Bcl-2 Immunostaining Research Articles

Relationship between expression of cyclin D1, Ki-67 and bcl-2 and biologic behavior in CD117-positive gastrointestinal stromal tumors

To investigate the clinicopathologic features of gastrointestinal stromal tumors (GISTs) and to identify reliable prognostic parameters. Fifty-nine GISTs were studied by immunostaining of CD117, CD34, SMA, desmin, S-100, bcl-2, and Ki-67. Histopathologic evaluations included tumor size, necrosis, histological growth patterns, mitotic activities and tumor lymphocytic infiltrate. The patients were clinically followed for 2 to 9 years. Univariate, multivariate and correlative statistical evaluations were used to analyze the data. Among the 59 patients, 40 were alive and 15 died of their tumors at follow-up, the remaining 4 patients died of other causes. Pathological parameters that correlated with prognosis included tumor sizes of more than 5 cm, tumor tissue necrosis, mitotic cell count equal or higher than 5 per 50 high power field, Ki-67 labeling index (LI) equal or higher than 5% and intense bcl-2 immunostaining. Multivariate analysis showed that the mitotic count and Ki-67 LI were independent prognostic indicators. There was a correlation between mitotic count and Ki-67 LI. Mitotic count and Ki-67 LI are the best predictors for a poor outcome of GIST after surgical treatment. Ki-67 immunostaining may substitute mitotic count as a useful prognostic parameter.

Age-Associated Changes in Mouse Oocytes During Postovulatory In Vitro Culture: Possible Role for Meiotic Kinases and Survival Factor BCL21

To elucidate molecular mechanisms underlying oocyte senescence, we investigated whether oocytes from female mice of advanced reproductive age exhibit a precocious postovulatory aging that, in turn, may be responsible for the precocious activation of an apoptotic program. During a 9-h in vitro culture, the frequency of oocytes showing MII aberrations, spontaneous activation, and cellular fragmentation increased in old oocytes (P < 0.05), whereas it did not change in the young group. In old oocytes, the activities of MPF (a complex of the cyclin-dependent kinase cdc2 and cyclin B1) and MAPK (mitogen-activated protein kinase) decreased precociously, showing a first drop as early as 3 h after the beginning of in vitro culture (P < 0.05). Immunoblotting and immunocytochemical analysis revealed that, in oocytes of the old group, reduction of BCL2 expression at protein level occurred earlier than in the young group (P < 0.05) and was not associated to the loss of BCL2 transcripts detected by RT-PCR. These changes are followed by an abrupt increase of the rate of TUNEL-positive oocytes after 24 h of culture to a value of 67% +/- 6%. Exposure of young oocytes to 20 microM roscovitine or 20 microM U0126, specific inhibitors of MPF and MAPK, resulted in the decreased percentage of oocytes showing positive immunostaining for BCL2 and in an increased rate of DNA fragmentation. Present results suggest that the developmental competence of oocytes ovulated by aging mice may be negatively influenced by a downregulation of MPF and MAPK activities that in turn induces the activation of a proapoptotic signaling pathway.

Transcription factor‐mediated proliferation and apoptosis in benign and malignant thyroid lesions

Transcription factors play an essential role in regulating both cell proliferation and programmed cell death. Proliferation and apoptosis-related transcription factor immunoexpression patterns were concomitantly investigated in tissue sections of normal thyroid, goiters, follicular adenomas and well-differentiated papillary and follicular carcinomas using antibodies against prothymosin alpha, E2F-1, p53, Bcl2, and Bax proteins. Proliferation and apoptotic indices were determined by Ki-67 immunoreactivity and the terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick-end labeling technique, respectively. Prothymosin alpha and E2F-1 immunoexpression levels were found to be significantly elevated in well-differentiated carcinomas compared to adenomas, goiters and normal tissues (P < 0.05). Both proteins were directly correlated with the proliferation index (P < 0.05). E2F-1 was additionally correlated with the apoptotic index (P < 0.05). The majority of cases were negative for p53 staining. Positive Bcl2 immunostaining was detected in all thyroid histotypes. None of the normal tissues showed Bax immunoreactivity, while positive accumulation differed significantly between hyperplastic and neoplastic histotypes. Direct correlations were observed between prothymosin alpha and Bcl2 as well as between E2F-1 and Bax immunoexpression (P < 0.05). These data demonstrate that prothymosin alpha and E2F-1 are strongly involved in the proliferation processes of thyroid neoplasias. Furthermore, prothymosin alpha may promote cell survival through the Bcl2 anti-apoptotic pathway, while E2F-1-induced apoptosis via p53-independent pathways may be associated with transcriptional activation of bax pro-apoptotic gene.

