, HTA28 is also applicable for retrospective investigations with archival materials because it has the advantage of allowing the cell proliferative status in tissue sections to be evaluated through an appropriate antigen retrieval technique without pretreatment.
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Acknowledgments |
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We thank Ms Hisayo Ban and Mr Tatsuya Wani for expert technical assistance.
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Footnotes |
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Literature Cited |
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