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Volume 51 (12): 1681-1688, 2003
Copyright ©The Histochemical Society, Inc.
Evaluation of Cell Proliferation in Rat Tissues with BrdU, PCNA, Ki-67(MIB-5) Immunohistochemistry and In Situ Hybridization for Histone mRNA
Charles River Laboratories (LM,JRL,EBH) and Division of Biometry and Risk Assessment of National Center for Toxicological Research (RLK), Jefferson, Arkansas
Correspondence to: Levan Muskhelishvili, PhD, Charles River Laboratories at National Center for Toxicological Research, 3900 NCTR Rd., MC 923, Jefferson, AR 72079. E-mail: lmuskhelishvili{at}nctr.fda.gov
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Summary |
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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.
(J Histochem Cytochem 51:1681–1688, 2003)
Key Words: proliferation • immunohistochemistry • in situ hybridization • labeling indices
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Introduction |
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EVALUATION of cell proliferation in tissues of experimental animals is essential for toxicology and carcinogenesis studies and also to assess the efficiency of cytotoxic and chemopreventive drugs in cancer research (Lucas et al. 1998
; Wardley et al. 1998; Fong and Magee 1999; Han et al. 2001). Detection of proliferating cells in tissue sections can be achieved by a number of methods. The gold standard has long been the in vivo labeling of DNA by the modified pyrimidine analogue, a halogenated derivative of thymidine, BrdU (Gratzner 1982; Alison 1995). BrdU can be administered to laboratory animals via IP injection for use in pulse-label experiments or via osmotic pumps for use in continuous-label studies (Goldsworthy et al. 1991). BrdU is readily incorporated into nuclei during the DNA synthetic phase of the cell cycle (S-phase) and is detected by IHC with an anti-BrdU antibody. BrdU IHC has proved useful for identifying S-phase cells. However, this method has several disadvantages: (a) it requires injecting living animals and additional animal handling; (b) BrdU is also incorporated into cell DNA during DNA repair; and (c) BrdU is a mutagen and is not suitable for genetic toxicology studies.
For many years, the most common method to identify proliferating cells in tissue sections without the use of BrdU has been IHC detection of PCNA. PCNA, initially described by Miyachi et al. (1978), is an auxiliary protein of DNA polymerases 
and 
, enzymes necessary for DNA synthesis (Kurki et al. 1986; Bravo et al. 1987; Wood and Shivji 1997). Expression of PCNA increases during the G1-phase, peaks at the S-phase, and declines during G2/M-phases of the cell cycle. These immunostaining characteristics allow the identification of cells in the different phases of the cycle (Foley et al. 1993). Although there have been conflicting reports on the correlation of anti-PCNA LI with other proliferation indices (Jain et al. 1991; Yu et al. 1991; Dervan et al. 1992; Leonardi et al. 1992; Rosa et al. 1992; Visakorpi 1992; Budke et al. 1994; Lardelli et al. 1994; Sarli et al. 1995), PCNA IHC has been extensively used for basic research and as a prognostic tool in surgical pathology (Eldridge and Goldsworthy 1996; Faderl et al. 2002; Kato et al. 2002; Lillo et al. 2002). However, the value of detection of proliferating cells with PCNA IHC has been consistently questioned because detectable levels of PCNA can vary significantly among different cell types, between cells in the malignant vs normal state, and depending on the fixatives and antigen retrieval solutions used (Morris and Mathews 1989; Hall et al. 1990; Coltrera and Gown 1991; Schwarting 1993; Scholzen and Gerdes 2000). PCNA is also involved in DNA repair (Celis and Madsen 1986; Toschi and Bravo 1988; Shivji et al. 1992; Wood and Shivji 1997), which suggests that PCNA may be expressed by cells that are not cycling.
Another IHC technique that has found application in the assessment of cell proliferation is the detection of Ki-67. The antigen Ki-67 is a ubiquitous human nuclear protein expressed in G1-, S-, and G2-phases of the cell cycle but not in the G0-phase (Gerdes et al. 1984; Gerlach et al. 1997a,b), and is therefore a measure of the growth fraction. The usefulness of Ki-67 IHC in tumor diagnostics has been well established for various types of malignancies (reviewed by Scholzen and Gerdes 2000). However, controversy exists with regard to Ki-67 protein expression and localization during the cell cycle, the protein's half-life, expression in different cell types, and its prognostic value and the protocols for fixation and staining (Littleton et al. 1991; Bruno and Darzynkiewicz 1992; Goldblum and Appelman 1995; Oka and Arai 1996; van Oijen et al. 1998; Scholzen and Gerdes 2000). Because monoclonal antibodies (MAb) raised against the human Ki-67 protein often have a limited cross-species reactivity, Ki-67 IHC in the beginning was applied mostly to human material. Recently, an MAb, MIB-5, was generated using bacterially expressed parts of the human Ki-67 cDNA (Schluter et al. 1993; Gerlach et al. 1997a,b). This antibody has the additional advantage of being able to react with the rodent-equivalent, cell cycle-related nuclear protein. It has been demonstrated that percentages of MIB-5- and anti-BrdU-immunostained cells correlated in rodent tissues (Ito et al. 1998; Birner et al. 2001; Enami et al. 2001; Fedrowitz et al. 2002).