Apoptosis in epithelial cells of apical radicular cysts

To investigate the occurrence of apoptotic cell death in the epithelium of radicular cysts and to compare its frequency in lesions presenting a distinct functional state. Twenty radicular cysts were selected and arranged into two groups with 10 lesions in each group: atrophic (quiescent) and hyperplastic (active) epithelium. Morphologic investigations of apoptosis were conducted by means of optic microscopy in haematoxylin and eosin slides. Immunohistochemical techniques to detect the bcl-2 protein were carried out by streptavidin-biotin-peroxidase assay. In both instances, 30 sequential high-power microscopic fields were observed to determine apoptotic (AI) and bcl-2 immunostaining (bcl-2I) indexes. The presence of AI and bcl-2I within the two groups was compared using the t-test. Correlation between the AI and the bcl-2I was investigated using the Spearman test. Apoptosis was detected in the epithelium of all cysts. Higher AI levels were found in lesions with an atrophic (0.17 +/- 0.19) rather than a hyperplastic (0.10 +/- 0.10) epithelium. The same was found for the bcl-2I levels (0.06 +/- 0.03 vs. 0.04 +/- 0.01, respectively). However, these differences were not statistically significant. A positive and significant correlation was found between AI and bcl-2I. Apoptosis was always present in the epithelium of the lesions and was more frequent in lesions with atrophic (quiescent) epithelium.

Follicular lymphoma subgrouping by fluorescence in situ hybridization analysis

The frequency of t(14;18) in follicular lymphoma (FL) in Japan has been reported to be low compared to North America and other European countries. Recently, it has also been reported that FL lacks t(14;18), mainly among histological grade 3b, and occasionally has a rearranged Bcl-6 gene. It is not known whether a difference in histology or immunostaining pattern exists between FL with and without t(14;18). We performed interphase fluorescence in situ hybridization (FISH) analysis to detect Bcl-2/IgH, Bcl-6 gene rearrangement, Bcl-2 gene amplification, and the cyclinD1/IgH gene in formalin-fixed paraffin embedded specimens from our FL archives. The correlation between morphological features, histological grades, immunohistochemical findings, and cytogenetical aberrations was studied. In total, we found that 28 of 47 cases (59.6%) had t(14;18). Bcl-6 gene rearrangement and extra Bcl-2 gene signals were found in five and two cases, respectively. Only one had cyclinD1/IgH fusion. Ten of 12 grade 1, nine of 17 grade 2, and 0 of two grade 3 cases had fusion signals, respectively. None of the above abnormalities were detected in 12 of 47 cases (25.5%). Our data confirmed a high frequency of t(14;18) in FL in grade 1, but a lower incidence among grade 2, that could be attributed to the lower incidence of the translocation in FL in Japan. Immunostaining of both Bcl-2 and CD10 was highly predictable for the presence of t(14;18); the positive predictive value was 75%, suggesting the usefulness of the staining.

Prevention of Endometrial Apoptosis: Randomized Prospective Comparison of Human Chorionic GonadotropinVersusProgesterone Treatment in the Luteal Phase

To study control of apoptosis in human endometrium, we examined late luteal-phase endometrial biopsies obtained in the late luteal phase for evidence of apoptosis and compared the effects of exogenous human chorionic gonadotropin (hCG) and progesterone on this process. Using a controlled, prospective, and randomized study design, 12 healthy, fertile, reproductive-age women (ages 20-34 yr) with regular menstrual cycles (range, 26-32 d) were recruited. Each underwent an endometrial biopsy 12 d after a urinary LH surge in a control and treatment cycle. After biopsy in a natural cycle, subjects were randomized to receive luteal doses of either 200 mg intravaginal progesterone (d 18-27) or a single im injection of 10,000 IU of hCG (d 19) followed by repeat endometrial biopsy and collection of serum on d 26. Apoptosis was assessed by DNA laddering, localizing apoptotic bodies using immunofluorescent labeling of DNA fragments (the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method), and immunohistochemical assessment of apoptosis markers bcl-2, bcl-x, and bax. Serum progesterone levels were compared between treatment groups. Evidence of apoptosis in control cycles was significantly reduced in endometrium after both luteal-phase treatments. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling results demonstrated significantly less apoptosis in the hCG treatment group compared with controls. Immunostaining for bcl-2 was higher in hCG- and progesterone-treated cycles, whereas bax expression was decreased and bcl-x immunostaining was not different between treatments. Serum progesterone levels were highest in the hCG-treated group, although statistical significance was not reached (P = 0.08). These results demonstrate that signs of apoptosis, already apparent by d 26 of the menstrual cycle can be reduced with either hCG or progesterone treatment. The clinical utility of these findings includes a rational use of luteal-phase support for treatment of women with infertility and/or recurrent pregnancy loss.

