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Apoptosis, the intrinsic programmed cell death process, is mediated by the Bcl-2 family members Bak and Bax. Activation via formation of symmetric core dimers and oligomerization on the mitochondrial outer membrane (MOM) leads to permeabilization and cell death. Although this process is linked to the MOM, the role of the membrane in facilitating such pores is poorly understood. We recently described Bak core domain dimers, revealing lipid binding sites and an initial role of lipids in oligomerization. Here we describe simulations that identified localized clustering and interaction of triacylglycerides (TAGs) with a minimized Bak dimer construct. Coalescence of TAGs occurred beneath this Bak dimer, mitigating dimer-induced local membrane thinning and curvature in representative coarse-grain MOM and model membrane systems. Furthermore, the effects observed as a result of coarse-grain TAG cluster formation was concentration dependent, scaling from low physiological MOM concentrations to those found in other organelles. We find that increasing the TAG concentration in liposomes mimicking the MOM decreased the ability of activated Bak to permeabilize these liposomes. These results suggest that the presence of TAGs within a Bak-lipid membrane preserves membrane integrity and is associated with reduced membrane stress, suggesting a possible role of TAGs in Bak-mediated apoptosis., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Despite the introduction of novel targeted therapies, chemotherapy still remains the primary treatment for metastatic melanoma in poorly funded healthcare environments or in case of disease relapse, with no reliable molecular markers for progression-free survival (PFS) available. As chemotherapy primarily eliminates cancer cells by apoptosis, we here evaluated if the expression of key apoptosis regulators (Bax, Bak, Bcl-2, Bcl-xL, Smac, Procaspase-9, Apaf-1, Procaspase-3 and XIAP) allows prognosticating PFS in stage III/IV melanoma patients. Following antibody validation, marker expression was determined by automated and manual scoring of immunohistochemically stained tissue microarrays (TMAs) constructed from treatment-naive metastatic melanoma biopsies. Interestingly and counter-intuitively, low expression of the pro-apoptotic proteins Bax, Bak and Smac indicated better prognosis (log-rank p < 0.0001, p = 0.0301 and p = 0.0227 for automated and p = 0.0422, p = 0.0410 and p = 0.0073 for manual scoring). These findings were independently validated in the cancer genome atlas (TCGA) metastatic melanoma cohort (TCGA-SKCM) at transcript level (log-rank p = 0.0004, p = 0.0104 and p = 0.0377). Taking expression heterogeneity between the markers in individual tumour samples into account allowed defining combinatorial Bax, Bak, Smac signatures that were associated with significantly increased PFS (p = 0.0002 and p = 0.0028 at protein and transcript level, respectively). Furthermore, combined low expression of Bax, Bak and Smac allowed predicting prolonged PFS (> 12 months) on a case-by-case basis (area under the receiver operating characteristic curve (ROC AUC) = 0.79). Taken together, our results therefore suggest that Bax, Bak and Smac jointly define a signature with potential clinical utility in chemotherapy-treated metastatic melanoma.

