Bcl-2 expression and its association with cell kinetics in human gastric carcinomas and intestinal metaplasia

The bcl-2 protooncogene was initially discovered at the t(14;18) chromosomal breakpoint in follicular lymphomas. It has been demonstrated that bcl-2 protein (Bcl-2) expression blocks apoptosis and plays an important role in cell development and maturation. In the present study, Bcl-2 expression was immunohistochemically examined in 103 cases of gastric carcinoma, as well as 64 cases of non-carcinous gastric mucosa, and its correlation with apoptosis, cell proliferation and p53 immunoreactivity was investigated. Bcl-2 was detected in 18.0% of differentiated-type gastric carcinomas (9 of 50) and 7.5% of the undifferentiated type (4 of 53). In adjacent intestinal metaplastic gastric epithelium, the incidence of Bcl-2 positivity in the incomplete type (21/23, 91.3%) was significantly higher than in the complete type (23/41, 56.1%) (P < 0.04). Double immunostaining for Bcl-2 and Ki-67 clearly revealed the majority of Bcl-2-positive cancer cells to be in a nonproliferating state, although some cancer cells expressed both proteins together. Statistical assessment demonstrated that the average Ki-67 labeling index and apoptotic labeling index in Bcl-2-positive foci were significantly lower than in Bcl-2-negative foci (P < 0.0001, P < 0.0003). In addition, a significant dissociation between Bcl-2 and p53 immunoreactivity was found in cancer tissues. These results indicate that aberrant Bcl-2 expression in gastric carcinomas possibly originates from intestinal metaplastic epithelium, and suggest a possible role in tumor development and growth.     

Automated and quantitative immunocytochemical assays of Bcl-2 protein in breast carcinomas

Expression of the bcl-2 gene was investigated in 218 human breast carcinomas by immunohistochemical analysis. Immunodetections were assessed using (1) frozen sections, (2) documented commercially available monoclonal antibody (bcl-2/124, Dako), (3) automation of immunoperoxidase technique (Ventana) and (4) quantitative evaluation of results by image analysis (SAMBA) and statistical analysis of quantitative data (BMDP software). Bcl-2 protein expression was correlated with current prognostic indicators and with molecular markers detected by the same procedure as for Bcl-2. It was shown that Bcl-2 expression is not related to patients' age, tumour size and type or lymph node status, but an inverse relationship was observed between Bcl-2 and tumour grade (P < 0.0001). An inverse relationship was also observed between Bcl-2 expression and p53 (P < 0.0001), Ki67/MIB1 antigen- (P = 0.0012), and P-gp- (P = 0.002) positive immunoreactions. In contrast, anti-Bcl-2 positive reaction was significantly associated with ER-positive (P < 0.001) and with ER/PR-positive or ER/PR/pS2-positive immunoreactions (P < or = 0.005). Bcl-2 expression was independent of CD31 and cathepsin D expression. Thus, Bcl-2 protein, thought to be antiapoptotic, exhibits parodoxical expression in human breast carcinomas. It is strongly detected in low-grade tumours (well-differentiated) with low (MIB1) growth fraction, but is independent of the tumour progression (size, node status, CD31, and cathepsin D). Bcl-2 acting on apoptosis is related to p53 gene abnormalities in breast carcinomas. Bcl-2 protein expression may also be involved in response to endocrine therapy (associated to ER/PR/pS2 positive immunoreactions) and probably with chemoresistance mechanisms (inverse relationship with P-gp).     

Frequent expression of Bcl-2 in renal-cell carcinomas carrying wild-type p53

Lunate excision alone is seldom utilized in the management of Kienbock's disease due to concerns about progressive carpal collapse following removal of this central carpal bone. We report a 32-year follow-up of a patient who underwent lunate excision only for treatment of Kienbock's disease with a successful outcome. Although lunate excision is thought to be associated with a high failure rate, a review of the literature suggests that success rates following lunate excision are comparable to those reported for other more conventional techniques such as radial shortening, ulnar lengthening, limited carpal fusions, and proximal row carpectomy. The current perception that lunate excision is associated with a high failure rate is not supported in the literature. As such, it may not be appropriate to assign this operation to the category of "historical interest only."     

