High Expression Of Bcl-2 Protein Research Articles

Specific Primer for Human Bcl-2-Protein Expression Analysis

Bcl-2 protein (B-cell lymphoma 2) is one of the proteins in the Bcl-2 protein family that are anti apoptotic and encoded by the BCL-2 gene. This protein controls the permeability of the outer membrane of the mitochondria, which plays a role in the release of cytochrome c and several other proteins that contribute to cell death. Bcl-2 protein plays a role in regulating cell death and regulating the balance between new and old cells. Several studies showed that changes in the structure and function of Bcl-2 play a role in cell apoptosis disorders, producing cancer cells. High expression of Bcl-2 protein is seen in lung, breast, lymph node and brain cancers. Analysis of Bcl-2 protein expression at the molecular level in cancer cell is needed to determine the expression level of this protein. This study aims to produce the Bcl-2 primer for qPCR method that specifically identifies the target gene, and can be used to see the BCL-2 gene expression level in cells. A BCL-2 sequence was obtained from the National Center for Biotechnology Information (NCBI). The next process is further analysis with Primer-BLAST (NCBI). Some primer candidates are produced and undergo another selection based on several criteria. Finally, one pair of potential primers was obtained for the reaction of qPCR to BCL-2. Primer specificity was tested with Basic Local Alignment Tools (BLAST) tools and result in specific primer for human BCL-2 gene. Annealing temperature optimization shows that the primer can react optimally at 59∘C.
 
 Keywords: Bcl-2 protein, BCL-2 gene, qPCR, primer, gene expression, apoptosis

Effect of plant based soya milk extender on Bcl-2 family proteins in cryopreserved Murrah buffalo spermatozoa

The study was designed to investigate the effect of plant based soya milk Tris (SMT)- extender used for cryopreservation of buffalo (Bubalus bubalis) spermatozoa on the expression of Bcl-2 family proteins that regulates the susceptibility of cells to apoptosis. Fresh buffalo semen (control without dilution) was cryopreserved in conventional EYT (20% egg yolk v/v in Tris) and SMT (25% soya milk v/v in Tris) extender and used for the assessment of expression of Bcl-2 family proteins. Proteins extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random were separated using SDS–PAGE followed by immunoblotting against Bcl-2, Bcl-xl, Bax and Bak. In addition, comet assay was performed to compare sperm DNA integrity in frozen thawed semen samples. The obtained results indicated a significant (p < 0.05) reduction in the expression of Bcl-2, Bax and Bak in cryopreserved semen samples. The results clearly demonstrated a significant (p<0.05) higher expression of Bcl-2 protein in SMT extender as compared to EYT extender indicating its better resistance to cryodamage.

Vulnerability of Small-Cell Lung Cancer to Apoptosis Induced by the Combination of BET Bromodomain Proteins and BCL2 Inhibitors.

Ten percent to 15% of all lung cancers are small-cell lung cancer (SCLC). SCLC usually grows and metastasizes before it is diagnosed and relapses rapidly upon treatment. Unfortunately, no new targeted agent has been approved in the past 30 years for patients with SCLC. The BET (bromodomain and extraterminal) proteins bind acetylated histones and recruit protein complexes to promote transcription initiation and elongation. BET proteins have been shown to regulate expression of key genes in oncogenesis, such as MYC, CCND2, and BCL2L1 Here, we demonstrate that approximately 50% of SCLC cell lines are exquisitely sensitive to growth inhibition by the BET inhibitor, ABBV-075. The majority of these SCLC cell lines underwent apoptosis in response to ABBV-075 treatment via induction of caspase-3/7 activity. ABBV-075 enhanced the expression of proapoptotic protein BIM and downregulated antiapoptotic proteins BCL2 and BCLxl to a lesser extent. Furthermore, BET inhibition increased BCL2-BIM complex, thus priming the cells for apoptosis. Indeed, strong synergy was observed both in vitro and in vivo when cotreating the cells with BET inhibitor and the BH3-mimetic, BCL2 inhibitor venetoclax (ABT-199). ABBV-075 interaction with venetoclax positively correlated with BCL2 expression. Taken together, our studies provide a rationale for treating SCLC with BET and BCL2 inhibitors in tumors with high BCL2 protein expression. Mol Cancer Ther; 16(8); 1511-20. ©2017 AACR.