Aims: The present study examined the role of microRNA-125b (miR-125b) in myocardial ischaemia/reperfusion (I/R) injury. We constructed lentivirus-expressing miR-125b (LmiR-125b) and developed transgenic mice with overexpression of miR-125b., Methods and Results: LmiR-125b was transfected into mouse hearts through the right common carotid artery. Lentivirus vector (LmiR-Con) served as vector control. Untreated mice served as I/R control. Sham operation served as sham control. Seven days after transfection, the hearts were subjected to ischaemia (45 min) followed by reperfusion (4 h). Myocardial infarct size was analysed by 2,3,5-triphenyltetrazolium chloride staining. In separate experiments, hearts were subjected to ischaemia (45 min) followed by reperfusion for up to 7 days. Cardiac function was measured by echocardiography before, as well as 3 and 7 days after myocardial I/R. Increased expression of miR-125b significantly decreased I/R-induced myocardial infarct size by 60% and prevented I/R-induced decreases in ejection fraction (EF%) and fractional shortening (%FS). Transgenic mice with overexpression of miR-125b also showed the protection against myocardial I/R injury. Increased expression of miR-125b attenuated I/R-induced myocardial apoptosis and caspase-3/7 and -8 activities. Western blot showed that increased expression of miR-125b suppresses p53 and Bak1 expression in the myocardium. In addition, transfection of LmiR-125b decreased the levels of TNF receptor-associated factor 6 (TRAF6) and prevented I/R-induced NF-κB activation., Conclusion: miR-125 protects the myocardium from I/R injury by preventing p53-mediated apoptotic signalling and suppressing TRAF6-mediated NF-κB activation., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Aim: To evaluate the expression of Bcl-xL, Bak, and Bax proteins in correlation with particular clinico-histopathological parameters, including tumor invasion front, in patients with colorectal cancer., Methods: The expression of these proteins was evaluated with the use of the immunohistochemical method in 50 primary tumors., Results: According to observations, a low expression of Bax and Bak proteins is related to the localization of the tumor in the rectum (P < 0.05 and P < 0.05 respectively), which may explain an increased incidence of colorectal cancer in this area. A positive expression of Bax protein also correlates with the presence of cancer cell infiltration to lymph and blood vessels (P < 0.05), which may suggest the participation of this protein in the early stages of colorectal cancer progression. Moreover, a positive expression of Bcl-xL protein correlated with a positive expression of Bak protein. This may suggest a greater participation of Bcl-xL protein in the inhibition of the proapoptotic Bak protein, but not the Bax protein., Conclusion: Bax protein is probably very significant in the cancerogenesis mechanism in the large intestine.

Glucocorticoids (GCs) are widely used to treat inflammatory diseases and cancers. A multitude of undesired side effects have been reported in GC-treated patients including decreased linear bone growth. We have previously reported that GCs activate the caspase cascade and trigger Bax-mediated mitochondrial apoptosis in growth plate chondrocytes causing growth retardation in young mice. To further explore the role of mitochondrial apoptosis in GC-induced bone growth retardation, a number of pro- and anti-apoptotic proteins were studied in ex vivo cultures of human growth plate cartilage and human HCS-2/8 proliferative chondrocytes exposed to dexamethasone. Dexamethasone was found to increase the pro-apoptotic proteins Bcl-xS, Bad, and Bak as well as the proteolysis of Bid. Anti-Bid small interfering RNA partially rescued the chondrocytes from dexamethasone-induced apoptosis. Taken together, our data suggest that GC treatment differentially regulates Bcl-2 family member proteins to facilitate mitochondrial apoptosis in proliferative chondrocytes thereby contributing to GC-induced bone growth impairment. Prevention of this imbalance between pro- and anti-apoptotic Bcl-2 family proteins may provide a new strategy to protect from adverse effects of GCs on bone growth., (Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

The proapoptotic BCL-2 family proteins BAX and BAK serve as essential gatekeepers of the intrinsic apoptotic pathway and, when activated, transform into pore-forming homo-oligomers that permeabilize the mitochondrial outer membrane. Deletion of Bax and Bak causes marked resistance to death stimuli in a variety of cell types. Bax(-/-)Bak(-/-) mice are predominantly non-viable and survivors exhibit multiple developmental abnormalities characterized by cellular excess, including accumulation of neural progenitor cells in the periventricular, hippocampal, cerebellar and olfactory bulb regions of the brain. To explore the long-term pathophysiological consequences of BAX/BAK deficiency in a stem cell niche, we generated Bak(-/-) mice with conditional deletion of Bax in Nestin-positive cells. Aged Nestin(Cre)Bax(fl/fl)Bak(-/-) mice manifest progressive brain enlargement with a profound accumulation of NeuN- and Sox2-positive neural progenitor cells within the subventricular zone (SVZ). One-third of the mice develop frank masses comprised of neural progenitors, and in 20% of these cases, more aggressive, hypercellular tumors emerged. Unexpectedly, 60% of Nestin(Cre)Bax(fl/fl)Bak(-/-) mice harbored high-grade tumors within the testis, a peripheral site of Nestin expression. This in vivo model of severe apoptotic blockade highlights the constitutive role of BAX/BAK in long-term regulation of Nestin-positive progenitor cell pools, with loss of function predisposing to adult-onset tumorigenesis.