Bcl-2 protein expression during murine development

Bcl-2 functions as a death repressor molecule in an evolutionarily conserved cell death pathway. To further explore the role of Bcl-2 in development, we assessed its pattern of expression during murine embryogenesis. Immunohistochemical analysis demonstrates that Bcl-2 is widely expressed early in mouse fetal development in tissues derived from all three germ layers and that this expression becomes restricted with maturation. Within epithelium, the E12.5 lung bud demonstrates a proximal to distal gradient of Bcl-2 expression which is enhanced by E18.5. Bcl-2 is expressed throughout the intestinal epithelium through E14.5, but by E18.5 only cells in the crypts and lower villi express Bcl-2. In the mesoderm-derived kidney, Bcl-2 is expressed in both the ureteric bud and metanephric cap tissue at E12.5. Tubular structures also express Bcl-2, although overall levels drop as the kidney matures. Retinal neuroepithelial cells uniformly express Bcl-2 until cells begin to differentiate and then display the topographic distribution maintained into adulthood. The developing limb provides a clear example where Bcl-2 is restricted to zones of cell survival; Bcl-2 is expressed in the digital zones but not in the interdigital zones of cell death. The wide distribution of Bcl-2 in the developing mouse suggests that many immature cells require a death repressor molecule or that Bcl-2 may have roles beyond regulating developmental cell death.     

Prognostic significance of Bcl-2 protein expression and Bcl-2 gene rearrangement in diffuse aggressive non-Hodgkin's lymphoma

The prognostic significance of Bcl-2 protein expression and bcl-2 gene rearrangement in diffuse large cell lymphomas (DLCL) is controversial. Bcl-2 protein expression prevents apoptosis and may have an important role in clinical drug resistance. The presence of a bcl-2 gene rearrangement in de novo DLCL suggests a possible follicle center cell origin and perhaps a distinct clinical behavior more akin to low-grade non-Hodgkin's lymphoma (NHL). The purpose of this study was to determine the impact of Bcl-2 protein expression and bcl-2 gene rearrangement (mbr and mcr) on survival of a cohort of patients with DLCL who were uniformly evaluated and treated with effective chemotherapy. Patients included the original MACOP-B cohort (n = 121) and the initial 18 patients treated with the VACOP-B regimen (total = 139). All patients had advanced-stage disease, were 16 to 70 years old, and corresponded to Working Formulation categories F, G, or H. No patients had prior treatment, discordant lymphoma, or human immunodeficiency virus seropositivity. Paraffin sections from diagnostic biopsies were analyzed for bcl-2 gene rearrangement including mbr and mcr breakpoints by polymerase chain reaction and Bcl-2 protein expression by immunohistochemistry. With a median follow-up of 81 months, overall (OS), disease-free (DFS), and relapse-free survival (RFS) were measured to determine the prognostic significance of these parameters. Analyzable DNA was present in 118 of 139 (85%) cases, with 14 demonstrating a bcl-2 rearrangement (11 mbr, 3 mcr). All 14 of these bcl-2 gene rearrangement-positive cases were found in the 102 patients with a B-cell immunophenotype, but the presence of this rearrangement had no significant influence on survival. Bcl-2 protein expression was interpretable in 116 of 139 (83%) cases, with immunopositivity detected in 54 of 116 (47%). Using a cut-off of greater than 10% Bcl-2 immunopositive tumor cells for analysis, positive Bcl-2 protein expression was seen in 28 of 116 (24%) patients and the presence of this expression correlated with decreased 8-year OS (34% v 60%, P < .01), DFS (32% v 66%, P < .001), and RFS (25% v 59%, P < .001). Bcl-2 protein expression remained significant in multivariate analysis that included the clinical international prognostic index factors and immunophenotype (P < .02). In conclusion, although bcl-2 gene rearrangement status could not be shown to have an impact on outcome, Bcl-2 protein expression is a strong significant predictor of OS, DFS, and RFS in DLCLs.     

