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Apoptosis profiles in benign prostatic hyperplasia: Close associations of cell kinetics with percent area density of histologic composition
BASIC SCIENCE
APOPTOSIS PROFILES IN BENIGN PROSTATIC HYPERPLASIA: CLOSE ASSOCIATIONS OF CELL KINETICS WITH PERCENT AREA DENSITY OF HISTOLOGIC COMPOSITION XIANGHUA ZHANG, QIAN ZHANG, ZHENG ZHANG, YANQUN NA,
AND
YINGLU GUO
ABSTRACT Objectives. To investigate the possible correlations of apoptosis and apoptosis-associated factors, including the apoptotic index (AI), proliferation index (PI), and expression of Bcl-2 and caspase 3, with the percent area density of epithelium and stroma in benign prostatic hyperplasia (BPH). Methods. A total of 60 patients with histologically determined BPH were investigated. The percent area density of epithelium and stroma was determined using a computerized image analysis system after Masson’s trichrome staining. Apoptosis was detected using the AI through the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay. Cell proliferation was determined using the PI through Ki-67 immunostaining. The expression of Bcl-2 and caspase 3 was immunohistochemically examined. Double-label immunofluorescent staining was performed to assess co-localization of Bcl-2 and caspase 3. Results. The stroma/epithelium ratio ranged from 4.6 to 6.3 (average 5.2) in BPH. A greater stromal PI was closely related to the percent area density of stroma (P ⬍0.01), and a greater epithelial AI was reversely related to the percent area density of the epithelium (P ⬍0.05). Bcl-2 and caspase 3 expression was detected in 50 (80%) and 48 (75%) of 60 BPH samples, respectively. The expression of Bcl-2 and caspase 3 was not related to PI, AI, or the percent area density of the BPH components (P ⬎0.05). Immunofluorescence analysis revealed co-localization of Bcl-2 and caspase 3 in the serial sections of BPH specimens that already showed either Bcl-2 or caspase 3 expression. Conclusions. The development of BPH may be associated with both stromal growth due to active stromal cell proliferation and epithelial growth due to reduced glandular apoptosis. UROLOGY 68: 905–910, 2006. © 2006 Elsevier Inc.
B
enign prostatic hyperplasia (BPH), the most frequent benign disease in men, is the result of proliferation of mesenchymal-stromal and glandular-epithelial compartments.1 Organ growth is highly dependent on the balance between cell proliferation and apoptotic cell death. Some studies have indicated that human BPH is associated with increased cell production and abnormal regulation of apoptosis.2–5 Disruption of the molecular mechanisms that regulate these two processes may unFrom the Department of Urology, Peking University First Hospital, Institute of Urology, Beijing, China Reprint requests: Xianghua Zhang, Ph.D., M.D., Department of Urology, Peking University First Hospital, Institute of Urology, Beijing 100034, China. E-mail: [email protected] Submitted: December 5, 2005, accepted (with revisions): May 19, 2006 © 2006 ELSEVIER INC. ALL RIGHTS RESERVED
derline the abnormal growth of the gland leading to BPH. Activation of the apoptotic process is regulated by several intracellular factors, such as Bcl-2 and caspase 3 proteins. Bcl-2 is an inner mitochondrial membrane protein, the main effect of which is to prolong cell survival by avoidance of apoptosis.6,7 Overexpression of Bcl-2 in mammalian cells results in delayed apoptosis.8 The caspase family of proteases is central to the cell death pathway.9 The extrinsic and intrinsic cell death pathways converge to activate caspase-3 for the final execution of apoptosis.10 Activation of caspase-3 cleaves a broad range of cellular substrates and promotes activation of a DNA endonuclease,11,12 required for internucleosomal DNA fragmentation, a widely accepted hallmark of apoptosis. Both Bcl-2 and 0090-4295/06/$32.00 doi:10.1016/j.urology.2006.05.013 905
caspase 3 expression have been observed in a variety of benign and malignant tumors, including BPH and prostate cancer.3,13–15 However, the roles of bcl-2 and caspase 3 expression in BPH development, particularly in the regulation of apoptosis induction, have yet to be described. Although the situations of cell turnover have been studied in BPH tissue, no study has investigated apoptosis and apoptosis-associated parameters in relation to the histologic composition of BPH. In the present study, we comparatively studied possible correlations of apoptosis and apoptosis-associated factors, including the apoptotic index (AI), proliferation index (PI), and expression of Bcl-2 and caspase 3, with the percent area density of epithelium and stroma in BPH. MATERIAL AND METHODS This study included 60 patients with BPH who underwent transurethral resection of the prostate or suprapubic prostatectomy. Their age range was 59 to 76 years (median age 66). None of these patients had undergone previous hormonal manipulation. The formalin-fixed and paraffin-embedded specimens were cut into 5-m-thick sections and placed on PolyL-lysine-treated glass slides. For histopathologic examination, each paraffin block was step sectioned and routinely stained with hematoxylin-eosin for the histopathologic study and with Masson’s trichrome for the determination of the percent area density of epithelium, stroma, and luminal space in BPH.