An alternative to the IHC approach to evaluate cell proliferation in tissue sections is to detect expression of histone mRNA. The synthesis of histone proteins is restricted to the S-phase of the cell cycle. During the S-phase the level of histone mRNA increases over 50-fold. When DNA synthesis is completed or inhibited, histone mRNAs are selectively and rapidly degraded with a half-life of 10 min (Heintz et al. 1983). Therefore, the presence of abundant quantities of histone mRNA provides a precise molecular marker of S-phase cells. The utility of ISH for histone mRNA to assess the number of S-phase cells has been demonstrated in some rat tissues and human tumors (Alison et al. 1994; Gown et al. 1996; Rautiainen et al. 1998; Murakami et al. 1999; Thomas et al. 2002). A methodological drawback to the application of this technique is that over-fixation or RNase contamination of specimens can reduce the signal and affect the accuracy of detection of labeled cells with ISH (Dijkman et al. 1996; Komminoth 1996).
An accurate method for evaluation of proliferative activity in tissues that can substitute for BrdU IHC when necessary is of great interest to investigators. Each of the candidate assays mentioned above—PCNA IHC, Ki-67(MIB-5) IHC, and ISH for histone mRNA—has its advantages and disadvantages. To determine the optimal choice, we analyzed the correlation of anti-PCNA, MIB-5, and histone mRNA LIs with anti-BrdU LI in rat renewing tissues.
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Materials and Methods |
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Animals
Male 12-week-old Sprague–Dawley rats were sacrificed by CO2 asphyxiation. At 2 hr before sacrifice, all rats (n=6) were given a single IP injection of BrdU (Sigma; St Louis, MO) at a dosage of 100 mg/kg body weight. The esophagus, duodenum, spleen, and a piece of dorsal skin were removed and separate samples of each tissue were fixed in 10% neutral buffered formalin for 24, 48, and 72 hr. After fixation the tissues were embedded in paraffin and serially sectioned. For histological evaluation the sections were stained with hematoxylin and eosin (H&E). The care of the animals and the experimental procedures were carried out according to the institutional guidelines.
Immunohistochemistry
To visualize BrdU incorporation, formalin-fixed, paraffin-embedded rat tissue sections were deparaffinized and hydrated. Endogenous peroxidase was inhibited by incubation with freshly prepared 3% H2O2 with 0.1% sodium azide. The sections were then treated with 0.1% trypsin (Sigma) in 0.1% calcium chloride at 37C for 15 min. DNA was denatured by an incubation with 95% formamide in 0.15 M sodium citrate at 70C for 45 min. Nonspecific staining was blocked with 0.5% casein for 20 min at room temperature (RT). The sections were then incubated with mouse monoclonal anti-BrdU antibody (Bu20a; DAKO, Carpinteria, CA) at a dilution of 1:150 for 1 hr at RT. After incubation with primary antibody, tissue sections were sequentially incubated with goat anti-mouse biotinylated IgG (no. B0529; Sigma) and ExtrAvidin-conjugated horseradish peroxidase (ExtrAvidin Peroxidase Staining Kit, Extra-2; Sigma) at a dilutions of 1:200 and 1:30, respectively. Staining was developed with diaminobenzidine (DAB; Sigma) substrate and sections were counterstained with hematoxylin.
For IHC demonstration of Ki-67 or PCNA, tissue sections were quenched for endogenous peroxidase as described above and placed in an antigen retrieval solution (0.01 M citrate buffer, pH 6.0) for 15 min in a microwave oven at 100C at 600 W. After incubation in the casein block, mouse MAb anti-rat Ki-67 (clone MIB-5; DAKO) or mouse MAb anti-PCNA (clone PC10; DAKO) was applied to the sections at dilutions of 1:50 and 1:5000, respectively. Incubations with primary antibodies lasted for 1 hr at RT. The same secondary detection system described above was used to visualize antibody binding. Staining was developed with DAB, slides were counterstained with hematoxylin, dehydrated, and mounted. For negative control in the IHC procedures performed, mouse 10% normal serum or PBS replaced the primary antibodies.
