The receptive endometrium is characterized by apoptosis in the glands

Apoptosis in the human endometrium up to now has been detected during the mid to late luteal phase and therefore connected to the onset of the menstrual shedding. However, there is increasing evidence that regulated apoptosis may be important during decidualization and implantation. To investigate a possible role for apoptosis in the human endometrium and its regulation, we correlated the immunolocalization of the apoptosis regulatory protein bcl-2 and the proliferation marker Ki67 to the in-situ nuclear DNA fragmentation - a key feature of apoptosis - detected by using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL) method during the menstrual cycle. Whereas proliferation and bcl-2-expression were predominantly detected in the glandular compartment during the proliferative phase, only single apoptotic cells could be shown during this period. During the transformation of the endometrium (days 15-19) proliferation and bcl-2 expression decreased markedly and there was no sign of apoptosis. At the beginning of the implantation window (days 19-20) we could detect the first signs of apoptosis in the glandular epithelia in the basalis, which extended to the functionalis during the luteal phase. Proliferation and bcl-2 expression are limited to the stromal compartment comprising the large granular lymphocytes - during this time, and extend in parallel with apoptosis from the basal to the functional layers. Apoptosis therefore may be related to the loss of the protective effect of bcl-2 and may have significance for the establishment of an endometrium adequately prepared for successful implantation.     

Extended survivability of prostate cancer cells in the absence of trophic factors: increased proliferation, evasion of apoptosis, and the role of apoptosis proteins

This project was undertaken to study the survival properties of various prostate cells, including normal (NHP), BPH (benign prostate hyperplasia), primary carcinoma (PCA), and metastatic prostate cancer cells (LNCaP, PC3, and Du145), in the absence of trophic factors. Cell proliferation and cell death were quantitated by enumerating the number of live cells using MTS/PMS kit and of dead (apoptotic) cells using 4',6-diamidino-2-phenylindole dihydrochloride nuclear staining. These cells demonstrated an overall survivability in the order of BPH < NHP < LNCaP < PC3 < PCA < Du145. Upon growth factor deprivation, NHP/BPH cells rapidly underwent apoptosis, leading to a decreased number of live cells. PCA/PC3/Du145 cells, in contrast, demonstrated an initial phase of aggressive growth during which apoptosis rarely occurred, followed by a "plateau" phase in which cell loss by apoptosis was compensated by cell proliferation, followed by a later phase in which apoptosis exceeded the cell proliferation. LNCaP cells demonstrated survival characteristics between those of NHP/BPH and PCA/PC3/Du145 cells. We concluded that the increased survivability in prostate cancer cells results from enhanced cell proliferation as well as decreased apoptosis. The molecular mechanisms for evasion of apoptosis in prostate cancer cells were subsequently investigated. Quantitative Western blotting was used to examine the protein expression of P53 and P21WAF-1, Bcl-2 and Bcl-X(L) (anti-apoptotic proteins), and Bax, Bak, and Bad (proapoptotic proteins). The results revealed that, upon trophic factor withdrawal, NHP and BPH cells upregulated wild-type p53 and proapoptotic proteins Bax/Bad/Bak and down-regulated the expression of P21. Furthermore, NHP and BPH cells endogenously expressed little or no Bcl-2. In sharp contrast, prostate cancer cells expressed nonfunctional P53 and various amounts of Bcl-2 proteins. Upon deprivation, these cancer cells up-regulated P21 and Bcl-2 and/or BclX(L), lost response to withdrawal-induced up-regulation of Bax/Bad/Bak or decreased or even completely lost Bax expression and expressed some novel proteins such as P25 and P54/55 complex. These data together suggest that prostate cancer cells may use multiple molecular mechanisms to evade apoptosis, which, together with increased proliferation, contribute to extended survivability of prostate cancer cells in the absence trophic factors.     