DETERMINATION OF PERCENT AREA DENSITY To determine the percent area density of epithelium and stroma in the BPH specimens, a computerized image analysis system, including a BX51 light microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP50 CCD color camera head, a Sony color monitor, and image analysis software (Win Roof, version 3.6, Mitani, Osaka, Japan), was used. As described previously,16 the percent area density of the epithelium, stroma, and luminal space of the prostate was quantitatively evaluated through the function of color-assisted image analysis of the Win Roof in the Masson’s trichrome-stained tissue sections. At least 10 different fields were examined at a magnification of 100⫻ from each tissue section, and the mean percent area density of the epithelium, stroma, and luminal space of BPH was determined.
IN SITU DETECTION OF APOPTOSIS Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining according to the procedures included in the ApopTag in situ apoptosis detection kit (Intergen, Purchase, NY). In brief, after deparaffinization and blocking of endogenous peroxidase with 0.3% hydrogen peroxide in methanol for 30 minutes at room temperature, incubation with 100 g/mL proteinase K (Sigma Chemical, St. Louis, Mo) was performed for 15 minutes. After prehybridization treatment, the sections were exposed to terminal deoxynucleotidyl transferase with digoxigenin-11-dUTP and dATP, with incubation in a moist chamber for 60 minutes at 37°C. Anti-digoxigenin-11-dUTP labeling for 30 minutes at room temperature was followed by exposure to diaminobenzidine solution. Counterstaining was achieved with 0.5% methyl green solution. The AI was obtained as the ratio of TUNELpositive cells relative to the total number of counted tumor 906
cells and calculated from observations of at least 1000 cells in each section.
IMMUNOSTAINING After deparaffinization, hydration, and a blocking step with 2.5% hydrogen peroxide in methanol, the sections were boiled in 0.01 M citrate buffer for 10 minutes and incubated with 5% normal blocking serum in Tris-buffered saline for 20 minutes. The sections were then incubated with the following antibodies: monoclonal mouse anti-Ki-67 (1/100 dilution, Zymed Laboratories, San Francisco, Calif); monoclonal mouse antiBcl-2 (1/100 dilution; Dako, Denmark), and polyclonal goat anti-caspase 3 (1/50 dilution; Santa Cruz Biotechnology, Santa Cruz, Calif), which, according to the company, detects both pro-caspase 3 and active caspase 3. The sections were next incubated with biotinylated anti-mouse IgG (1/200 dilution, Vector Laboratories) for 30 minutes. After incubation in an avidin-biotin-peroxidase complex for 30 minutes, the samples were exposed to diaminobenzidine tetrahydrochloride solution and counterstained with hematoxylin. To obtain specificity controls, the primary antibody was replaced with Trisbuffered saline, and all other steps were followed unchanged. The PI was obtained as the ratio of Ki-67-positive cells relative to the total number of counted tumor cells and calculated from observations of at least 1000 cells in each section. In evaluating bcl-2 and caspase 3 expression, the percentages of cells with strong reactivity were recorded after examining the area of the tissue samples. A reactivity of more than 10% was considered positive.
IMMUNOFLUORESCENCE ANALYSIS Co-expression of Bcl-2 and caspase 3 was detected through double-label immunofluorescent staining. To identify Bcl-2 protein, the specimens were incubated first with the mouse monoclonal anti-Bcl-2 antibody and then reacted with an antimouse Alexa Fluor-595 conjugate (1/200 dilution, Molecular Probes, Eugene, Ore) as the secondary antibody. To show caspase 3 localization, the specimens were incubated with the polyclonal goat anti-caspase 3 and then with anti-goat Alexa Fluor-488 conjugate (1/200 dilution, Molecular Probes). To visualize nuclei, the specimens were stained with 50 g/mL of 4,6-diamidino-2-phenylindole (Sigma Chemical), in which 100 g/mL of DNase-free RNase A was added to facilitate the examination of nuclei with a BX51 fluorescence microscope (Olympus).
STATISTICAL ANALYSIS The Student t test and Kruskal-Wallis test were used to analyze differences in the incidence of apoptosis and proliferation between the mean values in the various data sets. A P value less than 0.05 was considered to indicate statistically significant differences.