Fas, FasL, Bcl-2, and Bax in the endometrium of rhesus monkey during the menstrual cycle

To study possible role and regulation of apoptosis occurred in primate endometrium, the expression of apoptosis-related molecules, Fas, FasL, B cell lymphoma/leukaemia-2 (Bcl-2), and Bax were analyzed in relation to occurrence of apoptosis and proliferation in the cycling endometrium of the rhesus monkey using immunohistochemistry and Western blot. The cell apoptosis and proliferation were evaluated by means of in situ 3'-end labeling and Ki67 immunostaining, respectively. The results showed that the expressions of Fas, Fas ligand (FasL), Bcl-2, and Bax were co-localized predominantly in the epithelial cells of the endometrium. Modest Fas staining with no obvious change was detected throughout the menstrual cycle, while the levels of FasL and Bax protein in the epithelial cells increased in the secretory phase when apoptosis was most prevalent. In contrast, epithelial immunostaining for Bcl-2 was maximal during the proliferative phase and decreased in the secretory phase. Bcl-2 immunoreactivity was also detected in some immunocytes. The coordinated expression of Fas, FasL, Bcl-2, and Bax in the cycling endometrium of the rhesus monkey suggests that the cyclic changes in endometrial growth and regression may be regulated by the balance of these factors under the action of ovary steroids.

RENAL CELL CARCINOMA MIB-1, BAX AND BCL-2 EXPRESSION AND PROGNOSIS

Proliferation and programmed cell death (apoptosis) are key factors in oncogenesis and tumor progression. In carcinogenesis important regulators of apoptosis are members of the Bcl-2 family. In this family the Bcl-2 gene has an inhibitory effect on apoptosis, while Bax promotes cell death. In renal cell carcinoma (RCC) the associations between Bcl-2 proteins and RCC prognosis have been controversial. We evaluated Bax and Bcl-2 levels in RCC, and their associations with prognosis, proliferation and traditional prognostic factors. Our prospective study population comprised 138 consecutive patients who underwent radical nephrectomy for RCC. Immunostaining and semiquantitative indices for Ki-67 (MIB-1), Bax and Bcl-2 were estimated. Their associations with prognosis were explored. On univariate analysis according to survival statistically significant differences were achieved by Bax (positive vs negative HR 3.04, 95% CI 1.27 to 7.23), Bcl-2 (positive vs negative HR 0.43, 95% CI 0.23 to 0.81), MIB-1 (continuous HR 1.03, 95% CI 1.001 to 1.064), Fuhrman nuclear class (4 vs 1 plus 2 HR 8.15, 95% CI 3.13 to 21.20) and stage (4 vs 1 HR 60.04, 95% CI 13.99 to 257.68). Only stage (HR 47.96, 95% CI 10.85 to 212.03) and Fuhrman classification (HR 4.32, 95% CI 1.60 to 11.65) attained statistical significance on Cox regression multivariate analysis. In our prospective study Bax and Bcl-2 showed a statistically significant association with prognosis in RCC but did not achieve the status of independent prognostic factors. Further studies are needed to clarify the role of the apoptotic process in tumor progression and prognosis.