Targeted delivery of Bcl-2 conversion gene by MPEG-PCL-PEI-FA cationic copolymer to combat therapeutic resistant cancer

Deregulation of anti-apoptosis Bcl-2 protein expression was a key feature in human cancers with therapeutic resistance. Nuclear receptor Nur77 could induce the conformation change of Bcl-2 protein and converted it into an apoptosis inducer by “enemy to friend” strategy. However, the safe and effective delivery of this gene to combat therapeutic resistant cancer remained largely unexplored. In this report, we designed an amphiphilic cationic MPEG-PCL-PEI-FA copolymer, comprising biocompatible and hydrophilic methoxy-poly(ethylene glycol) (MPEG), biodegradable and hydrophobic poly(ε-caprolactone) (PCL), cationic poly(ethylene imine) (PEI) segments, and folic acid (FA) as targeting group, as a high efficient Nur77 gene carrier to folate receptor (FR) highly expressed and therapeutic resistant HeLa/Bcl-2 cancer cells. Interestingly, due to the incorporation of PCL and PEG segments, this MPEG-PCL-PEI-FA copolymer showed less toxicity but better gene transfection efficiency than non-viral gene carrier gold standard PEI (25kDa). This might be due to the formation of micelles to stabilize polyplex for enhanced gene transfection ability. More importantly, MPEG-PCL-PEI-FA copolymer exhibited excellent growth inhibition ability on therapeutic resistant HeLa/Bcl-2 cancer cells, which was FR overexpressed HeLa cervical cancer cells with high expression of Bcl-2 protein, thanks to its FA induced targeting ability, high gene transfection efficiency, and low cytotoxicity. This work signifies the first time that cationic amphiphilic MPEG-PCL-PEI-FA copolymers could be utilized for the gene delivery to therapeutic resistant cancer cells with high expression of anti-apoptosis Bcl-2 protein and the positive results are encouraging for the further design of polymeric platforms for combating drug resistant tumors.

Abstract 4692: Targeting the BET bromodomain proteins and anti-apoptotic protein BCL2 in small cell lung cancer

Abstract 10% to 15% of all lung cancers are small cell lung cancer (SCLC), named for the size of the cancer cells when seen under a microscope. SCLC often starts in the bronchi near the center of the chest. It usually grows and spreads quickly to distant parts of the body before it is diagnosed. Unfortunately, no new treatment has been identified in the past 30 years for patients with SCLC. The BET (bromodomain and extra-terminal) proteins bind acetylated histones and recruit protein complexes to promote transcription initiation and elongation. In hematologic cancers, BET proteins have been shown to regulate expression of MYC and other oncogenic transcription factors that drive disease pathology. Pharmacologic inhibition of BET binding to acetylated proteins has been shown to inhibit tumor growth in MYC-dependent cancers, such as acute myeloid leukemia, multiple myeloma and neuroblastoma. Here, we demonstrate that ∼40% of SCLC cell lines are exquisitely sensitive to growth inhibition by the BET inhibitor ABBV-075. Whereas most SCLC cell lines undergo cell cycle arrest with ABBV-075 treatment, a few cell lines undergo apoptosis. Sensitivity does not correlate with MYC status (amplification/expression). However, downregulation of anti-apoptotic protein BCLxL and BCL2 by ABBV-075 was observed in sensitive SCLC cells. Synergy was observed when co-treating BET inhibitor and BCL2 inhibitors, venetoclax (ABT-199) or navitoclax (ABT-263), and it positively correlated with BCL2 expression. Thus, high BCL2 protein expression could potentially be a biomarker predictive of these therapeutic combinations. The potential higher synergy with venetoclax may be explained by downregulation of BCLxL with BET inhibition and potent activity of venetoclax against BCL2. Our studies have provided evidence for treating SCLC by BET inhibition and/or combination with BCL2 inhibitors and a rationale for the clinical development of BET inhibitors in this disease with high unmet medical need. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Lloyd T. Lam, Xiaoyu Lin, Emily Faivre, Ziping Yang, Xiaoli Huang, Ricky Bellin, Xin Lu, Yu Shen, Tamar Uziel. Targeting the BET bromodomain proteins and anti-apoptotic protein BCL2 in small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4692.