Increased apoptosis accompanies neoplastic development in the human colorectum

A disturbance in the balance between cell proliferation and cell loss, or apoptosis, may underlie neoplastic development. Therefore, we determined spontaneous apoptotic and proliferative rates in normal, hyperplastic, adenomatous, and malignant colorectal epithelia. In paired sections, DNA strand breaks were detected using the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay, and apoptotic cells were also identified in H&E-stained slides by morphological criteria. Cell proliferation, bcl-2, and p53 expression were analyzed using specific monoclonal antibodies. In normal mucosa, luminal epithelial cells demonstrated higher rates of apoptosis compared to cells in the proliferative zone. Neoplastic transformation was associated with a significant increase in rates of apoptosis and proliferation. However, apoptosis, but not proliferation, decreased at the adenoma-to-carcinoma transition coincident with expression of mutant p53. In carcinomas, both mutant p53 and bcl-2 protein levels were associated with attenuated apoptotic rates. In conclusion, apoptosis is an important regulator of growth in normal and neoplastic colorectal epithelia. Increased apoptosis and proliferation accompany neoplastic transformation, suggesting that an alteration in apoptotic rates is an important event in colorectal carcinogenesis. Furthermore, the imbalance in these processes found in carcinomas may facilitate tumor growth and progression.     

T-cell-rich large-B-cell lymphomas contain non-activated CD8+ cytolytic T cells, show increased tumor cell apoptosis, and have lower Bcl-2 expression than diffuse large-B-cell lymphomas

The factor(s) responsible for the reduced B cell number and increased T cell infiltrate in T-cell-rich large-B-cell lymphomas (TCRBCLs) have not been well characterized. We studied 18 TCRBCLs and 12 diffuse large-B-cell lymphomas (DLBCLs) to compare the 1) predominant T cell subpopulation(s), 2) expression of cytotoxic granule proteins (TIA-1 and granzyme B), 3) level of tumor cell apoptosis (Apoptag system, Oncor, Gaithersburg, MD), and 4) expression of Ki-67 (Mib-1) and apoptosis-related proteins (fas (CD95), bcl-2, and p53). T cells in TCRBCLs and DLBCLs were predominantly CD8+ T cells expressing alphabeta T-cell receptors and TIA-1 (16 of 18 TCRBCLs with >50% TIA-1+ small lymphocytes) but lacking granzyme B (16 of 18 TCRBCLs with <25% granzyme B+ small lymphocytes). Scattered apoptotic tumor cells (confirmed with CD20 co-labeling) were present in 15 of 18 TCRBCLs, with 14 of 15 cases having <10% apoptotic cells. No apoptotic cells were seen in 12 of 12 DLBCLs. In 16 of 16 immunoreactive TCRBCLs, <25% tumor cells were bcl-2+, whereas 6 of 12 DLBCLs had >50% bcl-2+ tumor cells. CD95 (fas) expression was also lower, with 3 of 18 (16.7%) TCRBCLs versus 4 of 12 (33%) DLBCLs having >25% CD95+ tumor cells. TCRBCLs and DLBCLs had similar levels of p53 and Ki-67 (Mib-1) expression. Thus, T cells in TCRBCLs are non-activated cytotoxic T lymphocytes (TIA-1+, granzyme B-). Tumor cell apoptosis (perhaps cytotoxic T cell mediated) may partly account for the decreased number of large (neoplastic) B cells in TCRBCLs, but other factors (ie, decreased bcl-2 expression) may also be needed.     

Exchange of Ser-4 for Val, Leu or Asn in the sequon Asn-Ala-Ser does not prevent N-glycosylation of the cell surface glycoprotein from Halobacterium halobium

The aim of the present study was to gain a better understanding of the localization of apoptotic cells within the human endometrium during the menstrual cycle and to elucidate the relationships among the following for the human endometrium: apoptosis, p21 expression, and cell proliferation. Apoptosis and p21 expression were identified mainly in the glandular cells of the basal layer in the late secretory phase. In contrast, cells positive for Ki-67 were observed predominantly in the functional layer (in the proliferative phase in glandular cells and in the secretory phase in stromal cells). A very strong positive correlation (r = 0.81; P < 0.001) was demonstrated between the number of apoptotic cells and the number of p21-positive cells present among the glandular cells but, topographically, individual apoptotic cells were not coincident with p21-positive cells in serial sections. The results of this study suggest that the proliferation of the glandular cells of the basal layer is regulated by both apoptosis and p21 expression, particularly in the late secretory phase. Such regulation may be necessary to maintain a healthy population of glandular cells in the basal layer of the endometrium.     

Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo

Normal (nonneoplastic) human prostatic secretory epithelial cells do not express the bcl-2 protein. However, a recent immunohistochemical survey of neoplastic human prostate tissues showed that a fraction of primary untreated prostate adenocarcinoma cells expressed this apoptosis-suppressing oncoprotein at significant levels (Colombel et al., Am. J. Pathol., 143: 390-400, 1993). Additionally, a number of hormone-refractory prostatic adenocarcinomas obtained from hormonally-treated patients (subsequent to surgical or drug castration therapy) were examined and were found to be uniform in their elevated expression of bcl-2 oncoprotein. The results of this preliminary survey imply that bcl-2 expression distinguishes a subgroup of primary human prostate cancers and that the expression of this protein might be a factor enabling prostate cancer cells to survive in an androgen-deprived environment. The current study was undertaken to determine the degree to which overexpression of bcl-2 can protect human prostate cancer cells from apoptotic stimuli in vitro and in vivo. Human prostate cancer cells (LNCaP) were transfected with a neomycin-selectable eucaryotic expression vector containing cDNA encoding human bcl-2. Transfected clonal variants that express bcl-2 protein (LNCaP/bcl-2) were unaltered with regard to their basal growth rate in 10% serum-containing medium, or with regard to their expression of the differentiated human prostate cell gene products prostate-specific antigen or androgen receptor protein. The bcl-2-transfected clones were altered, however, with regard to their growth rate in charcoal-stripped serum lacking dihydrotestosterone. Additionally, in contrast to the parental or control-transfected cell lines, LNCaP/bcl-2 cells were highly resistant to a variety of apoptotic stimuli in vitro including serum starvation and 10 nM phorbol ester (phorbol 12-myristate 13-acetate) supplementation of the medium. Lastly, the overexpression of bcl-2 by these prostate cancer cells altered their tumorigenic potential in a nude mouse assay. s.c. injections of 10(6) LNCaP/bcl-2 cells into male nude mice resulted in earlier and larger tumor formation compared to an equivalent injection of parental or control-transfected LNCaP cells. When these variant cell lines were injected into castrated male nude mice, only the LNCaP/bcl-2-transformed cells gave rise to tumors. Moreover, LNCaP/bcl-2 tumors grown in intact male nude mice were refractory to the growth-inhibiting effects of castration demonstrated by parental LNCaP cells. Data obtained in this study demonstrate that the bcl-2 oncoprotein can protect prostate cancer cells from apoptotic stimuli in vitro and suggest that such protection correlates with the ability to form hormone-refractory prostate tumors in vivo.     

Secretion of insulin-like growth factors and their binding proteins by human normal and hyperplastic prostatic cells in primary culture

Benign prostatic hyperplasia (BPH) is the most common benign proliferative disorder of unknown etiology found in men. Because insulin-like growth factors (IGFs) with their binding proteins (IGFBPs) are involved in the control of cellular proliferation, differentiation, and metabolism, we compared their secretion by prostatic epithelial and stromal cells in primary culture from the four different zones of normal prostate and from hyperplastic tissue to assess their contributions to the hyperplastic development. IGF-I could not be detected in the conditioned medium from either epithelial or stromal cells from normal and BPH tissues. IGF-II concentrations were the same in the conditioned medium from the epithelial cells of the different zones of the normal prostate and that of BPH cells. IGF-II concentrations secreted in stromal cell culture medium, however, were higher in the periurethral zone than in the peripheral and central zones. Moreover, in the periurethral zone, stromal cells secreted higher concentrations of IGF-II than did epithelial cells. Also, BPH stromal cells secreted more IGF-II than did BPH epithelial cells. IGFBP-3, IGFBP-2, and IGFBP-4 were all secreted by both epithelial and stromal cells. In contrast, IGFBP-5 was only produced by stromal cells of the periurethral zone of the normal prostate and BPH tissue. IGFBP-3 was predominantly secreted by normal stromal cells of the transitional zone. We observed that BPH stromal cells presented the same pattern of IGF-II and IGFBP production as normal stromal cells of the periurethral zone. These data support the hypothesis that the periurethral zone is the main region of the prostate implicated in the development of BPH. They also suggest that the variability in both IGF-II secretion and the secreted forms of IGFBPs, depending on anatomical location within the organ, may be important for the autocrine regulation of normal and hyperplastic prostate growth.     