Bcl-2 protein expression: association with p53 and prognosis in colorectal cancer

In myocardial SPECT perfusion imaging, reorientation algorithms from transaxial image planes are used to generate short- and long-axis views of myocardial tracer uptake. We performed phantom experiments with 201Tl to delineate how image reorientation affects the results of quantitative image analysis. METHODS: Thirty consecutive patient studies were analyzed to characterize the distribution of the angle of reorientation in a clinical setting. Short-axis SPECT images of a cardiac phantom with and without a 180 degrees cold-spot insert were reconstructed with three different backprojection filters (ramp, Metz and Butterworth) and reoriented through different angles ranging from 45 degrees to 89 degrees. Four interpolation algorithms were used to calculate from the transaxial images the pixel values of the reoriented images: (a) a simple interpolator that averages the pixel values of the eight neighboring pixels of the transaxial image; (b) a three-dimensional linear interpolator; (c) a hybrid interpolator that combines a two-dimensional linear in-plane with a one-dimensional cubic across-plane interpolation; and (d) a three-dimensional cubic convolution interpolator. Images were reoriented twice with opposite angles so that the original and the reoriented images could be directly compared. Circumferential profile analysis was applied to determine the root mean square error of corresponding profiles and the difference of the extent and the severity of perfusion defects. Single and multivariate analyses of variance (ANOVA) were used to compare the effects of the reorientation angle, the backprojection filter and the interpolation algorithm. RESULTS: In the clinical studies, the angle between the transaxial and reoriented images was 75 degrees +/- 10 degrees (s.d.). In 48 phantom experiments, multivariate ANOVA demonstrated that the backprojection filter and the interpolation algorithm significantly affect the circumferential profiles and the extent and severity of a perfusion defect (p < 0.05). In contrast, the angle of reorientation was not a significant factor (p = ns). By univariate analysis, the three-dimensional cubic interpolator was associated with significantly (p < 0.05) less error than the simple and three-dimensional linear algorithms. Relative computation times (simple interpolator = 100%) were 119% for the three-dimensional linear, 136% for the hybrid and 243% for the three-dimensional cubic interpolator. CONCLUSION: For quantitative analysis of myocardial SPECT perfusion images, a Metz filter for filtered backprojection in combination with a three-dimensional cubic convolution interpolation for image reorientation appears to offer improved accuracy.     

Mucin antigen expression in gastric carcinomas of young and old adults

The striking differences in the histological features of gastric cancers in young and old adults have been thought to be related to differences in the biological behavior of these cancers. Recently, a new grading system (Goseki's classification) showed that the prognosis of the patient is particularly related to the mucin content of the carcinoma. In this study, we examined differences in mucin antigen expression in cancers from young and old adults and whether antigen expression is related to the clinical outcome. The expression of two mucin core proteins (DF3 antigen [MUC1 gene product] and MRP antigen [MUC2 gene product] and a mucin-type carbohydrate antigen [sialosyl-Tn; STn]) was examined immunohistochemically in gastric cancers from 69 young adults (30 to 39 years of age) and 110 old adults (80 to 89 years of age). The incidence rates of the three histological types (tubular adenocarcinoma, poorly differentiated adenocarcinoma, and signet-ring cell carcinoma) were different in the young and old adults. However, among the mucin antigens examined, only DF3 showed significantly higher frequency of expression in the old adults, and the difference was seen only in tubular adenocarcinomas (young, 43%; old, 68%) and poorly differentiated adenocarcinomas (young, 19%; old, 49%). In these two histological types, there was no difference in the frequency of MRP or STn expression between the young and old adults, although the old adults showed a high incidence of intestinal metaplasia that was positive for both antigens. Signet-ring cell carcinomas showed no significant difference in expression rates of the three antigens in young and old adults, but there were significantly higher expression rates in young patients for both MRP (young, 67%; old, 65%) and STn (young, 71%; old, 57%) and a lower rate of DF3 expression (young, 0%; old, 14%). In both young and old adults, the patients with DF3-positive carcinomas showed significantly poorer survival than those without DF3 expression, whereas there was not significant difference in the survival of the patient groups with positive and negative MRP or STn reactivity. In conclusion, the expression of DF3 was influenced by the age of patients and was related to the outcome. In contrast, MRP and STn expression was related more to the histological pattern of the tumor than to the age of the patient and did not correlate with the outcome.     