ISH for Histone mRNA
The Histone ISH Kit (NCL-HISTONE-513-K) and the histone probe alone (NCL-HISTONE-513) were obtained from Novocastra Laboratories (Newcastle, UK). The probe was a fluoroscein (FITC)-labeled oligonucleotide cocktail for detection of histone H2b, H3, and H4 mRNA sequences.
Paraffin sections were deparaffinized in xylene, hydrated in 100%, 95%, and 70% ethanol, and treated with 10 µg/ml proteinase K for 30 min at 37C. The sections then were washed in distilled water, dehydrated, and air-dried. The sections were incubated with probe solution for 2 hr at 37C. After washes in 0.1% Triton X-100 in Tris-buffered saline (TBS, pH 7.6), the sections were blocked in 10% normal rabbit serum for 10 min and then incubated with alkaline phosphatase-conjugated rabbit anti-FITC F(ab') (Novocastra) at a dilution of 1:100 for 30 min at RT. For demonstration of alkaline phosphatase activity, the sections were incubated overnight in the dark with 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitroblue tetrazolium (NBT) (Novocastra). The sections then were washed in distilled water and counterstained in hematoxylin for 10 sec. The sections were mounted with Crystal/Mount (Biomeda; Foster City, CA) and dried at 70C for 40–60 min. For negative control, corresponding serial sections were hybridized with a fluoroscein-labeled random oligonucleotide cocktail (Novocastra).
Measurement of LIs and Statistical Analysis
The slides were examined by light microscopy (BX40; Olympus, Tokyo, Japan) and the image was captured with a color video camera (World Video; Boyertown, PA). An Optimas image analysis system (Optimas Corp; WA) was used to quantify the numbers of anti-BrdU-, MIB-5-, anti-PCNA-labeled cells and histone mRNA-expressing cells.
In epidermis of the skin and stratified squamous epithelium of esophagus of each animal, LI was expressed as the percentage of labeled cells per 1000 basal cells. In the duodenum, epithelial cells were counted from the lowest point of the crypt to the tip of the villus and the percentage of positive cells was calculated. The mean value of the percentages of labeled cells in three randomly chosen villi per animal was used for further calculations. In the spleen, the numbers of labeled cells were calculated per 3 mm2. In anti-PCNA-immunostained sections, LIs for growth fraction (anti-PCNA LI) and S-phase (anti-PCNAS LI) were calculated separately by respectively counting either all labeled cells or only cells with dark brown nuclei. All cell counts were performed using the x40 magnification objective lens.
The correlation between anti-BrdU, MIB-5, anti-PCNA, anti-PCNAS, and histone mRNA LIs was analyzed using Pearson's coefficient of correlation (Snedecor and Cochran 1967). It was assumed that there were 24 independent observations on LIs (6 rats x 4 tissues). For each of the five LIs, the range of numerical values across all tissues was quite large in light of the use of different criteria to quantify cell proliferation in the different tissues. To eliminate the possible induction of a spurious correlation with such a wide data range, before analysis the observations were standardized for each tissue and method. Standardized indices were calculated by subtracting the tissue-by-method mean and dividing by the tissue-by-method standard deviation.
Anti-BrdU LI was paired with each of the other four LIs and Pearson's correlation coefficient was calculated for each pair. Each correlation coefficient was tested for statistical significance using an approximate t-test with 22 degrees of freedom. To assure that distribution assumptions for Pearson's correlation coefficient were adequately met, Spearman's rank correlation coefficient (Snedecor and Cochran 1967) was also calculated and tested and gave qualitatively similar results in all cases.
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Results |
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BrdU, MIB-5, PCNA IHC, and ISH for histone mRNA were performed on serial sections of rat paraffin-embedded tissues. In these sections, cells that incorporated BrdU displayed brown nuclei, with some variation in the strength of staining. Nuclear immunoreactivity revealed by MIB-5 usually had a dark brown granular appearance. In contrast, PCNA positivity was homogeneously dense, with nuclei showing variability in staining intensity and some proportion of cells displaying the immunoreactivity in cytoplasm as well. Histone mRNA-positive cells exhibited purple cytoplasm (Figure 1) . None of the negative controls showed any positive staining.