RESULTS PERCENT AREA DENSITY The average percent area density of epithelium, stroma, and lumen was 14.4% ⫾ 4.1%, 75.5% ⫾ 10.7%, and 10.1% ⫾ 5.9 %, respectively. The ratio of stroma to epithelium ranged from 4.6:1 to 6.3:1 (average 5.2:1) in BPH specimens. The percent area density of epithelium and stroma was not related to patient age or prostate volume. UROLOGY 68 (4), 2006
FIGURE 1. Observations of immunostaining and TUNEL staining in BPH. Original magnification ⫻100. (A) Ki-67positive cells in epithelium and stroma of BPH. (B) TUNEL-positive cells in epithelium and stroma of BPH. (C) Intense staining for Bcl-2 in epithelial cells of BPH. (D) Strong staining for caspase 3 in epithelium of BPH.
APOPTOTIC AND PROLIFERATION INDEXES Both TUNEL-positive cells and Ki-67-positive cells were detected in the glandular cells and stromal cells of BPH (Fig. 1A,B). The average PI and AI were 1.8 and 0.7 in the epithelium and 1.1 and 0.2 in the stroma of BPH tissues, respectively. Bcl-2 AND CASPASE 3 EXPRESSION The detection of Bcl-2 and caspase 3 expression was limited to the cytoplasm of the glandular epithelium in BPH (Fig. 1C,D). Of 60 BPH specimens, Bcl-2 and caspase 3 expression was detected in 50 (80%) and 48 (75%), respectively. Double-label immunofluorescence analysis revealed co-expression of Bcl-2 and caspase 3 in the serial sections of BPH specimens that had already shown either Bcl-2 or caspase 3 expression, respectively (Fig. 2). PERCENT AREA DENSITY AND CELL KINETICS A greater stromal PI was closely related to the percent area density of stroma in the BPH specimens (P ⬍0.01), and a significantly reverse relationship was noted between a greater epithelial AI UROLOGY 68 (4), 2006
and the percent area density of epithelium in the BPH specimens (P ⬍0.05; Fig. 3). The AI and PI were not related to patient age or prostate volume of BPH (P ⬎0.05). The expression of Bcl-2 and caspase 3 was not related to the PI, AI, or percent area density of the BPH components (P ⬎0.05). COMMENT It is known that human BPH is the result of proliferation of the stromal and epithelial compartments. This proliferation begins in the stroma, with proliferation of the glandular epithelium secondarily induced.1 A number of studies have shown close associations between the development and severity of clinical BPH and the cellular composition of the prostate.17,18 It is clear that the proportion of stromal components is absolutely greater than that of epithelial compartments in human BPH. However, it is unknown whether the cell kinetics in the proportion of stromal or epithelial components play different roles in the pathogenesis and development of BPH. 907
FIGURE 2. Double-label fluorescent immunostaining of Bcl-2 and caspase 3 in BPH tissue sections. Original magnification ⫻100. (A) Bcl-2 staining (green signal); (B) caspase 3 staining (red signal); (C) 4,6-diamidino-2phenylindole nuclear staining (blue signal); and (D) co-localization of Bcl-2 and caspase 3 (yellow signal).
Recently, scientific interest has focused on the reduced cell death theory in the prostate. It has been suggested that an imbalance between apoptosis and cell proliferation may result in the development of BPH.2 No doubt exists that the process of prostate growth, from natural growth into BPH, has been associated with increased cell proliferative activities. Regarding the frequency of apoptosis in human BPH, either increased or decreased apoptotic rate has been noted in the epithelium compared with normal tissues.2– 4 In agreement with previous studies, our quantitative image analysis study showed that the average ratio of stromal/epithelial components was 5.2, and the PI/AI ratio was 2.6 in the epithelium but 5.5 in the stroma of BPH specimens. These results may explain the continuous increase in BPH volume, particularly in the stromal compartments. Furthermore, our study demonstrated that a greater stromal PI was closely related to the percent area density of the stroma, and a greater epithelial AI was inversely related to the percent area density of the epithelium in BPH. It has been indicated that the development of BPH may be associated with both stromal growth, due to active mesenchy908
mal-stromal cell proliferation, and epithelial increment, due to reduced glandular apoptosis. Both Bcl-2 and caspase 3 are known to be involved in the regulation of apoptosis. In normal prostatic tissue, bcl-2 and caspase 3 expression was either undetectable or only weakly stained epithelial cells were observed.19,20 However, either bcl-2 or caspase 3 was strongly expressed in BPH and prostate cancer tissues.3,13–15 In accordance with these findings, our data showed that the expression of Bcl-2 and caspase 3 was frequently detected in the epithelial cells of the BPH specimens. Doublelabel immunofluorescent staining revealed that both caspase 3 and bcl-2 proteins were almost always co-expressed in the epithelial cells of the BPH tissue. Although the expression of bcl-2 and caspase 3 was not related to the frequency of cell proliferation and apoptosis in BPH, the present results may reflect the situation of lower apoptosis induction in BPH. The increased bcl-2 expression may balance against the death cascade by inhibiting the release of cytochrome c from the mitochondria that activate caspases. Therefore, it is suggested that the situation of a lower apoptosis rate in UROLOGY 68 (4), 2006
% of stroma
gen-dependent proliferation of prostatic ducts and surrounding stromal tissue. Many factors have been suggested to be involved in disease development, including the growth factors and hormonal activities of dihydrotestosterone and estrogen.21–23 It is clear that the level of androgens is lower with increasing age in men. However, neither our data nor the findings of previous studies have shown relationships between cell kinetics and patient age in patients with BPH.2,3
100 80 60 40 20
CONCLUSIONS 0
PI in stroma A
% of epithelium 30 25 20
This is the first study to examine correlations of apoptosis and apoptosis-associated factors with the percent area density of epithelial and stromal components in BPH. Our data showed a close relation of a greater PI to the percent area density of stroma and an inverse relation of a greater AI to the percent area density of epithelium in BPH tissue. The development of BPH may be associated with both stromal growth, due to active mesenchymalstromal cell proliferation, and epithelial growth, due to reduced glandular apoptosis.