Bcl-2 expression and apoptosis in nephrotoxic nephritis

The aim of this paper is to present a conceptual and practical framework of the case-control design in medical research. To illustrate this method, practical examples directed to clinicians and other health professionals interested in medical research are presented. The case-control method is very versatile and allows for multiple applications. Guidelines for the selection of cases and controls, and some considerations on sample size are presented. In the statistical analysis we use concrete examples of how to estimate odds ratios, confidence intervals, and methods to control for potential confounders, from stratified analysis to logistic regression.     

Toxin-induced activation of Rho GTP-binding protein increases Bcl-2 expression and influences mitochondrial homeostasis

It is now well established that apoptosis plays a pivotal role in several physiological and pathological situations. Consequently, the mechanisms controlling the cell fate are currently the subject of intense investigation. In this work, we report that an Escherichia coli protein toxin (Cytotoxic Necrotizing Factor 1, CNF1) which activates the Rho GTP-binding protein and prevent apoptosis in epithelial cells was able to: (i) influence the mitochondrial homeostasis and (ii) modulate the expression of proteins belonging to the Bcl-2 family. In particular, the content of the antiapoptotic products Bcl-2 and Bcl-XL resulted to be increased in treated cells, whereas the expression of the proapoptotic protein Bax remained unaltered. CNF1 induces cell spreading via activation of Rho and cell spreading has been reported to promote cell survival. Cytochalasin B, which provokes most of the morphological changes typical of CNF1, including cell spreading, but without the involvement of Rho, was unable to counteract apoptosis. Altogether our results suggest a link between the Rho GTP-binding protein and the regulation of the mitochondrial homeostasis via an effect on the antiapoptotic proteins of the Bcl-2 family.     

Subcellular localization of Bcl-2 protein in synovial sarcoma

The subcellular localization of Bcl-2 protein in surgically resected, fixed-frozen tissue specimens of seven tumors from six cases of synovial sarcoma and a synovial sarcoma cell line was examined using laser-scanning confocal microscopy and immunoelectron microscopy. Bcl-2 protein in synovial sarcoma cells was detected in the nuclear envelope, endoplasmic reticulum membrane, and mitochondrial circumference. Based on the finding of pre-embedding immunoelectron-microscopy observation, the distribution of Bcl-2 protein in such membranous organella was patchy. A computer-based image analysis failed to reveal any quantitative differences between the inner and the outer mitochondrial membranes. The tumorigenesis of synovial sarcoma is also discussed from the viewpoint of Bcl-2 overexpression.     