Expression of bcl-2, p53 and Ki-67 in arsenical skin cancers

To investigate the regulation of apoptosis and proliferation in arsenic-induced skin cancers, we examined the expression of bcl-2, p53, and Ki-67 using immunohistochemical staining. Thirty patients with Bowen's disease (BD), ten with basal cell carcinoma (BCC), eight with squamous cell carcinoma (SCC) and eleven of perilesional normal skin (PLN) of the non-sun exposure sites from endemic area were examined. The results showed that: 1) bcl-2 was expressed in all of the BCC homogeneously, in none of the SCC, and in 12/30 of the BD focally or homogeneously; 2) p53 was expressed in all of the arsenical skin cancers with a labelling index of 75 +/- 14% of BD, 50 +/- 17% of BCC, 61 +/- 15% of SCC, and also in all of the perilesional normal skin with a labelling index of 55 +/- 24%; 3) Ki-67 was expressed in all of the skin cancers with labelling index of 58 +/- 17% of BD, 12 +/- 7% of BCC, 47 +/- 21% of SCC, and in 9/11 of PLN with a labelling index of 41 +/- 24%. Expression of bcl-2 in BCC or BD is related to the phenotype of germinative basal cell. The constant expression of bcl-2 i early dysplastic cells of BD and the earliest expression of P53 in the basal cells of perilesional normal skin indicate that the initial step of arsenic-induced carcinogenesis is from the basal germinative cells. There is no mutual relationship between bcl-2, p53 or Ki-67 expression in any type of the arsenical skin cancers, but there is a positive correlation between p53 and Ki-67 expression identified in perilesional normal skin. BD had the highest labelling index of p53 and Ki-67.     

Abnormal bcl-2 and pRb expression are independent correlates of radiation response in muscle-invasive bladder cancer

The objective of this study was to determine whether the overexpression of bcl-2, a key protein governing the apoptotic response to radiation, adds to pRb status in estimating the propensity for radiation response in patients with muscle-invasive bladder cancer. Archival formalin-fixed, paraffin-embedded, pretreatment bladder tumor samples were available in 109 of 301 patients treated preoperatively with 50 Gy in 25 fractions followed by radical cystectomy 4-6 weeks later. Radiation response was assessed by clinical-to-pathological tumor downstaging or upstaging. Altered expression of bcl-2 (47% of 107 patients), p53 (56% of 109 patients), and pRb (30% of 98 patients) was assessed by immunohistochemical staining. Morphological criteria were used to calculate the percentage of apoptotic cells. bcl-2 staining correlated with tumor grade; all grade 2 tumors (n = 7) displayed normal bcl-2 expression (negative staining). No correlations between bcl-2 staining and pretreatment apoptosis levels, p53 staining, and pRb staining were observed. In terms of the radiation response parameters, univariate analyses revealed that bcl-2 overexpression was the only factor associated with upstaging. The main predictor of downstaging was the loss of pRb expression (negative staining). Multivariate logistic regression confirmed these findings and also showed that normal pRb expression (positive staining) was significantly related to upstaging. Patient outcome was adversely affected by bcl-2 overexpression, because these patients experienced significantly increased actuarial local failure rates. No difference in distant metastasis or survival rates by bcl-2 staining was seen. The strongest independent correlates of radiation response thus far identified in muscle-invasive bladder cancer are from bcl-2 and pRb immunohistochemical staining. The overexpression of bcl-2 and the normal expression of pRb seem to thwart the apoptotic response to radiation via independent mechanisms. Abnormalities in the expression of proteins that regulate apoptosis may prove to establish a molecular phenotype to characterize which patients should receive radiotherapy.     