15 10 5 0
AI in epithelium B FIGURE 3. Association study of cell kinetics with percent area density of stroma and epithelium in BPH. (A) Positive relation between greater PI and percent area density of stroma in BPH (P ⬍0.01). Average percent area density of stroma in three different PI groups was 72.1% ⫾ 2.514%, 75.0% ⫾ 3.590%, and 76.8% ⫾ 2.486%. (B) Inverse relation between greater AI and percent area density of epithelium in BPH (P ⬍0.05). Average percent area density of epithelium in three different AI group was 17.1% ⫾ 3.381%, 14.5% ⫾ 3.719%, and 11.6% ⫾ 3.373%.
BPH may be related to alterations in the balance between bcl-2 and caspase 3 expression. Regarding the detection of apoptosis in the stroma of BPH, an extremely low frequency of apoptosis or an absence of apoptotic cell death has been found in the stroma of human BPH.2,3 Our study also showed a lower rate of apoptosis in the stroma of BPH tissue. Because of an undetectable expression of bcl-2 and caspase 3 in the stromal compartments, the induction of apoptosis in the stromal cells may be regulated by life span instead of apoptotic signals such as occurs in the epithelial cells of BPH tissue. BPH is characterized by androUROLOGY 68 (4), 2006
REFERENCES 1. Foster CS: Pathology of benign prostatic hyperplasia. Prostate Suppl 9: 4 –14, 2000. 2. Claus S, Berges R, Senge T, et al: Cell kinetics in epithelium and stroma of benign prostatic hyperplasia. J Urol 158: 217–221, 1997. 3. Kyprianou N, Tu H, Jacobs SC, et al: Apoptotic versus proliferative activities in human benign prostatic hyperplasia. Hum Pathol 27: 668 – 675, 1996. 4. Colombel M, Vacherot F, Diez SG, et al: Zonal variation of apoptosis and proliferation in the normal prostate and in benign prostatic hyperplasia. Br J Urol 82: 380 –385, 1998. 5. Thompson TC, and Yang G: Regulation of apoptosis in prostatic disease. Prostate Suppl 9: 25–28, 2000. 6. Hockenbery D, Nunez G, Milliman C, et al: Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 22: 334 –336, 1990. 7. Bonkhoff H: Role of the basal cells in premalignant changes of the human prostate: a stem cell concept for the development of prostate cancer. Eur Urol 30: 201–205, 1996. 8. Hengartner MO, and Horvitz HR: C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2. Cell 76: 665– 676, 1994. 9. Yuan J, Shaham S, Ledoux S, et al: The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell 75: 641– 652, 1993. 10. Nunez G, Benedict MA, Hu Y, et al: Caspases: the proteases of the apoptotic pathway. Oncogene 17: 3237–3245, 1998. 11. Liu X, Zou H, Slaughter C, et al: DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89: 175–184, 1997. 12. Enari M, Sakahira H, Yokoyama H, et al: A caspase activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391: 43–50, 1998. 13. Shariat SF, Ashfaq R, Roehrborn CG, et al: Expression of survivin and apoptotic biomarkers in benign prostatic hyperplasia. J Urol 174: 2046 –2050, 2005. 14. Borre M, Stausbol-Gron B, and Overgaard J: p53 accumulation associated with bcl-2, the proliferation marker 909