Coexpression of MYC/Bcl-2 in Diffuse Large B-Cell Lymphoma: A Clinic Research

BackgroudDiffuse large B-cell lymphoma (DLBCL) is the most commonly occurred non-Hodgkin's lymphoma (NHL). With the development of chemotherapy and immunotherapy, some patients get a good prognosis, but still a considerable number of patients suffer from a strongly aggressive DLBCL with poor treatment effects. Due to the discovery of "double hit" of MYC/Bcl-2 genes, the coexpression of these two proteins has attracted more attention. Further investigations will be necessary to investigate the role of MYC/Bcl-2 co-expression in the clinical features of DLBCL as well as its relationship with prognosisto explore the significance of MYC/Bcl-2 protein coexpression in DLBCL.Methods89 confirmed DLBCL cases treated with the chemotherapy regimen of CHOP or R-CHOP between February 2008 and March 2014 were collected. Complete clinical data and paraffin-embedded tissues samples were available from these cases. immunohistochemistry analysis of MYC and Bcl-2 protein expression were carried out. In addition, the relationship between MYC/Bcl-2 protein coexpression and factors such as the age, gender, primary site, Ann Arbor staging, international prognostic index (IPI), immunohistochemical Hans type, and Ki-67 expression rate of patients were statistically analyzed in this paper. Furthermore, we investigated how the abovementioned indicators and whether or not Rituximab or MYC/Bcl-2 coexpression was applied were associated with the progression-free survival (PFS) and overall survival (OS) of patients.Results1. 89 cases with DLBCL were collected. The median age was 65 years with a range of 18-82. 54 cases were over 60 years old. 64 patients had a primary tumor in the lymph nodes .As per the IPI score, there were 30 low-risk cases (IPI<3). There were 27 cases at Ann Arbor stage I-II, 62 cases at stage III-IV, 31 GCB cases, 58 nonGCB cases. For 61 cases, the expression rate of Ki-67 was more than 70%.There were 41 cases treated with the chemotherapy regimen of R-CHOP and 48 cases treated with chemotherapy regimen of CHOP.MYC/Bcl-2 coexpression was associated with high-risk IPI score (P = 0.022).2. 37 cases had high MYC protein expression, accounting for 41.5%. Also, 51cases (57.3%) had high Bcl-2 protein expression. There were 29 cases (32.6%) of MYC/Bcl-2 co-expression Compared with non-coexpression group, the median PFS (16m VS 33m, P<0.001) of the co-expression group decreased significantly.but there was not a significantly difference of the median OS. Moreover, Compared the MYC or Bcl-2 single expression group with the non-expression group, there were no significant differences among them.3. Single factor analysis with other common risk factors indicated that in the high-risk group, including advanced aged (grouping by 60 years old)、treating without rituximab III-IV stages, and high IPI score, both PFS and OS decreased, and the difference was statistically significant (P<0.05); When grouping by immunohistochemical Hans classification, primary site and Ki-67 expression (divided by 70%), OS comparison of the two groups showed no significant difference (P> 0.05).4. In the multivariate regression analysis, It was found that MYC/Bcl-2 coexpression, IPI score, age, and use of rituximab, staging were independent prognostic factors to PFS (P <0.05), while IPI score, staging and age were independent prognostic risk factors to OS (P<0.05). However,MYC/Bcl-2 coexpression temporarily could not be considered as an independent risk factor for OS.ConclusionsBcl-2/MYC protein coexpression is significantly associated with IPI score of high-risk patients. Patients with MYC/Bcl-2 coexpression had a poorer prognosis than non-coexpression, with shortened PFS. They had a prognosis poorer than single high expressions of MYC or Bcl-2. The influence of MYC/Bcl-2 coexpression to the OS of DLBCL needs a longer follow-up time to study.MYC/Bcl-2 coexpression, IPI score, age, and use of rituximab, staging were independent prognostic factors to prognosis. DisclosuresNo relevant conflicts of interest to declare.