Development of a hammerhead ribozyme against bcl-2. I. Preliminary evaluation of a potential gene therapeutic agent for hormone-refractory human prostate cancer

BACKGROUND: The bcl-2 oncoprotein suppresses apoptosis and, when overexpressed in prostate cancer cells, makes these cells resistant to a variety of therapeutic agents, including hormonal ablation. Therefore, bcl-2 provides a strategic target for the development of gene knockout therapies to treat human prostate cancers. Towards this end, we have synthesized an anti-bcl-2 gene therapeutic reagent based on ribozyme technology and have tested its effectiveness against bcl-2 mRNA in vitro and in vivo. METHODS: A divalent hammerhead ribozyme was constructed by recombining two catalytic RNA domains into an antisense segment of the coding region for human bcl-2 mRNA. A disabled ribozyme lacking catalytic activity was also constructed as a control reagent for our experiments. The ribozymes were tested for endonucleolytic activity against synthetic and natural bcl-2 mRNAs. Simple transfection procedures were then utilized to introduce the ribozymes into cultured prostate cancer cells (LNCaP derivatives). We measured the effects of the ribozymes on endogenous expression of bcl-2 mRNA and protein in these cells as well as their ability to induce apoptosis. RESULTS: The functional but not the disabled ribozyme was able to rapidly degrade bcl-2 mRNA in vitro, without the requirement for any other cellular protein or factor. When directly transfected into LNCaP cell variants, it significantly reduced bcl-2 mRNA and protein levels within 18 hr of treatment. This activity was sufficient to induce apoptosis in a low-bcl-2-expressing variant of LNCaP, but not in a high-bcl-2-expressing LNCaP line. For the high-bcl-2-expressing variant, however, it did restore the ability to genetically respond to a secondary apoptotic agent, phorbol ester, as evidenced by the renewed ability of phorbol ester to induce NGF1A mRNA in these cells. CONCLUSIONS: This study supports the potential utility of an anti-bcl-2 ribozyme reagent for reducing or eliminating bcl-2 expression from hormone-refractory prostate cancer cells and for killing prostate cancer cells. As such, it is the first step toward an effective gene therapy against hormone-refractory human prostate cancers.     

Apoptotic regulation in primitive hematopoietic precursors

Bcl-2 and bcl-xL function as suppressors of programmed cell death. The expression of bcl-2 protein in vivo is associated with long-lived hematopoietic cells such as mature lymphocytes and early myeloid progenitors. Bcl-xL, a homologue of bcl-2, is also expressed in lymphocytes and thymocytes. In contrast, the bcl-2-related proteins (bax, bad, and bak) act by promoting apoptotic cell death as shown from their expression in hematopoietic cell lines. We analyzed the expression of bcl-2 and bcl-x proteins in hematopoietic precursors obtained from various cell sources in adult mobilized peripheral blood collected from 13 patients with solid tumors, 8 adult bone marrow, and 12 umbilical cord blood. The analysis was based on the expression of the proliferation and activation specific antigens, CD38 and class II (HLA-DR). Similarly, we analyzed the expression of bcl-2-related proteins bcl-xL, bax, bad, and bak before and during ex-vivo expansion. Hematopoietic precursors expressing strongly the CD34 antigen (CD34(s+)) and lacking CD38 or HLA-DR expression were analyzed by using three-color immunofluorescence staining. The majority of CD34(+) cells expressed bcl-2 and unexpectedly showed a bimodal distribution of low and high expression. More cells that lacked or expressed low density CD38 expressed low bcl-2 than the more differentiated counterparts (those with high density CD38). Immaturity (ie, little or no HLA-DR) is associated with the expression of low bcl-2 compared with HLA-DR+. However, HLA-DR-/low population contained a lower number of cells expressing low bcl-2 (30% to 40%) than CD38(-/low) in comparable samples. The hematopoietic precursors with bcl-2(low) and bcl-2(high) formed a homogeneous population of undifferentiated lymphoid-like cells having a similar forward scatter. These cells expressed strongly the bcl-xL protein (>95%) but were bax low (4% to 12%), bad low (0% to 0.8%), and bak low (0% to 3%). The expression of apoptosis specific protein (ASP) was also low (3.4% +/- 3.1%) as was Annexin V. In addition, the CD34(+)/CD38(-) showed low cell cycle activity (<2.2%). Induction of apoptosis by overnight incubation of CD34 cells in serum-deprived medium resulted in the upregulation of bcl-2 as a single population histogram. Thus, these results suggest that in quiescent hematopoietic precursors, the bcl-2 protein plays a less prominent role as a survival promoter than bcl-xL and that the low bcl-2 expression did not promote apoptosis. During day 10 of ex vivo expansion of CD34(+) cells in liquid culture containing stem cell factor, interleukin-3 (IL-3), IL-6, IL-1beta, and erythropoietin, the CD34(+)/CD38(-) cells expressed high bcl-2 as a single population histogram, and greater than 90% were bcl-xL high. However, the expression of pro- and apoptotic antigens increased: bax (10% to 15%), bad (5% to 8%), bak (6% to 14%), and ASP (6% to 10%). These results show the importance of monitoring the expression of these proteins when defining the culture conditions for ex vivo expansion.     