Immunohistochemical Study Of Bcl-2 Protein And Estrogen Receptor-Alpha Expression In Benign Prostatic Hyperplasia And Prostatic Carcinoma

The human prostate, a male sexual accessory tissue involved in seminal fluid production, has a remarkably high incidence of hyperplastic and neoplastic disease. The present study was carried out on one hundred and twenty (120) specimens divided into two groups; group 1: Included forty cases of benign prostatic hyperplasia (BPH) and group 2: Included sixty cases of prostatic adenocarcinoma (PC) (22 were low grade; GS: 2-6 and 38 were high grade; GS: 7- 10),in addition to twenty cases of histologically normal prostates taken as controls. Immunohistochemical technique was applied to detect Bcl-2 as well as ER α positivity in all specimens. Group 1 showed the following profile: ER α (+) in all cases (100%), Bcl-2 (-) in 95%, ERα (+) / Bcl-2 (+) in 95%, ERα (-) / Bcl-2 (+) in 0%, ERα (+) / Bcl-2 (-) in 5% and ERα (-) / Bcl-2 (-) in 0% of cases while group 2 showed the following profile: ERα (+) in 30%, Bcl-2 (+) in 21.7%, ERα (+) / Bcl-2 (+) in 15%, ERα (-) / Bcl-2 (+) in 6.7%, ERα (+) / Bcl-2 (-) in 15% and ERα (-) / Bcl-2 (-) in 70% of cases. The mean epithelial ER α -immunolabeling was, however, significantly increased in group 2 than in group 1 (P < 0.05) which, in turn, being higher than the normal cases (P<0.05 ) . Among group 2 , the mean ER α was significantly more in high grade than in low grade tumors (P < 0.05), however, the mean ER α immunolabeling revealed no significant correlation with T-stage (P = 0.219) or with the clinical stage (P = 0.391). In contrast, the Bcl-2 immunostaining was statistically higher in group 1 than in group 2 (P < 0.05) and showed a significant correlation with T stage (P < 0.05) although the study displayed no significant correlation between Bcl-2 immunopositivity and either Gleason score (P = 0.125) or the histologic grade (P = 0.146). In addition, combined ER α (+)/ Bcl 2 (+) immunoreactivity demonstrated the aggressive subgroup of PC cases more accurately than either ER α (+) or Bcl-2 (+) alone. Finally, multivariate analysis showed that the Bcl-2, proved to be an independent prognostic indicator (P < 0.05). Thus, the immunohistochemical expression of ER α and Bcl-2 protein in prostatic tissue may aid in better understanding the biology and genesis of both prostatic hyperplasia and carcinoma .

Immunohistochemical Study Of Bcl-2 Protein And Estrogen Receptor-Alpha Expression In Benign Prostatic Hyperplasia And Prostatic Carcinoma

The human prostate, a male sexual accessory tissue involved in seminal fluid production, has a remarkably high incidence of hyperplastic and neoplastic disease. The present study was carried out on one hundred and twenty (120) specimens divided into two groups; group 1: Included forty cases of benign prostatic hyperplasia (BPH) and group 2: Included sixty cases of prostatic adenocarcinoma (PC) (22 were low grade; GS: 2-6 and 38 were high grade; GS: 7- 10),in addition to twenty cases of histologically normal prostates taken as controls. Immunohistochemical technique was applied to detect Bcl-2 as well as ER α positivity in all specimens. Group 1 showed the following profile: ER α (+) in all cases (100%), Bcl-2 (-) in 95%, ERα (+) / Bcl-2 (+) in 95%, ERα (-) / Bcl-2 (+) in 0%, ERα (+) / Bcl-2 (-) in 5% and ERα (-) / Bcl-2 (-) in 0% of cases while group 2 showed the following profile: ERα (+) in 30%, Bcl-2 (+) in 21.7%, ERα (+) / Bcl-2 (+) in 15%, ERα (-) / Bcl-2 (+) in 6.7%, ERα (+) / Bcl-2 (-) in 15% and ERα (-) / Bcl-2 (-) in 70% of cases. The mean epithelial ER α -immunolabeling was, however, significantly increased in group 2 than in group 1 (P < 0.05) which, in turn, being higher than the normal cases (P<0.05 ) . Among group 2 , the mean ER α was significantly more in high grade than in low grade tumors (P < 0.05), however, the mean ER α immunolabeling revealed no significant correlation with T-stage (P = 0.219) or with the clinical stage (P = 0.391). In contrast, the Bcl-2 immunostaining was statistically higher in group 1 than in group 2 (P < 0.05) and showed a significant correlation with T stage (P < 0.05) although the study displayed no significant correlation between Bcl-2 immunopositivity and either Gleason score (P = 0.125) or the histologic grade (P = 0.146). In addition, combined ER α (+)/ Bcl 2 (+) immunoreactivity demonstrated the aggressive subgroup of PC cases more accurately than either ER α (+) or Bcl-2 (+) alone. Finally, multivariate analysis showed that the Bcl-2, proved to be an independent prognostic indicator (P < 0.05). Thus, the immunohistochemical expression of ER α and Bcl-2 protein in prostatic tissue may aid in better understanding the biology and genesis of both prostatic hyperplasia and carcinoma .