MIB-1 and survival in patients with prostate cancer subjected to watchful waiting. J Urol 164: 716 –721, 2000. 15. O’Neill AJ, Boran SA, O’Keane C, et al: Caspase 3 expression in benign prostatic hyperplasia and prostate carcinoma. Prostate 47: 183–188, 2001. 16. Zhang X, Na Y, and Guo Y: Biological feature of prostatic hyperplasia developed in spontaneously hypertensive rats. Urology 63: 983–988, 2004. 17. Shapiro E, Becich MJ, Hartanto V, et al: The relative proportion of stromal and epithelial hyperplasia is related to the development of symptomatic benign prostate hyperplasia. J Urol 147: 1293–1297, 1992. 18. Shapiro E, Hartanto V, and Lepor H: The response to alpha blockade in benign prostatic hyperplasia is related to the percent area density of prostate smooth muscle. Prostate 21: 297–307, 1992. 19. McDonnell TJ, Troncoso P, Brisbay SM, et al: Expression of the protooncogene bcl-2 in the prostate and its associ-
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ation with emergence of androgen-independent prostate cancer. Cancer Res 52: 6940 – 6944, 1992. 20. Krajewska M, Wang HG, Krajewski S, et al: Immunohistochemical analysis of in vivo patterns of expression of CPP32 (caspase-3), a cell death protease. Cancer Res 57: 1605–1613, 1997. 21. Boudon C, Rodier G, Lechevallier E, et al: Secretion of insulin-like growth factors and their binding proteins by human normal and hyperplastic prostatic cells in primary culture. J Clin Endocrinol Metab 81: 612– 617, 1996. 22. Burnett AL, Maguire MP, Chamness SL, et al: Characterization and localization of nitric oxide synthase in the human prostate. Urology 45: 435– 439, 1995. 23. Krieg M, Nass R, and Tunn S: Effect of aging on endogenous level of 5a-dihydrotestosterone, testosterone, estradiol, and estrone in epithelium and stroma of normal and hyperplastic human prostate. J Clin Endocrinol Metab 77: 375–381, 1993.
UROLOGY 68 (4), 2006
APOPTOSIS PROFILES IN BENIGN PROSTATIC HYPERPLASIA: CLOSE ASSOCIATIONS OF CELL KINETICS WITH PERCENT AREA DENSITY OF HISTOLOGIC COMPOSITION XIANGHUA ZHANG, QIAN ZHANG, ZHENG ZHANG, YANQUN NA,
AND
YINGLU GUO
ABSTRACT Objectives. To investigate the possible correlations of apoptosis and apoptosis-associated factors, including the apoptotic index (AI), proliferation index (PI), and expression of Bcl-2 and caspase 3, with the percent area density of epithelium and stroma in benign prostatic hyperplasia (BPH). Methods. A total of 60 patients with histologically determined BPH were investigated. The percent area density of epithelium and stroma was determined using a computerized image analysis system after Masson’s trichrome staining. Apoptosis was detected using the AI through the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay. Cell proliferation was determined using the PI through Ki-67 immunostaining. The expression of Bcl-2 and caspase 3 was immunohistochemically examined. Double-label immunofluorescent staining was performed to assess co-localization of Bcl-2 and caspase 3. Results. The stroma/epithelium ratio ranged from 4.6 to 6.3 (average 5.2) in BPH. A greater stromal PI was closely related to the percent area density of stroma (P ⬍0.01), and a greater epithelial AI was reversely related to the percent area density of the epithelium (P ⬍0.05). Bcl-2 and caspase 3 expression was detected in 50 (80%) and 48 (75%) of 60 BPH samples, respectively. The expression of Bcl-2 and caspase 3 was not related to PI, AI, or the percent area density of the BPH components (P ⬎0.05). Immunofluorescence analysis revealed co-localization of Bcl-2 and caspase 3 in the serial sections of BPH specimens that already showed either Bcl-2 or caspase 3 expression. Conclusions. The development of BPH may be associated with both stromal growth due to active stromal cell proliferation and epithelial growth due to reduced glandular apoptosis. UROLOGY 68: 905–910, 2006. © 2006 Elsevier Inc.
