Introduction:Benign prostatic hyperplasia (BPH) is a slowly progressive abnormal glandular enlargement with heterogeneous morphology. Disruption of apoptotic pathways has been suggested as an important regulatory mechanism in this common and significantly morbid disease.
Methods:Prostatic tissue from 20 patients with BPH and no prior or subsequent prostatic carcinoma was obtained by transurethral prostatectomy (TURP) at the University of California Davis. Apoptotic regulatory proteins: BCL2, BAX and p27 were analyzed by immunohistochemistry and evaluated for expression in four distinct histologic patterns: hyperplastic epithelium, nodules, dilated glands and atrophic/inflammatory glands.
Results:Particularly striking was the decreased expression of BAX and an abnormal BCL2 : BAX ratio within all nodules relative to expression in other epithelial patterns. p27 expression was decreased in 35% of the hyperplastic epithelial areas and 10% of the nodules.
Discussion:Overall, abnormal expression of BCL2, BAX and/or p27 was identified in the hyperplastic epithelium of 19 (90%) of specimens and all 12 (100%) of the hyperplastic nodules. The high frequency of abnormalities in apoptosis regulatory genes, suggests that alteration of apoptotic pathways is important for the development of this condition.
Benign prostatic hyperplasia (BPH) is a slowly progressive, abnormal enlargement that affects 50% of US men by the age of 50–60 y and 90% of men in their 80s.1 The enlarging gland results in significant urinary obstruction and frequently requires surgical and/or medical intervention to restore urinary health. It is estimated that BPH has an annual cost of $4 billion per year in the US.1
BPH is characterized by an increase in both epithelial and stromal components of the prostate and has a heterogeneous morphology with-mixture of hyperplastic, atrophic and dilated glands and increased stromal smooth muscle and collagen. Chronic inflammatory changes with a variable lymphocytic infiltrate are frequently observed. The most dramatic of the histologic changes is the formation of 1 mm–1 cm-sized nodules of hyperplastic glands.
We hypothesized that disruption of cell cycle and apoptotic pathways would be evident by abnormal protein expression in histologic sections of BPH. This study involved investigating the expression of Bcl-2, Bax and p27/Kip1 in BPH as detected by immunohistochemistry (IHC). We also described the differential expression of these proteins in distinct histopathologic patterns of BPH, that is, expression in hyperplastic, nonhyperplastic and nodular epithelial areas. In this work, we examined the morphologic pattern of multiple apoptosis regulators to generate an initial profile of genes involved in the complex pathways of cell cycle and apoptosis regulation in benign prostatic hyperplasia.
Prostatic tissue from 38 patients with BPH and no prior or subsequent prostatic carcinoma (CaP) was obtained by transurethral prostatectomy (TURP) at the University of California Davis. The patients had all given IRB- approved informed consent and were randomly selected. Apoptotic regulatory proteins: Bcl-2, Bax and p27 were analyzed by IHC and evaluated for expression in four distinct histologic patterns : hyperplastic epithelium, nodules, dilated glands and atrophic/inflammatory glands.
A nodule was defined as a cluster of hyperplastic glands having a circumscribed border. Immunohistochemistry was performed using standardized methods (#2) on 4-μm formalin-fixed, paraffin-embedded tissue sections from archival blocks using standardized avidin–biotin techniques. The commercial monoclonal antibodies are outlined in Table 1.
Duplicate sections subjected to all phases of staining except the primary antibody served as negative controls. Positive controls were paraffin-embedded sections of lymph node for Bax and Bcl-2 and a known positive CaP for p27. Expression of p27 was evaluated by two monoclonal antibodies with good concordance. Initially, we tested 15 samples with clone F-8 (Santa Cruz Antibodies) and after reviewing the findings of other investigators, we tested all 35 samples with clone DCS72 (Oncogene Science).
H&E-stained sections were analyzed by light microscopy for the presence and numbers of defined nodules as well as the percentage proportion of four distinct histologic areas : stroma, hyperplastic epithelium, atrophic and/or inflamed epithelium and dilated glands.