Distinction of basaloid carcinoma of the prostate from benign basal cell lesions by using immunohistochemistry for bcl-2 and Ki-67

The distinction of rare basaloid carcinomas (BC) of the prostate from more common basal cell hyperplasia may be difficult, because basal cell hyperplasia (BCH) may have prominent nucleoli and may appear infiltrative. Using immunohistochemistry, we studied bcl-2 and p53 expression and Ki-67 proliferation index in eight cases of typical BCH, eight cases of BCH with nucleoli, and six cases of BC. Bcl-2 expression (P < .0001) and Ki-67 index (P=.005) were elevated in BC compared with typical BCH or BCH with nucleoli, whereas there was no significant difference between typical BCH and BCH with nucleoli. P53 was not discriminative in separating benign from malignant basal cell lesions of the prostate. Bcl-2 may play a role in the pathogenesis of basal cell lesions of the prostate. Elevated expression of bcl-2 and higher Ki-67 index may aid in the diagnosis of basal cell proliferative lesions of the prostate.     

Detection of P2 precursors in the sperm cells of infertile patients who have reduced protamine P2 levels

Many chemotherapeutic agents are thought to exert their genotoxic effects through induction of programmed cell death (PCD) (apoptosis) in tumor cells. The bcl-2 is an anti-apoptotic oncoprotein and can confer a survival advantage to tumor cells by preventing apoptosis. Overexpression of bcl-2 may therefore be implicated in resistance to chemotherapy. We studied the significance of bcl-2 expression and the PCD index in pediatric acute lymphoblastic leukemia. Evaluation of bcl-2 by immunocytochemistry and PCD by an enzymatic end labelling technique using biotin-dUTP was carried out in a total of 55 cases and 40 controls. Bcl-2 was found to be expressed in 47% (26/55) of the acute lymphoblastic leukemia cases. The positive cells varied from 0-49% among individual samples. Pre-treatment (spontaneous) apoptosis was observed in 62% (34/55) cases. The mean pre-treatment PCD index was 8.27 1.3%, while the median PCD index was 5. The PCD value for the leukemic samples analyzed were then classified as either high apoptosis values ( 5) and low apoptosis values (<5). PCD index was high in 53% (29/55) and low in 47% (26/55). However, 23% (13/55) of cases did not show presence of either apoptosis or bcl-2. There was no association between clinical and laboratory parameters with the apoptotic index or bcl-2 protein expression. However, evaluation of apoptotic index and bcl-2 expression on day 7 of induction chemotherapy showed a borderline correlation between these markers and initial WBC count, presence of mediastinal mass and hepatosplenomegaly. Follow-up of these patients is being done to look for any association between treatment response and apoptosis.     

Evidence of significant apoptosis in poorly differentiated ductal carcinoma in situ of the breast

Following breast-conserving surgery for ductal carcinoma in situ (DCIS), the presence of comedo necrosis reportedly predicts for higher rates of post-operative recurrence. To examine the role of programmed cell death (apoptosis) in the aetiology of the cell death described as comedo necrosis, we studied 58 DCIS samples, using light microscopy, for morphological evidence of apoptotic cell death. The percentage of apoptotic cells (apoptotic index, AI) was compared between DCIS with and without evidence of 'comedo necrosis' and related to the immunohistochemical expression of the anti-apoptosis gene bcl-2, mitotic index (MI), the cellular proliferation antigen Ki67, nuclear grade and oestrogen receptor (ER) status. AI was significantly higher in DCIS samples displaying high-grade comedo necrosis than in low-grade non-comedo samples: median AI = 1.60% (range 0.84-2.89%) and 0.45% (0.1-1.31%) respectively (P < 0.001). Increasing nuclear grade correlated positively with AI (P < 0.001) and negatively with bcl-2 expression (P = 0.003). Bcl-2 correlated negatively with AI (P = 0.019) and strongly with ER immunoreactivity (P < 0.001). Cellular proliferation markers (MI and Ki67 immunostaining) correlated strongly with AI and were higher in comedo lesions and tumours of high nuclear grade (P < 0.001 in all cases). Thus, apoptosis contributes significantly to the cell death described in ER-negative, high-grade DCIS in which a high proliferative rate is associated with a high apoptotic rate. It is likely that dysregulation of proliferation/apoptosis control mechanisms accounts for the more malignant features typical of ER negative comedo DCIS.     