Background: Our previous studies showed that topical 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) is very effective for oral verrucous hyperplasia (OVH) and relatively less effective for oral leukoplakia (OL) lesions. Nevertheless, there has been no report on the association of the expression of apoptosis-related proteins in OVH and OL biopsy tissues prior to PDT with PDT treatment outcomes., Methods: This study used immunohistochemistry to evaluate whether the expression of Bak, Mcl-1, caspase-3, caspase-8, caspase-9, p53, p21, or PCNA protein in biopsy specimens of OVH and OL lesions could be used to predict the clinical outcomes of 18 OVH and 40 OL lesions treated with topical ALA-PDT. The marker labeling score (LS) was defined as labeling index (positive cells/total cells) multiplied by staining intensity. The lesions after ALA-PDT treatment were divided into complete response (CR) group and partial or no response (PR/NR) group., Results: The mean Bak LS and the mean Bak/Mcl-1 LS ratio were significantly higher in the CR group than in the PR/NR group. However, there was no significant difference in the Mcl-1, caspase-3, caspase-8, caspase-9, p53, p21, or PCNA protein LS between the CR and PR/NR groups., Conclusion: We conclude that the Bak LS or Bak/Mcl-1 LS ratio may be a useful biomarker to predict the clinical outcomes of OVH and OL lesions treated with topical ALA-PDT. Pre-PDT epithelial cell levels of Mcl-1, caspase-3, caspase-8, caspase-9, p53, p21, and PCNA may not have a significant influence on the clinical outcome of OVH and OL lesions treated with topical ALA-PDT., (© 2012 John Wiley & Sons A/S. All rights reserved.)

Abstract Protein-protein interactions play fundamental roles in most biological processes. Bimolecular fluorescence complementation (BiFC) is a promising method for its simplicity and direct visualization of protein-protein interactions in cells. This method, however, is limited by background fluorescence that appears without specific interaction between the proteins. We report here a point mutation (V150L) in one Venus BiFC fragment that efficiently decreases background fluorescence of BiFC assay. Furthermore, by combining this modified BiFC and linear expression cassette (LEC), we develop a simple and rapid method (LEC-BiFC) for protein interaction analysis that is demonstrated by a case study of the interaction between Bcl-X(L) and Bak BH3 peptide. The total analysis procedure can be completed in two days for screening tens of mutants. LEC-BiFC can be applied easily in any lab equipped with a fluorescence microscope.

Bcl-2 proteins represent a rheostat that controls cellular viability. Obatoclax, a BH3-mimetic, has been designed to specifically target and counteract anti-apoptotic Bcl-2 proteins. We evaluated the biological effects of obatoclax on the anti-tumour activity of rituximab and chemotherapy agents. Obatoclax induced cell death of rituximab/chemotherapy-sensitive (RSCL), -resistant cell lines (RRCL) and primary tumour-cells derived from patients with B-cell lymphomas (N=39). Obatoclax also enhanced the activity of rituximab and had synergistic activity when combined with chemotherapy agents. The ability of Obatoclax to induce PARP cleavage varied between patient samples and was not observed in some RRCL. Inhibition of caspase activity did not affect obatoclax activity, suggesting the existence of caspase-independent death pathways. Autophagy was detected by LC3 conversion and/or electron microscopy in RRCL and in patient-derived tumour cells. Moreover, obatoclax activity was inhibited by Beclin-1 knockdown. In summary, obatoclax is an active Bcl-2 inhibitor that potentiates the activity of chemotherapy agents and, to a lesser degree, rituximab. Defining the molecular events triggered by obatoclax is necessary to further its clinical development and identify potential biomarkers that are predictive of response., (© 2011 Blackwell Publishing Ltd.)