High expression of Bcl-2 protein predicts favorable outcome in non-small cell lung cancer: evidence from a systematic review and meta-analysis.

The prognostic value of Bcl-2 protein expression in non-small cell lung cancer (NSCLC) is under debate. We therefore systematically reviewed the evidence for Bcl-2 protein effects on NSCLC survival to elucidate this issue. An electronic search in Pubmed and Embase complemented by manual searches in article references were conducted to identify eligible studies to evaluate the association between Bcl-2 protein expression and overall survival (OS) as well as disease free survival (DFS) of NSCLC patients. Combined hazard ratios (HRs) with corresponding 95% confidence intervals (95%CIs) were pooled using the random-effects model. A total of 50 trials (including 52 cohorts) encompassing 7,765 patients were pooled in the meta-analysis regarding Bcl-2 expression and OS of NSCLC patients. High expression of Bcl-2 protein had a favorable impact (HR=0.76, 95%CI=0.67-0.86). In the group of Bcl-2 expression and DFS, 11 studies including 2,634 patients were included. The synthesized result indicated high expression of Bcl-2 protein might predict good DFS (HR=0.85, 95%CI=0.75-0.95). Our present meta-analysis demonstrated favorable prognostic values of Bcl-2 expression in patients with NSCLC. Further prospective trails are welcomed to validate the utility of assessing Bcl-2 in NSCLC patient management.

Effects of hypoxia preconditioning on inflammatory responses and apoptosis in the rat model after spinal cord injury

Objective To observe the effects of hypoxia preconditioning on inflammatory cytokines and apoptosis after spinal cord injury in the rat model.Methods 154 adult female wistar rats were randomly divided into three groups,including the spinal cord injury group,the spinal cord injury ± hypoxia precondition (HP) group,the sham operation group.Immunohistochemistry was performed to determine the expressions of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β)in spinal cord injury area at five time points (2,6,12 h,1,3 d).BBB score was used to evaluate the function recovery of hind limb.The method of immunohistochemistry and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) were adopted to detect the level of B cell lymphoma/leukemia-2 (bcl-2) protein and the apoptotic cells at five time points (6 h,1,3,7,14 d)after spinal cord injury.Results The integral absorbance (IA) of TNF-α in HP group was decreased at 6 h and 12 h,compared with that in spinal cord injury group (5.187±0.628 vs.7.962±2.185; 3.219±0.724 vs.5.361 ±1.246,P＜0.05).The IA ofIL-1β in HP group was lower than spinal cord injury group at three time points (6,12 h,1 d) (3.845 ± 1.669 vs.6.674 ± 1.537 ; 6.241 ± 1.142 vs.9.961 ± 2.814 ; 2.264 ± 1.476 vs.4.418 ± 0.973,P ＜ 0.05).In HP group,the IA of bcl-2 protein was significantly higher than spinal cord injury group at any time point (4.335 ±0.563 vs.3.256 ± 1.024,P ＜0.05; 6.873 ±2.032 vs.5.029 ± 1.537,P ＜0.01 ; 11.869 ± 2.560 vs.6.893 ± 2.164,P ＜ 0.01 ; 10.340 ± 1.469 vs.8.074 ± 2.175,P ＜ 0.05 ; 8.143 ± 2.376 vs.5.238 ± 1.467,P ＜0.05).The apoptosis index (AI) of HP group was decreased on 1 d,3 d,7 d,14 d (20.63 ±5.86 vs.26.37 ±4.76,P＜0.05; 29.85 ±8.12 vs.37.96 ±3.81,P＜0.05; 31.56 ±4.01 vs.41.74 ±6.69,P ＜0.01 ; 22.66 ±5.24 vs.32.87 ±9.90,P ＜0.05),compared with the spinal cord injury group.The BBB scores of HP group was higher than spinal cord injury group on 7 d and 14 d (5.68 ±1.83 vs.3.06 ±1.21; 10.21 ±2.64 vs.6.45 ±1.97,P＜0.05).Conclusion Hypoxiapreconditioning can inhibit the inflammatory response of local tissue damage and reduce apoptosis through the high expression of bcl-2 protein,thus promote the neural functional recovery after spinal cord injury. Key words: Hypoxia precondition ; Spinal cord injury; Inflammation ; Apoptosis

Antiapoptotic and antioxidant effects of carvedilol and vitamin E protect against diabetic nephropathy and cardiomyopathy in diabetic Wistar albino rats.