Prognostic value of bcl-2 oncoprotein expression in stage II colon carcinoma

The bcl-2 proto-oncogene encodes a Mr 25,000 protein that has been shown to prevent apoptosis or programmed cell death. The bcl-2 protein is detectable in basal cells of normal colonic epithelium, and an altered topographic distribution of this protein is found in colonic neoplasms. However, the clinical significance of abnormal bcl-2 expression in colon carcinomas remains unknown. We examined the prognostic value of the bcl-2 protein in TNM stage II colon carcinomas and its relationship to DNA ploidy, cell proliferation indices, p53 expression, and clinicopathological features. We analyzed 119 resected and otherwise untreated, paraffin-embedded stage II colon carcinomas for bcl-2 and p53 protein expression using immunohistochemistry. DNA ploidy and proliferative index (% S-phase + % G2-M) were determined by flow cytometry, and tumor grade and vascular microinvasion were assessed on histological sections. Cytoplasmic expression of the bcl-2 protein was detected in 72 (66%) of 110 carcinomas, and a high level of expression was significantly correlated with diploid DNA content (P = 0.02) and low proliferative activity (P = 0.005). bcl-2 was not associated with nuclear p53 expression. In a univariate analysis, a higher fraction of bcl-2-positive tumor cells was associated with better relapse-free survival (P = 0.02) and overall survival (P = 0.05) rates. Moreover, a high level of bcl-2 expression was an independent predictor of better relapse-free survival (P = 0.04), but not overall survival (P = 0.14), after adjustment for other variables, including proliferative index, DNA ploidy, and race. In conclusion, bcl-2 overexpression is associated with favorable prognostic features and may predict clinical outcome in stage II colon carcinomas.     

Apoptotic myonuclei in human Duchenne muscular dystrophy

The current view that apoptosis precedes necrosis in death of dystrophin-deficient muscle fibers of the mdx mouse has been well substantiated. Moreover, apoptotic myonuclei have been reported to increase in dystrophin-deficient mice 2 days after spontaneous exercise. To investigate the role of apoptosis in human muscular dystrophy, muscles from 11 patients of different ages with Duchenne muscular dystrophy were analyzed for apoptosis. The amount of apoptosis was assessed by terminal deoxynucleotidyl transferase assay, and the expression of bcl-2 and bax was examined by immunohistochemistry. Although very rare in normal muscles (less than 0.1%), apoptotic nuclei were detected in dystrophic muscles, particularly at the interstitial level. Nevertheless, few dystrophin-deficient myofibers with centrally located nuclei showed a positive reaction for DNA fragmentation. A mosaic pattern of bcl-2/bax-positive myofibers characterized dystrophic muscles, thus the relative proportion of pro- and antiapoptotic proteins differs among muscle fibers in correlation with the presence of apoptotic myonuclei. In the interstitium, apoptotic cells were identified as macrophages and activated satellite cells. This is the first study to show an apoptotic process in adult muscle fibers of patients with Duchenne muscular dystrophy, thereby shedding new light on muscle damage and its progression in dystrophinopathies.     