Expression of the three apoptotic regulatory proteins was analyzed by light microscopy in each histologic region as well as in the defined nodules. Protein expression within an individual cell was defined as uniform nuclear staining for p27 and cytoplasmic staining for Bax. For Bcl-2, both nuclear and/or cytoplasmic staining was included. The histologic regions were then rated as abnormal for protein expression utilizing the criteria listed in Table 1.
A data analysis of abnormal protein expression by age group utilized a two-sided Fisher's exact test to examine the statistical significance. The Fisher's exact test was used rather than the usual χ2-test because of the small sample size.
Benign prostatic hyperplasia showed marked histologic heterogeneity (Table 2). In all, 25 (71%) specimens had all four histopathologic patterns : hyperplastic epithelium, dilated epithelium, inflamed/atrophic epithelium and stroma. The proportion of these patterns within a given specimen was quite variable. The stromal component ranged from 20 to 70% with an average of 37%. Hyperplastic epithelium comprised 0–70% with an average of 27%. A total of 33 discrete hyperplastic nodules were found in 21 specimens, thus the majority of hyperplastic epithelium was in a non-nodular distribution. Dilated glands constituted 0–50% of tissues with an average composition of 17%, while the pattern of inflamed and/or atrophic glands comprised 0–55% of specimen tissues and averaged 19% per specimen.
Overall (Table 3), abnormal expression of at least one cell cycle or apoptotic regulatory gene (BCL2, BAX and/or P27/KIP1) was identified in the secretory cells of hyperplastic epithelium of 27 (77%) of the BPH specimens and 21 (75%) of the hyperplastic nodules. Particularly striking was the decreased expression of Bax resulting in an increased Bcl-2 : Bax ratio within most hyperplastic nodules relative to expression in other epithelial patterns (Figure 1). Abnormal or decreased Bax was noted in 25 (72%) of the hyperplastic epithelial cells and in 20 (71%) of the hyperplastic epithelial nodules. Decreased Bax was present in all dilated glands and most areas of inflamed/atrophic epithelium (89%).
The patients ranged in age from the youngest at 52 y to the oldest at 82 y with a median age of 71 y. Three patients were under 60 y, 13 between 60 and 69 y, 17 between 70 and 79 y and two over 80 y. There was no statistical significance to the distribution of hyperplastic nodules or to the ratio of stroma to epithelium over age. There was, however, an interesting decrease in the frequency of nodules in the 60–69 y group, which also had a high stromal : epithelial ratio. Overall, 83% of patients had abnormal Bax and/or Bcl-2 expression and 34% had abnormal p27 expression. There was no significant difference in the proportion of the Bax : Bcl-2 abnormalities among the age groups. This generally held when considering Bax or Bcl-2 abnormalities separately as well. Although the proportion of patients with p27 abnormalities is slightly higher for patients under 70 y compared to those 70 y or older, this did not achieve statistical significance (P=0.42). However, an interesting finding was that no p27 abnormalities were found in patients who had normal Bax and Bcl-2 expression, while 41% of patients with Bax : Bcl-2 abnormalities had p27 abnormalities. This trend approaches statistical significance with a P-value of 0.07 (Figure 2).
The etiology of BPH is uncertain but one hypothesis proposes that this paradoxical growth in the aging prostate is the result of growth stimulatory factors.2, 3 Measurements of proliferation in BPH, analyzing Ki-67 (MIB-1) expression by IHC, have shown rates only slightly higher than normal prostate tissues.4, 5, 6, 7 This modestly increased proliferation is seemingly insufficient to explain the exuberant glandular enlargement, apparent in 60% of patients with BPH, which results in such profound clinical symptoms.