Carvedilol is a novel β-adrenoreceptor blocker, with antioxidant properties inhibiting lipid peroxidation and preventing the depletion of endogenous antioxidants. Moreover, carvedilol was reported to enhance the expression of Bcl-2 gene, which has antioxidant and antiapoptotic effects. There are few researches testing the protective effect of carvedilol on the development of diabetic cardiomyopathy and nephropathy. In this study, we induced diabetes mellitus in male Wistar albino rats. We investigated carvedilol, as well as vitamin E, administrated in healthy and diabetic rats for 6 weeks to compare their effects on biochemical parameters and the expression of Bcl-2 protein in both myocardial and renal tissues by immunohistochemistry. The study showed that the diabetic rats not only had renal dysfunction and more myocardial damage, but also showed lower expression of Bcl-2 protein. Carvedilol and vitamin E treatments were associated with better renal function and less myocardial damage, lower blood glucose, and lipid peroxidation, higher antioxidant capacity, better serum lipids, and higher expression of Bcl-2 protein in diabetic rats. These results indicate that carvedilol and vitamin E treatments partly protect against myocardial and renal damage probably via their antioxidant and antiapoptotic properties in diabetic rats.

MicroRNA-34a Targets Bcl-2 and Sensitizes Human Hepatocellular Carcinoma Cells to Sorafenib Treatment

MiR-34a, a direct target of p53, has been shown to target several molecules associated with the cell cycle and cell survival pathways, and its dysregulation is implicated in cancer drug resistance or sensitivity in several human cancers. However, the correlation between miR-34a expression and chemoresistance has not been explored in HCC. In this study, we confirmed that miR-34a was significantly down-regulated in HCC tissues and HCC cell lines by qRT-PCR. HCC tissues with lower miR-34a expression displayed higher expression of Bcl-2 protein than those with high expression of miR-34a; therefore, an inverse correlation is evident between the miR-34a level and Bcl-2 expression. Moreover, patients with lower miR-34a expression had significantly poorer overall survival. Bioinformatics and luciferase reporter assays revealed that miR-34a binds the 3'-UTR of the Bcl-2 mRNA and represses its translation. Western blotting analysis and qRT-PCR confirmed that Bcl-2 is inhibited by miR-34a overexpression. Functional analyses indicated that the restoration of miR-34a reduced cell viability, promoted cell apoptosis and potentiated sorafenib-induced apoptosis and toxicity in HCC cell lines by inhibiting Bcl-2 expression. This study is the first to demonstrate that miR-34a induces sensitivity to the anti-tumor effect of sorafenib in human HCC cells, suggesting a potential role of miR-34a in the treatment of HCC.

Study on inhibitory effect of &lt;i&gt;Spica Prunellae&lt;/i&gt; extract on T Lymphoma cell EL-4 tumor

The objective of the study was to investigate the in-vivo anti-tumour activity of Spica prunellae extract, and to preliminarily explore the possible mechanism of in-vivo anti-tumour effect of Spica prunellae extract. Tumour inhibition rate and tumour apoptosis-related protein status were determined using the mice model of transplanted T lymphoma cell EL-4 tumour, and by immunohistochemical method. The results revealed that Spica prunellae extract showed certain tumour inhibitory effect, and compared with the model group. Tumour weight in Spica prunellae high-dose group was highly significantly different (P<0.01). Tumour weight in Spica prunellae medium-dose group was also significantly different (P<0.05) compared with the model group. Spica prunellae high-dose group enabled the high expression of Bcl-2 protein (47.54%) and low expression of Bax protein (13.14%). The study concluded that Spica prunellae extract has certain in vivo anti-tumour effect, which may be achieved through regulation of apoptosis related proteins.