The precursor form of the human kallikrein 2, a kallikrein homologous to prostate-specific antigen, is present in human sera and is increased in prostate cancer and benign prostatic hyperplasia

Prostate-specific antigen (PSA, hK3) is a diagnostic marker for prostatic cancer but lacks the specificity to sufficiently distinguish between prostatic cancer and benign prostatic hyperplasia (BPH). Human glandular kallikrein 2 (hK2) has been proposed as a potential diagnostic marker for prostate cancer that could complement the current PSA test. Recently we demonstrated that proPSA is present in prostate cancer sera. This study examines the expression of prohK2 in prostate cells and its presence in human sera. Western blot analysis was used to assess prohK2 expression in the human carcinoma cell line, LNCaP. A highly specific and sensitive dual monoclonal immunoassay for prohK2 was developed and used to assess the presence of prohK2 in human sera. prohK2 was detected in the spent media of LNCaP cells. Furthermore, prohK2 was present at immunodetectable concentrations in human sera, and its concentration was increased in prostatic cancer and BPH. These results indicate for the first time that prohK2 is secreted by human prostate cells and is a major component of uncomplexed (free) hK2 in human sera. In addition, prohK2 in human sera is associated with prostate disease and thus may be a useful marker for prostatic cancer and BPH.     

Absence of bcl-2 expression by activated CD45RO+ T lymphocytes in acute infectious mononucleosis supporting their susceptibility to programmed cell death

bcl-2 proto-oncogene encodes an inner mitochondrial membrane protein that blocks programmed cell death (apoptosis). There is now increasing evidence that regulation of bcl-2 expression is a determinant of life or death in normal lymphocytes. We have recently described that activated (CD45RO+) CD4+ and CD8+ T cells in acute infectious mononucleosis (IM) undergo apoptotic cell death on culturing, indicating an activation-driven cell death of mature T cells. In this work, we examine bcl-2 expression by activated T cells in acute IM using a flow-cytometric analysis with an anti-bcl-2 monoclonal antibody (MoAb). It was consistently observed that most T cells from acute IM patients displayed only much less bcl-2, while normal T cells expressed bcl-2 relatively strongly. Multicolor analysis showed that bcl-2-lacking T cells in acute IM were restricted to the CD45RO+ (activated) populations of CD4+, as well as CD8+ T cells. In contrast, the relatively intense levels of bcl-2 were expressed in both CD45RO+ and CD45RO- T-cell populations from normal subjects. This marked difference in bcl-2 expression of CD45RO+ T cells between acute IM and normal controls was also confirmed by Western blot analysis. Activated (CD45RO+) T cells with low bcl-2 expression, but not bcl-2-expressing CD45RO- T cells, in acute IM patients were found to die easily when cultured without added growth factors. However, in normal individuals, both CD45RO+ and CD45RO- T cells were relatively stable on culturing. These findings suggest that lack of bcl-2 expression by activated (CD45RO+) T cells in acute IM might be associated with their susceptibility to programmed cell death.     

Sex-role reversal and the absence of extra-pair fertilization in Wilson''s phalaropes

Human endometrial leukocytes undergo regular cyclical changes during the menstrual cycle, with a striking increase in the phenotypically unusual population of CD56+ CD16- endometrial granulated lymphocytes (eGLs) in the late secretory phase and early pregnancy. The factors that regulate this increase in eGL numbers are unclear; their unusual morphology, however, has led to the suggestion that they undergo apoptosis at the end of the menstrual cycle. Apoptosis, bcl-2 expression, and proliferative activity were examined in the stroma of normal cycling, progesterone-treated, and early-pregnancy endometrium. The expression of bcl-2 and the Ki67 proliferation marker by highly purified (> 98% CD56+) eGLs from endometrium during the menstrual cycle and from first-trimester decidua was also studied. Apoptotic cells were rarely observed in the endometrial stroma of any of the samples examined. Stromal bcl-2 expression, however, increased from the proliferative to the premenstrual phase, and double immunohistochemical labeling demonstrated large numbers of bcl-2+ CD56+ eGLs. In contrast, Ki67 expression was high in the endometrial stroma during the proliferative phase, fell during the secretory phase, and rose again premenstrually, because of expression by eGLs. Isolated CD56+ eGLs also showed high bcl-2 and Ki67 expression at the end of the menstrual cycle. Unlike premenstrual endometrium, progesterone-treated endometrium and first-trimester decidua contained few proliferating cells, expressed high levels of bcl-2, and showed no evidence of apoptosis. Thus, eGLs do not undergo apoptosis in premenstrual endometrium, and their regulatory mechanisms remain to be clarified.