Programmed cell death or apoptosis is an important negative regulator of tissue growth by its efficient, noninflammatory process of eliminating damaged and/or senescent cells. Disruption of apoptosis leads to a pathological accumulation of cells and a perpetuation of abnormal gene expression. Major regulatory apoptotic proteins that have been identified include the antiapoptotic factor, Bcl-2 and the proapoptotic factor, Bax. These are cofunctional entities whose ratio determines whether stressed cells are protected or eliminated by apoptosis. In normal prostate epithelium, Bcl-2 is observed only in basal cells, which is consistent with their function to serve as a stem cell component within prostatic acini. BPH epithelium shows a similar phenotype with consistent basal cell expression of Bcl-2. However, benign prostatic epithelium that has been subjected to various stresses such as radiation therapy or androgen ablation demonstrate strong expression of Bcl-2 in the surviving cells, which probably functions to block apoptosis induced through the p53 pathway.8, 9 Our prior study of Bcl-2 expression in benign and malignant prostatic tissues subjected to androgen ablation demonstrated a 30% incidence of Bcl-2 overexpression in the secretory cell layer, which may contribute to cell survival and to gland enlargement.10
Kyprianou has investigated the role of Bcl-2 in benign prostatic hyperplasia, and reported expression of the antiapoptotic Bcl-2 protein concurrently with decreased apoptosis in BPH compared to normal prostatic epithelium.6 Bax has been recognized as a potent proapoptotic regulator and the intracellular balance of Bax and Bcl-2 described as a ‘rheostat’ of apoptosis sensitivity in that the ratio of these proteins influences a cell's ability to respond to apoptotic signals.11, 12, 13 Both Bax and Bcl-2 are noted to form homo- and heterodimers and this ratio of these dimeric forms propels the cells towards survival or apoptosis.11 Two investigators have assessed the expression of BAX and Bcl-2 in in vivo models of castration-induced prostate apoptosis. Perlman et al14 observed a transient but marked increase in the BAX : Bcl-2 gene expression ratio in prostatic tissue that peaked on the 2nd and 3rd days after castration of adult male rats.14 Bruckheimer et al15 reported that modulation of Bax and Bcl-2 each might contribute to androgen withdrawal-induced apoptosis in prostatic glandular cells and suggested that the presence of Bcl-2 may be a more important indicator than a deficiency of Bax.15 The significance of the Bax : Bcl-2 ratio in BPH has not been determined and the effect, if any, of dimerization on IHC results is unknown.
Other mechanisms of apoptosis are likely to play an equally important role in the maintenance of cellular populations. The cyclin-dependent kinase inhibitor p27/Kip1 is a negative regulator of the transition from G1 to S phase of the cell cycle. Evidence for the role of p27 in prostate disease includes the observation that knockout mice with a p27−/− genotype demonstrate histologic benign prostatic hyperplasia.16 Kyprianou's group has shown loss of p27 in prostatic carcinoma as well as the important observation that this loss is associated with higher proliferative activity and higher tumor grade.17 Cordon-Cardo et al18 have demonstrated loss of p27 in prostatic carcinoma, as well as diffuse p27 staining in normal prostate epithelium and a distinct absence of staining in BPH nodules.
Our study found increased Bcl-2 expression in 17% of hyperplastic regions and only 7% of distinct nodules suggesting that abnormal expression of this protein may be important in only a small subset of patients with BPH. Only one dilated gland had increased Bcl-2 while none were abnormal for Bax or p27, which is consistent with the interpretation that these are essentially inactive glands with little potential for renewed growth. The atrophic and inflamed epithelial cells, in contrast, showed strong Bcl-2 expression with a generally decreased or absent Bax expression and virtually no p27 expression that may represent a mechanism for cell survival during the stress of inflammation.
Renewed interest in the interaction of inflammatory stress and the prostate has focused on the role of inactivation of the π-glutathione S transferase gene (GSTP1) via somatic ‘CpG island’ methylation changes. The hypothesis is that chronic inflammation results in oxidative stress via free radical formation. This may result in genomic damage and lead to neoplasia.19 The findings of increased methylation abnormalities in prostatic inflammatory atrophy (PIA), clearly offer a link between a subset of patients with BPH to PIN and prostatic cancer. We hypothesize that similar mechanisms may be functioning in benign prostatic hyperplasia with resulting alteration of apoptotic mechanisms and we will explore this in future studies.