B
enign prostatic hyperplasia (BPH), the most frequent benign disease in men, is the result of proliferation of mesenchymal-stromal and glandular-epithelial compartments.1 Organ growth is highly dependent on the balance between cell proliferation and apoptotic cell death. Some studies have indicated that human BPH is associated with increased cell production and abnormal regulation of apoptosis.2–5 Disruption of the molecular mechanisms that regulate these two processes may unFrom the Department of Urology, Peking University First Hospital, Institute of Urology, Beijing, China Reprint requests: Xianghua Zhang, Ph.D., M.D., Department of Urology, Peking University First Hospital, Institute of Urology, Beijing 100034, China. E-mail: [email protected] Submitted: December 5, 2005, accepted (with revisions): May 19, 2006 © 2006 ELSEVIER INC. ALL RIGHTS RESERVED
derline the abnormal growth of the gland leading to BPH. Activation of the apoptotic process is regulated by several intracellular factors, such as Bcl-2 and caspase 3 proteins. Bcl-2 is an inner mitochondrial membrane protein, the main effect of which is to prolong cell survival by avoidance of apoptosis.6,7 Overexpression of Bcl-2 in mammalian cells results in delayed apoptosis.8 The caspase family of proteases is central to the cell death pathway.9 The extrinsic and intrinsic cell death pathways converge to activate caspase-3 for the final execution of apoptosis.10 Activation of caspase-3 cleaves a broad range of cellular substrates and promotes activation of a DNA endonuclease,11,12 required for internucleosomal DNA fragmentation, a widely accepted hallmark of apoptosis. Both Bcl-2 and 0090-4295/06/$32.00 doi:10.1016/j.urology.2006.05.013 905
caspase 3 expression have been observed in a variety of benign and malignant tumors, including BPH and prostate cancer.3,13–15 However, the roles of bcl-2 and caspase 3 expression in BPH development, particularly in the regulation of apoptosis induction, have yet to be described. Although the situations of cell turnover have been studied in BPH tissue, no study has investigated apoptosis and apoptosis-associated parameters in relation to the histologic composition of BPH. In the present study, we comparatively studied possible correlations of apoptosis and apoptosis-associated factors, including the apoptotic index (AI), proliferation index (PI), and expression of Bcl-2 and caspase 3, with the percent area density of epithelium and stroma in BPH. MATERIAL AND METHODS This study included 60 patients with BPH who underwent transurethral resection of the prostate or suprapubic prostatectomy. Their age range was 59 to 76 years (median age 66). None of these patients had undergone previous hormonal manipulation. The formalin-fixed and paraffin-embedded specimens were cut into 5-m-thick sections and placed on PolyL-lysine-treated glass slides. For histopathologic examination, each paraffin block was step sectioned and routinely stained with hematoxylin-eosin for the histopathologic study and with Masson’s trichrome for the determination of the percent area density of epithelium, stroma, and luminal space in BPH.
DETERMINATION OF PERCENT AREA DENSITY To determine the percent area density of epithelium and stroma in the BPH specimens, a computerized image analysis system, including a BX51 light microscope (Olympus, Tokyo, Japan) equipped with an Olympus DP50 CCD color camera head, a Sony color monitor, and image analysis software (Win Roof, version 3.6, Mitani, Osaka, Japan), was used. As described previously,16 the percent area density of the epithelium, stroma, and luminal space of the prostate was quantitatively evaluated through the function of color-assisted image analysis of the Win Roof in the Masson’s trichrome-stained tissue sections. At least 10 different fields were examined at a magnification of 100⫻ from each tissue section, and the mean percent area density of the epithelium, stroma, and luminal space of BPH was determined.
IN SITU DETECTION OF APOPTOSIS Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining according to the procedures included in the ApopTag in situ apoptosis detection kit (Intergen, Purchase, NY). In brief, after deparaffinization and blocking of endogenous peroxidase with 0.3% hydrogen peroxide in methanol for 30 minutes at room temperature, incubation with 100 g/mL proteinase K (Sigma Chemical, St. Louis, Mo) was performed for 15 minutes. After prehybridization treatment, the sections were exposed to terminal deoxynucleotidyl transferase with digoxigenin-11-dUTP and dATP, with incubation in a moist chamber for 60 minutes at 37°C. Anti-digoxigenin-11-dUTP labeling for 30 minutes at room temperature was followed by exposure to diaminobenzidine solution. Counterstaining was achieved with 0.5% methyl green solution. The AI was obtained as the ratio of TUNELpositive cells relative to the total number of counted tumor 906
cells and calculated from observations of at least 1000 cells in each section.