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The strongest signal for mRNA staining was observed in the tissue samples fixed for 24 hr. In all the tissues fixed for 48 hr in formalin, the numbers of labeled cells were significantly lower (more than 50% less). The signal was completely undetectable in tissue samples fixed for 72 hr (Table 1). Therefore, all counts for the correlation study were performed using serial sections obtained only from the tissue samples fixed for 24 hr in formalin.
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Anti-BrdU, MIB-5, anti-PCNA, and histone mRNA LIs in the tissues studied are shown in Table 2. On calculating Pearson's correlation coefficient (Pearson's r) on the standardized values in Table 2, the correlation between anti-BrdU and histone mRNA LIs was highly significant, with r=0.5712 and p=0.004. The correlation between anti-BrdU and MIB-5 LIs was also statistically significant at the 1% level (r=0.5159, p=0.01). There was no statistically significant correlation between anti-BrdU and anti-PCNA LIs (r=0.1023, p=0.63) or between anti-BrdU and anti-PCNAS LIs (r=0.0035, p=0.88) (Figure 2) .
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Discussion |
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TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
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The strong correlation between anti-BrdU and histone mRNA LIs throughout all the tissues observed in the study suggests that detectable levels of histone mRNA have no dependence on the cell type and that ISH for histone mRNA is a sensitive and valuable technique for evaluation of proliferating activity in tissue sections. These results are in conformity with the data obtained by other investigators (Alison et al. 1994
; Gown et al. 1996; Rautiainen et al. 1998; Murakami et al. 1999; Thomas et al. 2002). However, a limitation for the use of ISH for histone mRNA is optimal fixation time. According to our results, it must not exceed 24 hr in formalin. After this period of time the signal gradually decreases and becomes undetectable at around 72 hr of fixation.
The correlation between anti-BrdU and MIB-5 LIs suggests that MIB-5 can reliably detect the growth fraction in rat paraffin-embedded tissues and confirms the results of recent studies on mouse and rat tissues (Ito et al. 1998; Birner et al. 2001; Enami et al. 2001; Fedrowitz et al. 2002). However, statistical analysis showed that the correlation coefficient between anti-BrdU and MIB-5 was lower and the p value was higher (r=0.5159, p=0.01) compared to those between anti-BrdU and ISH for histone mRNA, which indicates stronger correlation between the latter two indices (Figure 2).
Absence of a correlation between anti-BrdU and anti-PCNA LIs or between anti-BrdU and anti-PCNAS LIs in the present study is in agreement with some previous reports in which no correlation between anti-PCNA LI and other indices of proliferation was observed (Jain et al. 1991; Yu et al. 1991; Leonardi et al. 1992; Rosa et al. 1992; Visakorpi 1992). Possible explanations include the long half-life of PCNA, leading to its continuous expression in some cells that are not actively dividing (Morris and Mathews 1989; Scott et al. 1991), and immunostaining of cells containing the nucleoplasmic population of PCNA, which is not directly associated with DNA replication sites (Bravo and Macdonald–Bravo 1987). The same reasons, along with the involvement of PCNA in DNA repair (Celis and Madsen 1986; Toschi and Bravo 1988; Shivji et al. 1992; Wood and Shivji 1997), could also have contributed to the fact that in all tissues anti-PCNA labeled a significantly larger proportion of cells than did MIB-5 (Table 2; Figure 1).
Comparison of S-phase LIs between control and treated animals is a commonly used endpoint in cell proliferation studies (Lucas et al. 1998; Wardley et al. 1998; Fong and Magee 1999; Sakamoto et al. 1999; Cuenca et al. 2001; Han et al. 2001). The absence of statistically significant correlation between anti-BrdU and anti-PCNAS LIs in the present study suggests that making a clear distinction of S-phase cells from non-S-phase cells using PCNA IHC can be difficult and inaccurate, especially for tissues in which the proliferative activity is high and the cells are small, e.g., spleen. Therefore, when experimental conditions require a critical assessment of the S-cell fraction in vivo, e.g., in situations when effects on G1
S transition have to be evaluated, PCNA IHC may not be the best choice. In contrast to PCNA, histone mRNA appears to be a valuable marker for S-phase cells.
In conclusion, the results of the present study suggest that both ISH for histone mRNA and IHC with MIB-5 are preferable techniques for assessment of proliferative activity in rat paraffin-embedded renewing tissues compared to PCNA IHC. They can substitute for BrdU IHC when necessary. However, each of them suffers from specific shortcomings: MIB-5 does not label S-phase cells distinctly, and a limitation to the use of ISH for histone mRNA is optimal fixation time.
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Footnotes |
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Received for publication May 22, 2003; accepted July 30, 2003
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