Dioscorealide B is a pharmacologically active compound from the rhizome of the Thai medicinal plant Dioscorea membranacea. Here, we demonstrated that in vitro treatment of dioscorealide B resulted in a cytotoxic effect on MCF-7 human breast cancer cells (IC50 = 2.82 microM). To determine whether this compound induces apoptosis in MCF-7, the Annexin V assay was performed. The data showed that the number of apoptotic cells were increased 7-12 folds over that of the control cells after treatment with various concentrations of dioscorealide B (3, 6 and 12 microM) for 24 hours. Dioscorealide B-induced apoptosis was associated with modulation of the multidomain Bcl-2 family members Bax, Bak and Bcl-2. After treatment with 3 microM dioscorealide B, acceleration of the level of proapoptotic proteins Bax and Bak were observed at 6 hours and 12 hours, respectively, while the decrease in the expression of antiapoptotic protein Bcl-2 was observed 3 hours after the treatment. These effects of dioscorealide B might result in the activation of caspase-8, -9 and -7, which lead to apoptosis in MCF-7 cells. Taken together, the results of this study provide evidence that dioscorealide B possesses an antitumor property against human breast cancer cells and thus provide the molecular basis for the further development of dioscorealide B as a novel chemotherapeutic agent for breast cancer treatment.

Objectives: alpha-Tocopheryl succinate (alpha-TOS) is thought to be toxic only for cancer cells. We ascertained in vitro alpha-TOS effects on pancreatic cancer (PC) and normal cell growth and verified whether the combination of nontoxic alpha-TOS and 5-fluorouracil (5-FU) doses causes cancer cell death and whether alpha-TOS effects are mediated by the proapoptotic proteins Bax/Bak and/or SMAD4/DPC4 status., Methods: Five PC cell lines, myoblasts, normal monocytes, wild-type (WT) and Bax/Bak double knockout mouse embryonic fibroblast (MEF) cells, and permanently SMAD4/DPC4-transfected PSN1 cells were cultured in 1% and 10% fetal calf serums (FCSs), without or with alpha-TOS (5-500 micromol/L). Nontoxic 5-FU (0.0001 mmol/L) and alpha-TOS alone or in combination were also evaluated., Results: Only PSN1 PC cell line, which had SMAD4/DPC4 homozygous deletion, was sensitive to nontoxic alpha-TOS doses (5 micromol/L in 1% FCS and 50 micromol/L in 10% FCS). A 20-micromol/L alpha-TOS inhibited MEF-WT, not MEF-double knockout growth. Only PSN1 cells were sensitive to nontoxic 5-FU and alpha-TOS combination. SMAD4/DPC4 transfection restored PSN1 resistance to the effects of combined 5-FU and alpha-TOS effects., Conclusions: Only a minority of PC cells are sensitive to the antiproliferative effects of alpha-TOS, any sensitivity appearing to be correlated with SMAD4/DPC4 homozygous deletion and Bax/Bak expression.

Paracoccidioidomycosis presents a variety of clinical manifestations and Paracoccidioides brasiliensis can reach many tissues, most importantly the lungs. The ability of the pathogen to interact with host surface structures is essential to its virulence. The interaction between P. brasiliensis and epithelial cells has been studied, with particular emphasis on the induction of apoptosis. To investigate the expression of different apoptosis-inducing pathways in human A549 cells, we infected these cells with P. brasiliensis Pb18SP (subcultured) and 18R (recently isolated from cell culture and showing a high adhesion pattern) samples in vitro. The expressions of Bcl-2, Bak and caspase 3 were analysed by flow cytometry and DNA fragmentation using the TUNEL technique. Apoptosis of human A549 cells was induced by P. brasiliensis in a sample and time-dependent manner. Using an in vitro model, our data demonstrates that caspase 3, Bak, Bcl-2 and DNA fragmentation mediate P. brasiliensis-induced apoptosis in A549 cells. The overall mechanism is a complex process, which may involve several signal transduction pathways. These findings could partially explain the efficient behaviour of this fungus in promoting tissue infection and/or blood dissemination.