Channel formation by antiapoptotic protein Bcl-2

Bcl-2 is the prototypical member of a large family of apoptosis-regulating proteins, consisting of blockers and promoters of cell death. The three-dimensional structure of a Bcl-2 homologue, Bcl-XL, suggests striking similarity to the pore-forming domains of diphtheria toxin and the bacterial colicins, prompting exploration of whether Bcl-2 is capable of forming pores in lipid membranes. Using chloride efflux from KCl-loaded unilamellar lipid vesicles as an assay, purified recombinant Bcl-2 protein exhibited pore-forming activity with properties similar to those of the bacterial toxins, diphtheria toxin, and colicins, i.e., dependence on low pH and acidic lipid membranes. In contrast, a mutant of Bcl-2 lacking the two core hydrophobic alpha-helices (helices 5 and 6), predicted to be required for membrane insertion and channel formation, produced only nonspecific effects. In planar lipid bilayers, where detection of single channels is possible, Bcl-2 formed discrete ion-conducting, cation-selective channels, whereas the Bcl-2 (Deltah5, 6) mutant did not. The most frequent conductance observed (18 +/- 2 pS in 0.5 M KCl at pH 7.4) is consistent with a four-helix bundle structure arising from Bcl-2 dimers. However, larger channel conductances (41 +/- 2 pS and 90 +/- 10 pS) also were detected with progressively lower occurrence, implying the step-wise formation of larger oligomers of Bcl-2 in membranes. These findings thus provide biophysical evidence that Bcl-2 forms channels in lipid membranes, suggesting a novel function for this antiapoptotic protein.     

P53 protein immunohistochemical expression in colonic adenomas with and without associated carcinoma

On the basis of the positive outcome of animal experiments, several large placebo-controlled trials are underway and aiming for the first time at the prevention of an immune-mediated disease, type 1 diabetes. The first of these trials, The Deutsche Nicotinamide Intervention Study (DENIS), evaluated the clinical efficacy of high doses of nicotinamide in children at high risk for IDDM. Nicotinamide has been shown to protect beta-cells from inflammatory insults and to improve residual beta-cell function in patients after onset of IDDM. Individuals at high risk for developing IDDM within 3 years were identified by screening the siblings (age 3-12 years) of patients with IDDM for the presence of high titer (> or =20 Juvenile Diabetes Foundation [JDF] U) islet cell antibodies. Probands (n = 55) were randomized into placebo and nicotinamide (slow release, 1.2 g x m(-2) x day(-1)) receiving groups and followed prospectively in a controlled clinical trial using a sequential design. Rates of diabetes onset were similar in both groups throughout the observation period (maximum 3.8 years, median 2.1 years). This sequential design provides a 10% probability of a type II error against a reduction of the cumulative diabetes incidence at 3 years from 30 to 6% by nicotinamide. The trial was terminated when the second sequential interim analysis after the eleventh case of diabetes showed that the trial had failed to detect a reduction of the cumulative diabetes incidence at 3 years from 30 to 6% (P = 0.97). The group receiving nicotinamide exhibited decreased first-phase insulin secretion in response to intravenous glucose (P = 0.03). No other side effects were observed. We conclude that in this subgroup of diabetes-prone individuals at very high risk and with an assumed rapid disease progression, nicotinamide treatment did not cause a major decrease or delay of diabetes development. However, the data do not exclude the possibility of a less strong, but potentially meaningful, risk reduction in this cohort, or a major clinical effect of nicotinamide in individuals with less risk of progression to IDDM than studied here.     

Estradiol upregulates Bcl-2 expression in adult brain neurons

Bcl-2, a protein which negatively modulates apoptosis, is up-regulated by estrogen in several tissues. To determine the effect of estradiol on Bcl-2 in the adult brain, its immunoreactive distribution was examined in the hypothalamic arcuate nucleus of female rats under different endocrine conditions. The number of Bcl-2-immunoreactive neurons was significantly increased (p < 0.001) on the day of estrus compared with proestrus, diestrus and metestrus, was decreased by ovariectomy and showed a dose-response increase after estradiol administration to ovariectomized rats. Progesterone, when injected simultaneously with estradiol, reduced the effect of estradiol. These findings indicate that ovarian hormones regulate Bcl-2 in hypothalamic neurons and suggest that this protein may be involved in the neuroprotective effects of estrogen.     