Time-dependent mitochondrial-mediated programmed neuronal cell death prolongs survival in sepsis.

To investigate whether apoptosis is a possible mechanism of brain dysfunction occurring in septic syndrome. Experimental prospective study. Laboratory of Surgical Research at the University of Athens. Male pathogen-free Wistar rats. Rats (n = 112) were subjected to sepsis by cecal ligation and puncture. Sham-operated animals (n = 40) underwent the same procedure but without ligation or puncture. Septic animals were either randomly divided (n = 62) in six groups and studied at 6, 12, 24, 36, 48, and 60 hrs after the operation or monitored (n = 50) for 48 hrs as a survival study group. Sham-operated animals were killed at 6, 12, 24, 36, 48, and 60 hrs after the procedure. Brain and cecum were then removed and postfixed in paraffin sections. Apoptosis was evaluated by light microscopy in hematoxylin and eosin-stained specimens and by transmission electron microscopy. In paraffin-embedded sections, immunostaining for bax, bcl-2, cytochrome c, and caspase-8 was done. In septic rats, increased apoptosis was detected in neurons of the CA1 region of the hippocampus, in choroid plexus, and in Purkinje cells of the cerebellum. Bax immunopositivity was found decreased after the septic insult (p =.03). Bax immunoreactivity was altered as the septic syndrome evolved; it was up-regulated in the early stages (6-12 hrs) and progressively decreased in the late phases (p =.001). Cytochrome c presented a similar regional pattern of expression and was found to be the sole gene marker carrying an independent prognostic role (p =.03). Both bcl-2 and caspase-8 expression remained at constant levels at all times evaluated. There is evidence that more neurons undergo apoptosis during sepsis than in normal brain tissue in certain sites where the blood-brain barrier is compromised. In this phenomenon, mitochondrial gene regulators such as bax and products such as cytochrome c seem to play important regulating and prognostic roles, respectively.

EXPRESSION OF HEPATOCYTE GROWTH FACTOR AND ITS RECEPTOR C-MET IN ACQUIRED RENAL CYSTIC DISEASE ASSOCIATED WITH RENAL CELL CARCINOMA

The development of renal cell carcinoma (RCC) is a critical problem in acquired renal cystic disease (ARCD) in patients with end stage renal disease, particularly males on chronic hemodialysis. We examined the expression of hepatcyte growth factor (HGF) and its receptor c-met in ARCD associated with RCC to elucidate the role of the HGF/c-met pathway in renal cyst development and subsequent tumor transformation. Immunohistochemical expression of HGF, c-met and Bcl-2 was examined in 15 normal tissue samples of kidneys obtained from nephrectomy for localized RCC and 19 with ARCD associated with RCC from 16 patients on dialysis. The expression of HGF mRNA was examined by reverse transcriptase-polymerase chain reaction. In normal kidneys immunostaining for HGF was scarcely detected in renal tubular cells. Bcl-2 and c-met immunostaining was observed mainly in distal tubular cells. In nontumor areas of kidneys with ARCD associated RCC the expression of HGF and c-met was up-regulated in tubular and the cyst epithelial cells. Hyperplastic cysts with multilayer epithelium and micropapillary projection were the predominant cysts stained with HGF and c-met. Immunostaining for HGF and c-met was also detected in RCC regions. The Bcl-2 immunostaining pattern was similar to that of HGF and c-met in nontumor and RCC regions. On reverse transcriptase-polymerase chain reaction HGF mRNA expression was up-regulated in nontumor areas and RCC regions in ARCD. These results suggest that up-regulation of HGF and its receptor c-met may be involved in renal cyst formation and subsequent tumor transformation in patients with end stage renal disease. Increased Bcl-2 expression may promote this process through the inhibition of apoptosis.

Chronic inflammation in benign prostate hyperplasia is associated with focal upregulation of cyclooxygenase-2, Bcl-2, and cell proliferation in the glandular epithelium.