Objective: To investigate the effects of resveratrol and tannic acid on apoptosis, and Bcl-2 homologous antagonist/killer (Bak) and fas associated death domain (FADD) proteins in the CaCo-2 cell line., Methods: In the present study, resveratrol and tannic acid were administrated in the CaCo-2 cell line at doses of 25, 50, and 100 microM. The CaCo-2 cells were grown and cultured in the Medical Biology Department, Eskisehir Osmangazi University, Eskisehir, Turkey in 2007. The effects of these agents on apoptotic index were determined by Apop Taq peroxidase kit and their effects on the ratios of Bak and FADD proteins by the immunohistochemical staining method at 24, 48, and 72 hours. Stained and non-stained cells in 30 separate areas of the 3 separate chamber slides, prepared for each group, were counted. The percentage of apoptosis, and Bak and FADD proteins was calculated with the control. Mean +/- standard error values were calculated for the 3 experiments., Results: Apoptotic index, Bak protein percentage ratio, and FADD protein percentage ratio values in all groups that received tannic acid and resveratrol increased when compared within the groups. This increase was found to be time and dose independent in all parameters., Conclusion: Cells undergo apoptosis in 2 pathways (mitochondrial and death receptor) in resveratrol and tannic acid induced CaCo-2 cells.

Follicular Lymphoma International Prognostic Index-FLIPI is an established clinical predictor for outcome in follicular lymphoma. The role of molecular abnormalities in blood and bone marrow of follicular lymphoma patients including t(14;18) is less clear. Seventy-five patients from a single institution diagnosed with follicular lymphoma between1999 and 2005 were included into the study. Diagnosis was based on lymph node biopsy in 62 cases (83%). Thirty-nine patients (52%) had G1 histological grade and 47 (63%) had entirely follicular growth pattern, as well as 9 patients (12%) had systemic symptoms and 33 (44%) were assigned to a good risk according to FLIPI. Median age of patients was 53 years. During a median observation time of 3 years 63 patients (84%) required initiating anti-lymphoma treatment. Seventy-five samples of peripheral blood and 65 samples of bone marrow were collected at the diagnosis. Bcl2 rearrangements including major breakpoint region and minor breakpoint cluster region were investigated using nested polymerase chain reaction technique. The primary end points of the study were time to first line lymphoma treatment and progression-free survival. Cells carrying t(14;18) were found in 31 cases (41%) including 29 samples of peripheral blood and 26 samples of bone marrow. Detection of t(14;18) in blood and bone marrow at diagnosis had no influence on clinical outcome. Age, follicular growth pattern systemic symptoms, and FLIPI score above 1 were predictive for initiation of the first lymphoma therapy. Follicular growth pattern, initial nodal involvement, serum LDH level, and FLIPI score above 1 were predictive for longer progression-free survival.

Particle-induced osteolysis is a major cause of aseptic loosening after total joint replacement. The purpose of the current study was to identify various apoptosis-related pathways in the cellular response to wear debris. Fas receptor, BAK and caspase-3 cleaved were evaluated immunohistochemically in capsules and interface membranes from patients with aseptic hip implant loosening. Moreover, we investigated local cellular proliferation, documented by the presence of Ki-67, to evaluate the proportion of apoptosis in relation to the proliferation in the different cells. We detected a strong expression of caspase-3 cleaved, Fas and BAK in macrophages, giant cells and T-lymphocytes. The fibroblasts showed caspase-3 cleaved and BAK, but no Fas staining. Demonstrated by Ki-67 staining, we found increased proliferation of macrophages and fibroblasts. Statistical analysis showed a significant positive correlation (p<0.001) between the above mentioned results and the presence of wear debris. The intensity of apoptosis and proliferation differed, depending on the extent of osteolysis. Overall, four different patterns of immunoreactivity were identified. We think, however, that in particle-induced osteolysis apoptosis is pathologically increased - a phenomenon also seen in other diseases. In these instances, the number and degree of apoptotic reactions are so great that the resulting cell remains cannot be completely removed. This leads to an increased excretion of fibrogenic mediators that could be responsible for increased proliferation of fibroblasts in spite of the increased apoptosis. Moreover, it leads to an increased excretion of cytokines which could be responsible for the activation of osteoclasts.