胃癌组织中HER2基因状态与p21蛋白表达的关系研究

Objective:We aimed to analysis the HER2 gene status and its relationship with p21 protein expression in gastric carcinoma.Methods:Fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC) techniques were used to detect HER2 gene status and p53 protein in 59 cases of gastric cancer.Results:FISH detection of HER2 gene amplification rate was 16.9% (10/59),HER2 gene amplification in 49 cases without copy number gain and gene amplification were a total of 49.2% (29/59).HER2 protein expression was 42.4% (25/59),HER2 gene amplification rates in patients with +++,++ HER2 protein expression were 3/3 and 5/8,while in patients with + HER2 protein expression,it was 2/14,there was significant difference (P < 0.05).p21 protein expression rate was 49.2% (29/59),HER2 gene amplification rates and p21 protein expression had significant difference in tumor invasion depth,lymph node metastasis (P < 0.05);had no statistical significance in histological type,age,gender differences (P > 0.05).Conclusion:HER2 gene amplification rate and gene copy number had positively correlation with p21 protein expression,HER2 gene status and expression of p21 protein combined detection can provide a reference value in gastric cancer metastasis,patient's condition development and prognosis,it also can guide clinical development of individual treatment.     

Bcl-2 expression and glucocorticoid-induced apoptosis of leukemic and lymphoma cells

The lytic response of lymphoid cells to glucocorticoid hormones (GC) is prototypical of the induction of apoptosis: a special form of cellular demise for the removal of unwanted or redundant cells. Initiation and execution of a death programme are therefore major checkpoints in GC-sensitivity. Although Bcl-2 protein can prevent or delay apoptosis of lymphoma and leukemia cells, exposed to multiple cytotoxic agents, its antagonism of GC-induced apoptosis appears most critical in conferring resistance to corticosteroids. Moreover, Bcl-2 may modulate GC-signalling to apoptosis through its association with fundamental cellular processes such as energy state, Ca2+ homeostasis and transmembrane transport. However, this signalling pathway can also be interrupted by Bcl-2- independent mechanisms. This review discusses the various cellular and oncogenetic factors that control GC sensitivity of leukemia/lymphoma cells and proposes a hypothesis of how GC may induce a death programme, sensitive to blockade by Bcl-2.     

Different KGF expression in squamous cell carcinomas of the head and neck and in normal mucosa

Inducible nitric oxide synthase (iNOS) and TGF-beta were localized by immunocytochemistry in skin lesions from patients across the leprosy spectrum, and from patients undergoing reversal reaction. iNOS expression was highest at the tuberculoid pole of the spectrum, and increased during reversal reaction. TGF-beta was observed throughout the leprosy spectrum, but was highest at the lepromatous pole. Levels of TGF-beta decreased during reversal reaction. Reduced levels of TGF-beta may contribute to unregulated inflammatory responses during reactional episodes.     

Human Bcl-2 expression delays ultraviolet-induced apoptosis in marsupial cells

We have introduced the human bcl-2 gene under the control of the human metallothionein MTIIA promoter into the rat kangaroo PtK2 cell line. Two independent clones were obtained in which the levels of Bcl-2 protein expression can be controlled by the addition of metals in the culture medium. These cell lines were employed to investigate the effects of this protein in UV-induced apoptosis. Overexpression of Bcl-2 in PtK2 cells resulted in a delay in the appearance of apoptosis markers, such as chromatin condensation and internucleosomal DNA fragmentation. However, colony survival after UV was not affected, suggesting that Bcl-2 did not impose a definitive block for cell death. The elimination of cyclobutane pyrimidine dimers through photoreactivation 24 h after irradiation in cells overexpressing Bcl-2 did not affect apoptosis. This indicates that irreversible events in the signaling pathway of apoptosis occur in the period between irradiation and photoreactivation even in the presence of high levels of Bcl-2 protein can delay the onset of UV-induced apoptosis in these marsupial cells, early events triggered by the pyrimidine dimers, upstream from the Bcl-2 action, lead the cell to a state committed to die.