Chronic inflammation has been suggested to be linked to the development and progression of prostate cancer. An inflammatory microenvironment may support the development of malignancy by upregulation of proinflammatory cytokines and cyclooxygenase-2 (COX-2). Recent studies have suggested that COX-2 is upregulated in cancer and in proliferative inflammatory atrophy (PIA) of the prostate. Immunohistochemistry (IHC) was used to investigate the expression of COX-2 in prostate epithelium. The relationships between COX-2 expression and inflammatory cells, proliferation (proliferating cell nuclear antigen (PCNA) and Ki-67), and apoptosis (Bcl-2) were studied in 45 BPH samples. COX-2 expression was detected in prostate luminal epithelial cells in all 45 samples studied. The overall percentage of COX-2 positive glands was 7.5%, distributed as 0.2% positive glands in normal prostate tissue, 25.7% in postatrophic hyperplasia (PAH), and 11.9% in simple atrophy (SA). The highest proliferation index was found in COX-2 positive stained epithelium. COX-2 expression was associated with Bcl-2 immunostaining in atrophic lesions (P < 0.0001). T lymphocytes and macrophages were the predominant inflammatory cells related to the COX-2 expression in prostate epithelium. The data demonstrate that T lymphocytes and macrophages appeared to play an important role in the induction of COX-2 expression in prostate epithelium, which was associated with increased cell proliferation and possibly, due to overexpression of Bcl-2, apoptotic resistance. This suggests that pro-inflammatory cytokines released by adjacent inflammatory cells may induce COX-2 in the epithelial cells in prostate atrophic lesions. In addition, COX-2 expressing cells may be involved in the pathogenesis of prostate cancer.

Short-term lithium treatment promotes neuronal survival and proliferation in rat striatum infused with quinolinic acid, an excitotoxic model of Huntington's disease

We assessed the ability of lithium to reduce neurodegeneration and to stimulate cell proliferation in a rat model of Huntington's disease in which quinolinic acid (QA) was unilaterally infused into the striatum. LiCl (0.5-3.0 mEq/kg) was injected subcutaneously 24 h before and 1 h after QA infusion. At 7 days after QA injection, lithium significantly diminished the loss of neurons immunostained for Neuronal Nuclei (NeuN) in the injured striatum, but failed to prevent the reduction of NADPH-diaphorase-positive striatal interneurons. Lithium also reduced the number of neurons showing DNA damage or activated caspase-3. This neuroprotection was associated with an upregulation of Bcl-2 protein levels in the striatal tissue and an increase in the number and density of Bcl-2 immunostaining in striatal neurons. Bromodeoxyuridinie (BrdU) labeling in the lithium-treated injured striatum revealed the presence of large numbers of proliferating cells near the QA-injection site, with a reduction of BrdU-labeled cells in the subventricular zone (SVZ). All BrdU-labeled cells in the SVZ and the majority of BrdU-labeled cells near the QA-injection site were negative for either NeuN or glial fibrillary acidic protein (GFAP), suggesting that they are undifferentiated progenitor cells. However, a small number of BrdU-positive cells found in the QA-injected and lithium-treated striatum site were positive for either NeuN or GFAP. Our results suggest that lithium is neuroprotective in the QA-injection model of Huntington's disease not only due to its ability to inhibit apoptosis but also because it can stimulate neuronal and astroglial progenitor proliferation in the QA-injected striatum or their migration from the SVZ.

Significance of p53, Bcl-2, and HER-2/neu protein expression in Omani Arab females with breast cancer.

Racial disparity in the presentation of breast cancer and the outcome of its treatment is well established. However, the causes remain unexplained. The scarcity of reports about the prognostic significance of p53, bcl-2, and HER-2/neu in Arab females with breast cancer has been the impetus to this study. We evaluated the prognostic significance of altered expression of p53, bcl-2, HER-2/neu in Omani Arab females with non-metastatic breast cancer with correlation to other established prognostic factors. We have retrospectively analyzed the immunohistochemical expression of p53, HER-2/neu and bcl-2 in paraffin embedded blocks of 72 females diagnosed with invasive breast cancer between 1992 and 2002. The expression of the above proteins was correlated with other prognostic factors and univariate and multivariate analysis was carried out for all prognostic factors. Overexpression of p53 significantly correlated with younger age (<40), pre-menopausal status, poor differentiation with inverse correlation with bcl-2 expression. Expression of bcl-2 immunopostivity significantly correlated to low histological grade and positive estrogen and progesterone receptor status. On univariate and multivariate p53 overexpression and lack of bcl-2 immunostaining resulted in worse survival outcome, but not Her-2/neu overexpression. Expression patterns of p53 and bcl-2 are independent predictors of survival in Omani Arab population which may help to stratify these patients into different risk groups.