Candidate gene and pathway approaches, and unbiased gene expression profiling, have identified marker signatures predictive of tumor phenotypes, such as drug sensitivity and invasive or metastatic potential. However, application of such information to evaluation of tumors in the clinic is limited by cell heterogeneity in the tumor. We have developed a novel method of fluorescence in situ hybridization (FISH) that can detect transcriptional activation of individual genes at their site in single cells in the interphase nucleus. A major obstacle in the treatment of colorectal cancer is relative insensitivity to the chemotherapeutic agent 5-Fluorouracil (5-FU). Here, we have developed a sensitive approach to predict relative sensitivity of colorectal cancer cells to 5-FU, using FISH with probes targeted to nascent mRNAs to measure the number of individual cells with active transcription sites for a panel of candidate genes. These results reveal that the transcriptional status of four key genes, thymidylate synthase (TYMS), MORF-related gene X (MRGX), Bcl2-antagonist/killer (BAK), and ATPase, Cu(2+) transporting beta polypeptide (ATP7B), can accurately predict response to 5-FU. As proof of principle, we show that this transcriptional profile is predictive of response to 5-FU in a small number of patient colon tumor tissues. This approach provides a novel ability to identify and characterize unique minor cell populations in the tumor that may exhibit relative resistance to chemotherapy.

The aim of this study was to evaluate pro-apoptotic Bak expression in the germinal centers of adenoid in children on the assumption of the potential usefulness of Bak as adenoid function marker. The study involved 95 children undergoing adenoidectomy; divided into three age groups: aged up to 5 years (25 children), 5-10 years (54 children) and over 10 years (16 children). The analyzed material was adenoids removed on the ground of hypertrophy. Immunohistochemical analyses were carried out using goat polyclonal Bak antibodies (DAKO) directed against human Bak protein. The presence of Bak positive lymphocytes within germinal centers and Bak immunostaining were scored. The immunohistochemical staining showed the Bak positive lymphocytes mainly within the germinal centers of the lymphoid follicles. The Bak reactivity was also present in hyperplastic lymphoid tissue within the subepithelial B lymphocytes. We have not found statistically significant correlation between Bak expression and clinical status and change in Bak expression level according to age. The apoptotic presence within the germinal centers are the manifestation of which is Bak expression and its lack in the mantle zone, what we confirmed in our former study by describing Bcl-2 expression, seems to be a proper B cells maturation marker within lymphoid follicles. Our finding shows that these processes are not influenced by age and supports our thesis that adenoid involution is rather the effect of changes in the number of lymphoid follicles that changes in them.

A number of methods have been developed to examine the morphologic, biochemical, and molecular changes that happen during the DNA damage response that may ultimately lead to death of cells through various mechanisms that include apoptosis. When cells are exposed to ionizing radiation or chemical DNA-damaging agents, double-stranded DNA breaks (DSB) are generated that rapidly result in the phosphorylation of histone variant H2AX. Because phosphorylation of H2AX at Ser 139 correlates well with each DSB, phospho-H2AX is a sensitive marker to used to examine the DNA damage and its repair. Apoptotic cells are characterized on the basis of their reduced DNA content and morphologic changes, including nuclear condensation, which can be detected by flow cytometry (sub-G1 DNA content), trypan blue, or Hoechst staining. The appearance of phosphatidylserine on the plasma membrane with annexin V-fluorochrome conjugates indicates the changes in plasma membrane composition and function. By combining it with propidium iodide staining, this method can also be used to distinguish early versus late apoptotic or necrotic events. The activation of caspases is another well-known biochemical marker of apoptosis. Finally, the Bcl-2 family of proteins and the mitochondria that play a critical role in DNA damage-induced apoptosis can be examined by translocation of Bax and cytochrome c in and out of mitochondria. In this chapter, we discuss the most commonly used techniques used in our laboratory for determining the DNA damage response leading to apoptosis.