Clinical significance of bcl-2 protein expression and classification algorithm in diffuse large B-cell lymphoma

To investigate the clinical significance of bcl-2 protein expression and three classification algorithms including Hans model, Chan model and Muris model in patients with diffuse large B-cell lymphoma (DLBCL). Two-hundred and thirty-seven cases were collected. Standard two-step EnVision method of immunohistochemical staining was used to assess the expression of Ki-67, CD3, CD45RO, CD20, CD79a, bcl-2, bcl-6, CD10, MUM-1, GCET-1, and FOXP-1. The phenotypic classifications were assessed according to the standard of the three models. The male (131 cases) to female (106 cases) ratio was about 1.24:1, the average age was 52.6 years. Seventy-five cases (31.6%, 75/237) showed primarily lymph node involvement. Gastrointestinal tract (71 cases) was the most commonly involved extra-nodal organ. All cases expressed one or more pan B cell markers such as CD20 (99.1%, 231/233). All patients with complete clinical follow-up data survived from 1 - 120 months. The expression of bcl-2 protein indicated an adverse prognosis (P = 0.019). Two-hundred and thirty cases were classified according to Hans model, with ninety five GCB cases and one-hundred and thirty five non-GCB cases. Survival analysis showed no difference between GCB and non-GCB subtypes (P = 0.102). According to the Chan's algorithm, sixty eight case of one-hundred and eighty one were belong to GCB group, with one-hundred and thirteen non-GCB cases. GCB subtype showed much better prognosis than non-GCB subtype according to survival analysis (P = 0.031). Additionally, bcl-2 protein expression in non-GCB subtype showed the worst survival. In Muris' model, 154 of 218 cases were classified as Group 1, while 64 cases were classified as Group 2. Group 1 showed better prognosis than Group 2 (P < 0.05). Non-GCB group is the more common type of DLBCL in China. High expression of bcl-2 protein is detected in the non-GCB group. Not all subgroups classified with different classification models indicate different prognosis. Bcl-2 expression combined with Chan's algorithm may be the best tool to predict outcome.

The cooperating mutation or “second hit” determines the immunologic visibility toward MYC-induced murine lymphomas

AbstractIn Eμ-myc transgenic animals lymphoma formation requires additional genetic alterations, which frequently comprise loss of p53 or overexpression of BCL-2. We describe that the nature of the “second hit” affects the ability of the immune system to contain lymphoma development. Tumors with disrupted p53 signaling killed the host more rapidly than BCL-2 overexpressing ones. Relaxing immunologic control, using Tyk2−/− mice or by Ab-mediated depletion of CD8+ T or natural killer (NK) cells accelerated formation of BCL-2–overexpressing lymphomas but not of those lacking p53. Most strikingly, enforced expression of BCL-2 prolonged disease latency in the absence of p53, whereas blocking p53 function in BCL-2–overexpressing tumors failed to accelerate disease. This shows that blocking apoptosis in p53-deficient cells by enforcing BCL-2 expression can mitigate disease progression increasing the “immunologic visibility.” In vitro cytotoxicity assays confirmed that high expression of BCL-2 protein facilitates NK and T cell–mediated killing. Moreover, we found that high BCL-2 expression is accompanied by significantly increased levels of the NKG2D ligand MULT1, which may account for the enhanced killing. Our findings provide first evidence that the nature of the second hit affects tumor immunosurveillance in c-MYC–driven lymphomas and define a potential shortcoming of antitumor therapies targeting BCL-2.