Immunohistochemical status of p53, MDM2, bcl2, bax, and ER in invasive ductal breast carcinoma in Tunisian patients.

TP53 gene alterations have been associated with sporadic breast cancer. To assess the role of p53 in invasive ductal carcinoma (IDC) of the breast among Tunisian patients, p53 protein status was studied by immuno-histochemical analysis. The p53 protein was expressed in 41 of 70 (58%) tumors. Study of the status of its target gene expression showed that MDM2 was overexpressed in 43 tumors (61%), bcl2 in 29 (41%), and bax in only 9 (12%). Estrogen receptor (ER) was detected in 38 tumor tissues (54%). The accumulated p53 was significantly associated with MDM2-positive, bcl2-negative, and ER-negative tumors (P = 0.024, P = 0.000027, and P = 0.000008, respectively), whereas with bax the correlaton was not significant. Bcl2 immunostaining displayed a positive correlation with ER (P = 0.001). A significantly higher fraction of p53-positive cells was observed in ER-negative SBRII-SBRIII tumors than in ER-positive SBRI-SBRII tumors (P = 0.000066). bcl2-positive tumors were significantly correlated with ER-positive/SBRI-SBRII tumors (P = 0.007), but negatively correlated with p53/bax (P = 0000004). MDM2 immunostaining displayed the same phenotype as p53 in the correlation with bcl2 and ER (P = 0.003), strengthened by significant associations between MDM2-positive/p53-positive and bcl2-negative or ER-negative, respectively (P = 0.00005 and P = 0.000001, respectively). MDM2-positive cells were significantly correlated with the p53-positive/bax-negative phenotype (P = 0.04). These results suggest that p53 accumulated in these tumor tissues is associated with bad prognostic markers (ER-negative, SBRIII) of IDC. MDM2 overexpression might be responsible for the accumulated p53 value in IDC. Regulation of the apoptotic process is involved in IDC; bcl2 is associated with a good prognostic marker (ER-positive and SBRI-II), whereas the regulation of bax is complex and does not necessarily correlate with the overexpression of p53.

Expression of cellular apoptosis susceptibility protein in serous ovarian carcinoma: a clinicopathologic and immunohistochemical study

Objective. This study investigated the expression of cellular apoptosis susceptibility protein (CAS) in serous ovarian carcinoma by immunohistochemistry and compared it with topoisomerase IIα (topo IIα), bcl-2, bcl-x, the frequency of apoptotic bodies (ABI), mitotic activity, and c-erbB-2 with regard to clinicopathologic variables. Methods. Formalin-fixed, paraffin-embedded archival tissue sections of 10 benign serous cystadenomas, 10 serous neoplasms of low malignant potential (LMP), and 41 serous ovarian carcinomas were immunostained with antibodies to CAS, bcl-x, topo IIα, bcl-2, and c-erbB-2. Immunostaining for CAS, bcl-x, and bcl-2 was scored concerning approximate percentage of positive tumor cells and relative staining intensity. For c-erbB-2, at least 10% of tumor cells had to display membrane staining. Topo IIα-labeling indices were quantitated as the percentage of positively stained nuclei in 1000 tumor cells. ABIs were reported as the number of apoptotic bodies in 1000 tumor cells, mitotic activity as mitotic figures per 10 high power fields. Results. CAS expression was negative in serous cystadenomas and LMP. In contrast, moderate or strong immunostaining was observed in 34 of 41 cases (83%) of serous carcinomas. CAS immunoreactivity was positively related with ABI ( P = 0.0170), mitotic activity ( P < 0.0001), c-erbB-2 ( P = 0.0153), grade ( P = 0.0107), and adverse outcome ( P = 0.0035), but not with FIGO stage, topo IIα, bcl-2, and bcl-x. Other variables indicating outcome were topo IIα, c-erbB-2, and ABI. Conclusions. CAS is frequently upregulated in serous ovarian carcinomas, correlated with apoptosis and mitotic activity, and relevant prognostically. CAS protein expression may serve as a marker of aggressive tumor behavior in serous ovarian carcinomas.