IMMUNOSTAINING After deparaffinization, hydration, and a blocking step with 2.5% hydrogen peroxide in methanol, the sections were boiled in 0.01 M citrate buffer for 10 minutes and incubated with 5% normal blocking serum in Tris-buffered saline for 20 minutes. The sections were then incubated with the following antibodies: monoclonal mouse anti-Ki-67 (1/100 dilution, Zymed Laboratories, San Francisco, Calif); monoclonal mouse antiBcl-2 (1/100 dilution; Dako, Denmark), and polyclonal goat anti-caspase 3 (1/50 dilution; Santa Cruz Biotechnology, Santa Cruz, Calif), which, according to the company, detects both pro-caspase 3 and active caspase 3. The sections were next incubated with biotinylated anti-mouse IgG (1/200 dilution, Vector Laboratories) for 30 minutes. After incubation in an avidin-biotin-peroxidase complex for 30 minutes, the samples were exposed to diaminobenzidine tetrahydrochloride solution and counterstained with hematoxylin. To obtain specificity controls, the primary antibody was replaced with Trisbuffered saline, and all other steps were followed unchanged. The PI was obtained as the ratio of Ki-67-positive cells relative to the total number of counted tumor cells and calculated from observations of at least 1000 cells in each section. In evaluating bcl-2 and caspase 3 expression, the percentages of cells with strong reactivity were recorded after examining the area of the tissue samples. A reactivity of more than 10% was considered positive.
IMMUNOFLUORESCENCE ANALYSIS Co-expression of Bcl-2 and caspase 3 was detected through double-label immunofluorescent staining. To identify Bcl-2 protein, the specimens were incubated first with the mouse monoclonal anti-Bcl-2 antibody and then reacted with an antimouse Alexa Fluor-595 conjugate (1/200 dilution, Molecular Probes, Eugene, Ore) as the secondary antibody. To show caspase 3 localization, the specimens were incubated with the polyclonal goat anti-caspase 3 and then with anti-goat Alexa Fluor-488 conjugate (1/200 dilution, Molecular Probes). To visualize nuclei, the specimens were stained with 50 g/mL of 4,6-diamidino-2-phenylindole (Sigma Chemical), in which 100 g/mL of DNase-free RNase A was added to facilitate the examination of nuclei with a BX51 fluorescence microscope (Olympus).
STATISTICAL ANALYSIS The Student t test and Kruskal-Wallis test were used to analyze differences in the incidence of apoptosis and proliferation between the mean values in the various data sets. A P value less than 0.05 was considered to indicate statistically significant differences.
RESULTS PERCENT AREA DENSITY The average percent area density of epithelium, stroma, and lumen was 14.4% ⫾ 4.1%, 75.5% ⫾ 10.7%, and 10.1% ⫾ 5.9 %, respectively. The ratio of stroma to epithelium ranged from 4.6:1 to 6.3:1 (average 5.2:1) in BPH specimens. The percent area density of epithelium and stroma was not related to patient age or prostate volume. UROLOGY 68 (4), 2006
FIGURE 1. Observations of immunostaining and TUNEL staining in BPH. Original magnification ⫻100. (A) Ki-67positive cells in epithelium and stroma of BPH. (B) TUNEL-positive cells in epithelium and stroma of BPH. (C) Intense staining for Bcl-2 in epithelial cells of BPH. (D) Strong staining for caspase 3 in epithelium of BPH.
APOPTOTIC AND PROLIFERATION INDEXES Both TUNEL-positive cells and Ki-67-positive cells were detected in the glandular cells and stromal cells of BPH (Fig. 1A,B). The average PI and AI were 1.8 and 0.7 in the epithelium and 1.1 and 0.2 in the stroma of BPH tissues, respectively. Bcl-2 AND CASPASE 3 EXPRESSION The detection of Bcl-2 and caspase 3 expression was limited to the cytoplasm of the glandular epithelium in BPH (Fig. 1C,D). Of 60 BPH specimens, Bcl-2 and caspase 3 expression was detected in 50 (80%) and 48 (75%), respectively. Double-label immunofluorescence analysis revealed co-expression of Bcl-2 and caspase 3 in the serial sections of BPH specimens that had already shown either Bcl-2 or caspase 3 expression, respectively (Fig. 2). PERCENT AREA DENSITY AND CELL KINETICS A greater stromal PI was closely related to the percent area density of stroma in the BPH specimens (P ⬍0.01), and a significantly reverse relationship was noted between a greater epithelial AI UROLOGY 68 (4), 2006
and the percent area density of epithelium in the BPH specimens (P ⬍0.05; Fig. 3). The AI and PI were not related to patient age or prostate volume of BPH (P ⬎0.05). The expression of Bcl-2 and caspase 3 was not related to the PI, AI, or percent area density of the BPH components (P ⬎0.05). COMMENT It is known that human BPH is the result of proliferation of the stromal and epithelial compartments. This proliferation begins in the stroma, with proliferation of the glandular epithelium secondarily induced.1 A number of studies have shown close associations between the development and severity of clinical BPH and the cellular composition of the prostate.17,18 It is clear that the proportion of stromal components is absolutely greater than that of epithelial compartments in human BPH. However, it is unknown whether the cell kinetics in the proportion of stromal or epithelial components play different roles in the pathogenesis and development of BPH. 907
FIGURE 2. Double-label fluorescent immunostaining of Bcl-2 and caspase 3 in BPH tissue sections. Original magnification ⫻100. (A) Bcl-2 staining (green signal); (B) caspase 3 staining (red signal); (C) 4,6-diamidino-2phenylindole nuclear staining (blue signal); and (D) co-localization of Bcl-2 and caspase 3 (yellow signal).