Bax expression is generally ubiquitous throughout both secretory and basal cells of normal prostate. The most striking of our findings was the high frequency of loss of Bax expression in hyperplastic epithelium, particularly within hyperplastic epithelial nodules. This dramatically altered Bcl-2 : Bax ratio suggests that this epithelium is resistant to apoptosis and has the capability for continued expansion. This reinforces the view that in the evaluation of apoptosis, it is necessary to examine expression of both the positive and negative regulators.20 While approximately one-third of our patients showed decreased staining for p27 in hyperplasia, the effect was more prominent in defined nodules (43%), which is consistent with the findings of other investigators.18
The process of apoptosis protects the integrity of the genome by eliminating cells with extensive DNA damage or those undergoing uncontrolled proliferation.11 For instance, during development, RB knockout mice did not experience uncontrolled cellular growth as expected because apoptotic processes eliminated abnormally cycling cells.21 Similarly, transfection of the G1 cell cycle regulator Cyclin D1 into rat embryo cells, which also disrupts the RB pathway, resulted in increased apoptosis.20 To circumvent these protective feedback loops, hyperplastic and potentially malignant cells of many tissue types must acquire defects in apoptotic pathways. Thus, loss of both the apoptosis-regulatory machinery and proliferative control are common occurrences in cancer and it may be that apoptosis abnormalities must occur prior to or at the same time as cell cycle abnormalities. The data presented here are consistent with the paradigm that mutations that disrupt apoptotic processes precede or occur simultaneously to disruption of cell cycle regulatory control in BPH.
p27, a cyclin-dependent kinase inhibitor, has a significant role in the control of cell cycling. A decrease of p27 is required for activation of cyclin-dependent kinase 2 (Cdk2) which regulates progression from late G1 into the S phase of the cell cycle. Thus, alterations in apoptosis-associated genes such as BCL2 and BAX are likely to precede or occur simultaneously with cell cycle gene alterations such as p27. Consistent with this paradigm, although not reaching statistical significance (=0.07), no patient sample in this study had p27 abnormalities without the occurrence of a Bax or Bcl-2 abnormality. However, p27 alterations that increase proliferation and occur after a disrupted Bcl-2 : Bax ratio may result in more florid disease and/or clinical symptoms at a younger age. Consistent with this hypothesis was the finding of a higher proportion of patients under 70 y of age who had abnormal p27 expression.
The elucidation of disrupted apoptosis regulation in benign prostatic hyperplasia may have significant implications for therapeutic intervention. For example, Kyprianou et al22, 23 have reported that α1-adrenoreceptor antagonists induce apoptosis in BPH without affecting cell proliferation. There may be value in therapy that focuses more directly on the regulators of apoptosis :Bcl-2, Bax and p27 for an enhanced tissue shrinkage and relief of obstructive symptoms.
Now that we have confirmed the occurrence of molecular abnormalities in BPH and that these differences are altered in the different patterns of BPH, more extensive studies will follow. While Bcl-2 and Bax are the classic markers of apoptotic control, additional markers such as survivin are worthy of further investigation. Survivin has been described in a number of urologic conditions and may be operational in BPH.24 Recently, the overexpression of the Skp2 protein has been associated with a decreased p27 expression in prostate cancer.25 It is hypothesized that this protein may affect the cell cycle by degrading p27. We intend to explore the expression of both of these proteins in our future investigations of BPH.
Although we have focused on epithelial cell abnormalities, it is important to acknowledge that a significant proportion of gland enlargement is due to an increase in smooth muscle and connective tissues. Our patients had an average stromal component of 40%. An examination of the regulation of stromal cell proliferation and apoptosis may need to be addressed to comprehensively treat BPH.
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Our work was supported by a grant from the National Institute for Aging (AG15404).
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Gandour-Edwards, R., Mack, P., deVere-White, R. et al. Abnormalities of apoptotic and cell cycle regulatory proteins in distinct histopathologic components of benign prostatic hyperplasia. Prostate Cancer Prostatic Dis 7, 321–326 (2004). https://doi.org/10.1038/sj.pcan.4500749
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