サルコイドーシス患者のBALF細胞におけるアポトーシス抑制因子Bcl-2, Bcl-xLの蛋白量の検討

サルコイドーシス患者のBALF細胞におけるアポトーシス抑制因子Bcl-2, Bcl-xLの蛋白量の検討

BH3 mimetic ABT-737 neutralizes resistance to FLT3 inhibitor treatment mediated by FLT3-independent expression of BCL2 in primary AML blasts

FLT3 defines a promising target for the treatment of acute myeloid leukemia (AML). In contrast to their efficacy in cell lines, FLT3-specific inhibitors as single agents have only modest clinical activity in patients with AML. As demonstrated here, overexpression of anti-apoptotic proteins of the BCL2 family leads to resistance against FLT3 inhibitors in a hematopoietic cell line model with activating FLT3 mutations. The susceptibility to FLT3 inhibition could be restored by treatment with the novel BH3 mimetic ABT-737. Primary AML samples tested in our study showed a high expression of BCL2 protein, but not of BCL-xL or MCL1. BCL2 protein levels were not reduced after dephosphorylation of FLT3 and its downstream target STAT5 in patient samples with FLT3 internal tandem duplications. Interestingly, treatment with ABT-737 caused apoptotic cell death in all primary AML samples at submicromolar level and synergized efficiently with FLT3 inhibition in AML samples with activating FLT3 mutations. In contrast to AML cell lines, BCR-ABL transformed human cells showed resistance to ABT-737, which might be due to the induction of MCL1 by BCR-ABL. Inhibition of BCL2 family members might define a novel highly efficient and specific strategy in the combined or monotreatment of AML.

Differential expression of apoptosis related proteins and nitric oxide synthases in Epstein Barr associated gastric carcinomas

To determine the incidence of Epstein Barr virus associated gastric carcinoma (GC) in Brazil and compare the expressions of apoptosis related proteins and nitric oxide synthases between EBV positive and negative gastric carcinoma. In situ hybridization of EBV-encoded small RNA-1 (EBER-1) and PCR was performed to identify the presence of EBV in GCs. Immunohistochemistry was used to identify expressions of bcl-2, bcl-xl, bak, bax, p53, NOS-1, NOS-2, and NOS-3 proteins in 25 EBV positive GCs and in 103 EBV negative GCS. 12% of the cases of GC (25/208) showed EBER-1 and EBNA-1 expression. The cases were preferentially of diffuse type with intense lymphoid infiltrate in the stroma. EBV associated GCs showed higher expression of bcl-2 protein and lower expression of bak protein than in EBV negative GCs. Indeed, expressions of NOS-1 and NOS-3 were frequently observed in EBV associated GCs. Our data suggest that EBV infection may protect tumor cells from apoptosis, giving them the capacity for permanent cell cycling and proliferation. In addition, EBV positive GCs show high expression of constitutive NOS that could influence tumor progression and aggressiveness.

Biological features of sporadic colorectal carcinoma with high-frequency microsatellite instability: special reference to tumor proliferation and apoptosis.

We evaluated the relationship between biological behavior and microsatellite instability (MSI) status, with or without p53 status, in sporadic colorectal carcinoma. MSI was analyzed with regard to biological features such as cellular proliferation and apoptotic cell death, in addition to clinicopathological features, in 87 patients with sporadic colorectal carcinoma. Fourteen (16.1%) of 87 tumors showed instability at two or more of the five loci examined (high-frequency MSI [MSI-H]). Four demonstrated instability at one locus (low-frequency MSI [MSI-L]), and 69 showed no instability (microsatellite-stable [MSS]). The MSI-H tumors tended to be located in the proximal colon and more often were mucinous carcinoma. The MSI-H tumors also tended to be in patients with multiple colorectal carcinomas and to demonstrate, rarely, an infiltrating growth pattern or venous invasion. The incidence of p53 protein overexpression in the MSI-H tumors was significantly lower than that in the MSI-L/MSS tumors (21% vs 54%). There was no significant difference in the proliferating-cell nuclear antigen (PCNA) labeling index (PI) or apoptotic index (AI) between the MSI-H and MSI-L/MSS tumors. The AI in the MSI-H tumors with p53 overexpression was significantly lower than that in the MSI-H tumors without p53 overexpression, and was also significantly lower than that in the MSI-L/MSS tumors with p53 overexpression. In the MSI-H tumors with p53 overexpression, no expression of BAX protein was found, and there was high expression of bcl-2 protein, resulting in a low BAX/bcl-2 ratio. In sporadic colorectal carcinoma, an MSI-H tumor with p53 protein overexpression may display aggressive biological features.