Recently, scientific interest has focused on the reduced cell death theory in the prostate. It has been suggested that an imbalance between apoptosis and cell proliferation may result in the development of BPH.2 No doubt exists that the process of prostate growth, from natural growth into BPH, has been associated with increased cell proliferative activities. Regarding the frequency of apoptosis in human BPH, either increased or decreased apoptotic rate has been noted in the epithelium compared with normal tissues.2– 4 In agreement with previous studies, our quantitative image analysis study showed that the average ratio of stromal/epithelial components was 5.2, and the PI/AI ratio was 2.6 in the epithelium but 5.5 in the stroma of BPH specimens. These results may explain the continuous increase in BPH volume, particularly in the stromal compartments. Furthermore, our study demonstrated that a greater stromal PI was closely related to the percent area density of the stroma, and a greater epithelial AI was inversely related to the percent area density of the epithelium in BPH. It has been indicated that the development of BPH may be associated with both stromal growth, due to active mesenchy908
mal-stromal cell proliferation, and epithelial increment, due to reduced glandular apoptosis. Both Bcl-2 and caspase 3 are known to be involved in the regulation of apoptosis. In normal prostatic tissue, bcl-2 and caspase 3 expression was either undetectable or only weakly stained epithelial cells were observed.19,20 However, either bcl-2 or caspase 3 was strongly expressed in BPH and prostate cancer tissues.3,13–15 In accordance with these findings, our data showed that the expression of Bcl-2 and caspase 3 was frequently detected in the epithelial cells of the BPH specimens. Doublelabel immunofluorescent staining revealed that both caspase 3 and bcl-2 proteins were almost always co-expressed in the epithelial cells of the BPH tissue. Although the expression of bcl-2 and caspase 3 was not related to the frequency of cell proliferation and apoptosis in BPH, the present results may reflect the situation of lower apoptosis induction in BPH. The increased bcl-2 expression may balance against the death cascade by inhibiting the release of cytochrome c from the mitochondria that activate caspases. Therefore, it is suggested that the situation of a lower apoptosis rate in UROLOGY 68 (4), 2006
% of stroma
gen-dependent proliferation of prostatic ducts and surrounding stromal tissue. Many factors have been suggested to be involved in disease development, including the growth factors and hormonal activities of dihydrotestosterone and estrogen.21–23 It is clear that the level of androgens is lower with increasing age in men. However, neither our data nor the findings of previous studies have shown relationships between cell kinetics and patient age in patients with BPH.2,3
100 80 60 40 20
CONCLUSIONS 0
PI in stroma A
% of epithelium 30 25 20
This is the first study to examine correlations of apoptosis and apoptosis-associated factors with the percent area density of epithelial and stromal components in BPH. Our data showed a close relation of a greater PI to the percent area density of stroma and an inverse relation of a greater AI to the percent area density of epithelium in BPH tissue. The development of BPH may be associated with both stromal growth, due to active mesenchymalstromal cell proliferation, and epithelial growth, due to reduced glandular apoptosis.
15 10 5 0
AI in epithelium B FIGURE 3. Association study of cell kinetics with percent area density of stroma and epithelium in BPH. (A) Positive relation between greater PI and percent area density of stroma in BPH (P ⬍0.01). Average percent area density of stroma in three different PI groups was 72.1% ⫾ 2.514%, 75.0% ⫾ 3.590%, and 76.8% ⫾ 2.486%. (B) Inverse relation between greater AI and percent area density of epithelium in BPH (P ⬍0.05). Average percent area density of epithelium in three different AI group was 17.1% ⫾ 3.381%, 14.5% ⫾ 3.719%, and 11.6% ⫾ 3.373%.
BPH may be related to alterations in the balance between bcl-2 and caspase 3 expression. Regarding the detection of apoptosis in the stroma of BPH, an extremely low frequency of apoptosis or an absence of apoptotic cell death has been found in the stroma of human BPH.2,3 Our study also showed a lower rate of apoptosis in the stroma of BPH tissue. Because of an undetectable expression of bcl-2 and caspase 3 in the stromal compartments, the induction of apoptosis in the stromal cells may be regulated by life span instead of apoptotic signals such as occurs in the epithelial cells of BPH tissue. BPH is characterized by androUROLOGY 68 (4), 2006
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