The Bcl-2 protein expression in germinal centers of hypertrophied adenoids in children

The aim of this study was to evaluate Bcl-2 expression in the germinal centers of adenoid in children on the assumption that it can be treated as a marker of adenoidal tissue function. The study involved 95 children. The patients were divided into three age groups: aged up to 5, 5–10 and above 10 years. The analysed material were adenoids removed on the grounds of hypertrophy. Immunohistochemical analyses were carried out using monoclonal antibodies (DAKO/Bcl-2, No. M0887) directed against human Bcl-2 protein. The presence of Bcl-2 positive lymphocytes within the lymphoid follicles and Bcl-2 immunostaining were scored. The immunohistochemical staining showed Bcl-2 positive lymphocytes in the examined sections of adenoidal tissue and their characteristic location, mainly within the mantle zone. Bcl-2 reactivity was widespread in hyperplastic lymphoid tissue, including germinal centers surroundings, scattered cells within the germinal centers and the T-cell areas in general. We have not found statistically significant correlation between Bcl-2 expression and clinical status and change in Bcl-2 expression level according to age. The apoptosis presence within the germinal centers and its lack in mantle zone, the manifestation of which is Bcl-2 expression, is a sign of a proper lymphocytes maturation within lymphoid follicles. It seems that these processes are not influenced by age, so the adenoid involution is probably associated with the changes in the number of lymphoid follicles rather than change or inhibition of the processes that take place in them.

The clinical relevance of Bcl-2, Rb and p53 expression in advanced non-small cell lung cancer

Purpose: The aim of this study was to evaluate the impact of Bcl-2, retinoblastoma (Rb) and p53 proteins on overall survival of 102 patients with locally advanced and metastatic NSCLC who underwent cisplatin-based chemotherapy. Materials and methods: Paraffin-embedded bronchial biopsy and fine-needle biopsy specimens were evaluated by an immunostaining method. Results: Median age of analyzed patients was 61 years. Male/female ratio was 88/14. There were 10 (10%) patients with stage IIIA, 37 (36%) with stage IIIB and 55 (54%) with stage IV NSCLC. Only 15 (15%) tumor specimens had no detectable alterations for analyzed factors. Forty-six samples (45%) had positive immunostaining for p53, 61 (60%) had negative immunostaining for Rb and 8 (8%) had positive immunostaining for Bcl-2. Median and 5-year survival of analyzed population was 12 months and 6%, respectively. In univariate analysis Bcl-2 overexpression, stage (III versus IV) and normal lactate dehydrogenase (LDH) serum levels were associated with better overall survival ( P<0.02, 0.001 and 0.03). In multivariate analysis, only stage was identified as an independent predictive factor. Conclusion: High frequency of Rb and Bcl-2 loss was detected in patients with advanced NSCLC. P53, Rb and Bcl-2 have not been shown to be independent predictors of survival even if Bcl-2 might have a particular relevance in patients with advanced NSCLC and should be better explored in this setting.

Premature keratinocyte death and expression of marker proteins of apoptosis in human skin after UVB exposure.

Epidermal keratinocytes undergo a process of terminal differentiation or cornification that in many aspects resembles apoptosis. It is characterized by the elimination of cell nuclei within the granular layer, whereas the cytoplasm is transformed into horn cells. Premature death of keratinocytes can be induced by extrinsic factors such as UV irradiation. We investigated the time-dependent expression of apoptotic marker proteins in the skin of one healthy human volunteer after irradiation with a fourfold minimal erythema dose (MED) of UVB. The data were supplemented by including healthy skin areas of biopsies from patients UVB-irradiated for therapeutic reasons. Punch biopsies were analysed by in situ end-labelling (ISEL) for DNA strand breaks and by immunohistochemistry for expression of p53, bcl-2, active caspase-3 and its proform, and deoxyribonuclease I (DNase I). Keratinocytes with pyknotic nuclei were first detected 6 h after UVB exposure, and apoptotic keratinocytes (sunburn cells) 12 h after exposure. These aggregated to sunburn bodies after 24 h. In control skin, nuclei with DNA strand breaks were only occasionally detected in the granular layer but 6 h after UVB irradiation in the spinous layer. After 12 h, many sunburn cells were ISEL-positive and positively stained for active caspase-3, P53, and DNase I. Morphometric evaluation of the immunohistochemical data demonstrated that maximal upregulation of P53, DNase I and activation of caspase-3 occurred 12 h after irradiation and in advance of the peak of apoptotic cell death reached after 24 h as verified by ISEL. In contrast, strong Bcl-2 immunostaining appeared restricted to presumed melanocytes and basal cells but was not increased after UVB irradiation.