Apoptosis and expression of its regulatory proteins in soft tissue sarcomas

Information for the occurrence and extent of apoptosis in soft tissue sarcoma (STS) and their clinical implication are limited. In 102 cases of STSs, apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method and expression of Bcl-2, Bax, MIB-1 and p53 protein was examined immunohistochemically. The apoptotic index of the STSs ranged from 0 to 15% with an average value of 1.9%. The mean values of positive cell staining for Bcl-2, Bax, MIB-1 and p53 protein were 32.1, 40.8, 17.0 and 20.3%, respectively. The extent of apoptosis and expression of Bcl-2 protein were correlated to the histologic types of the tumor. Synovial sarcoma had a significantly higher expression of Bcl-2 protein, and lower incidence of apoptosis. STS underwent apoptosis at a constitutional level. There were no significant relationships between extent of the apoptosis, expression of its regulatory proteins and prognosis of the patients.

Protease inhibitors restore radiation-induced apoptosis to Bcl-2-expressing lymphoma cells.

The proteasome pathway is important for the turnover of many regulatory proteins. This pathway has recently become a target for antitumor agents and several research groups have demonstrated that inhibitors with specificities for the proteasome are potent apoptosis-inducing agents. Many mechanisms by which proteasome inhibitors exert their effects have been suggested, including inhibition of NF-kappa B activity and stabilization of the p53 tumor suppressor protein. We investigated the ability of inhibitors with specificities for the proteasome and for another protein degradation enzyme, calpain, to sensitize a murine B-cell lymphoma with constitutive NF-kappa B1 homodimer activity and high expression of Bcl-2 protein to radiation-induced apoptosis. Protease inhibitors tested were calpain inhibitor I, calpain inhibitor II, calpeptin, MG132, and Lactacystin. All five inhibitors induced apoptosis and sensitized cells to radiation despite the maintenance of Bcl-2 protein levels throughout the course of treatment. An electrophoretic migration shift assay for NF-kappa B1 activity provided evidence that reversal of NF-kappa B activity was not required for induction of cell death; however, p53 levels were elevated for all inhibitors tested. HL-60 cells, devoid of p53, could not be sensitized to radiation by MG132 treatment, suggesting that p53 was important for cell death induced by combined treatment with protease inhibitors and radiation. We concluded that protease inhibitors are capable of overcoming the protective effects of Bcl-2 to induce apoptosis and suggest that protease inhibitor treatment, when combined with ionizing radiation, leads to p53-mediated apoptosis.

Cadmium chloride-induced dysplastic changes in the ventral rat prostate: an immunohistochemical and quantitative study.

Cadmium chloride is an environmental toxic that might be implicated in human prostate carcinogenesis. The study was directed: 1) to evaluate the immunoexpression of markers for cell proliferation, apoptosis, and resistance to apoptosis, and 2) to estimate the size of premalignant cell population in the preneoplastic changes induced in ventral prostates of rats treated with cadmium chloride administered in drinking water. The following parameters were calculated in the ventral prostatic lobe of normal rats and rats that received cadmium in drinking water during 18 months: total volume, epithelial volume, total number of epithelial cells, numerical density of epithelial cells, percentage of cells that immunostained to the proliferating cell nuclear antigen (PCNA), percentage of apoptotic cells (evaluated by a DNA fragmentation method), and absolute volume and volume fraction of immunostaining to bcl-2. The percentage of PCNA immunoreactive nuclei, the bcl-2 expression, and the numerical density of epithelial cells were significantly (P < 0.05) increased in the dysplastic prostatic acini of treated rats in comparison with the normal acini of treated rats and control animals. The percentage of apoptotic nuclei from ventral dysplastic acini was significantly (P < 0.05) decreased in comparison with that of normal acini. A negative correlation between proliferation and apoptosis was found in dysplastic lesions. Prostate epithelial dysplasia induced in rats by cadmium presents an increased proliferative activity and high expression of bcl-2 protein, as was described in human prostate intraepithelial neoplasia. However, the rate of apoptosis in rat dysplasia was importantly decreased, in contrast to that observed in some human preneoplastic changes. This decrease might be related to the increase of bcl-2 expression.