Copper and two molecules of diethyl dithiocarbamate [DEDTC] form the Cu[DEDTC](2) complex, which shows cytotoxicity against melanoma and carcinoma cells, making it a potentially useful anti-cancer agent. The differential response to Cu[DEDTC](2) in susceptible human SKBR3 carcinoma and C8161 melanoma cell variants of moderate and high resistance to this organometallic complex was evaluated in this study. Both cell lines underwent apoptosis-associated PARP cleavage, changes in expression of nuclear NFkB p65, p21WAF1 and cyclin A, with loss of clonogenicity in response to this agent. However, a threefold greater concentration [IC(50) 0.6 microM DEDTC: 0.3 microM Cu] was required to kill moderately resistant C8161 melanoma compared to highly susceptible SKBR3 cells. Decreased susceptibility to Cu[DEDTC](2) in C8161 melanoma correlated with greater levels of glutathione peroxidase and catalase, and a fourfold lower requirement for N-acetyl cysteine (1mM) to overcome toxicity. Whereas melanoma cells selected for resistance to [0.8 microM DEDTC: 0.4 microM Cu] showed persistent catalase and GPx activity, melanoma cells with moderate susceptibility showed decreased catalase and Gpx when responding to treatment. Cytotoxic response in moderately susceptible C8161 melanoma cells involved an early accumulation of pro-apoptotic Bax in the G2 cell cycle phase, followed by an increased ratio of pro-apoptotic Bak to anti-apoptotic Mcl-1 in mitochondria. Our data suggests that Cu[DEDTC](2) toxicity is mediated through an increase in pro-apoptotic Bak/Bax via disruption of the peroxide and thiol metabolism.

The expression of various bcl2 family proteins has been reported in Hodgkin and Reed-Sternberg cells, but the proteins bad, bid, and bim have not been analyzed in classical Hodgkin's lymphomas (HLs). This study aimed to investigate the expression of the proteins bcl2, bcl-xl, mcl1, bax, bak, bad, bid, bim, and active caspase 3, and the TUNEL (terminal deoxynucleotidyl transferase-mediated in situ labeling) index to gain further insight into the apoptosis profile of classical HLs. A high expression of the proteins bcl2, bcl-xl, mcl1, bax, bak, bad, bid, and bim in HRS cells was found in 27 of 101 (27%), 95 of 101 (94%), 27 of 97 (29%), 73 of 95 (77%), 37 of 102 (36%), 85 of 94 (90%), 19 of 109 (17%), and 43 of 91 (47%) cases, respectively. The high expression of bcl-xl, bax, and bad in HRS cells in most classical HLs indicates that these proteins may play predominant roles in the regulation of apoptosis in classical HLs. Active caspase 3-positive and TUNEL-positive Reed-Sternberg cells were detected in 47 of 70 (67%; range, 0%-12%) and 60 of 71 (85%; range, 0%-19%) cases, respectively. Significant positive correlations were found between bax/bcl2 (P = .002), bad/bcl2 (P = .020), bad/bcl-xl (P = .003), and bim/mcl1 (P = .036). Based on these findings, it could be hypothesized that the antiapoptotic proteins bcl2, bcl-xl, and mcl1 may counteract the expression of the proapoptotic proteins bax, bad, and bim, thereby contributing to the survival of Reed-Sternberg cells.

Prognosis of primary melanoma is presently based on morphological parameters, mainly tumor thickness. However, more reliable prognostic markers are needed that allow a better stratification of patients, especially with regard to therapeutic options. Here, a retrospective study was performed on patients with primary superficial-spreading melanoma (SSM, n=44) or nodular melanoma (n=16) of 1.5-4 mm thickness. Thirty patients had survived the follow-up of 10 years, whereas the other 30 patients developed metastases. Tumor sections were analyzed by immunohistochemistry for the expression of regulators of the cell cycle (p21; retinoblastoma protein (pRb)), of the intrinsic or extrinsic proapoptotic pathways (p53; murine double minute gene 2 protein; tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-R1/DR4; TRAIL-R2/DR5) and of Bcl-2-related proteins (Bcl-2, Mcl-1, Bax, Bak, Bok), which regulate the common mitochondrial apoptotic pathway. In SSM, decrease of Bax and Bak was significantly correlated with a poor prognosis: high Bax was associated with 10-year survival rates of 68%, whereas low Bax resulted in only 26% survival, and high Bak was associated with 10-year survival rates of 62%, whereas low Bak resulted in only 10% survival. Regulators of apoptosis may therefore candidate for independent prognostic markers for primary melanomas. The study underlines the particular role of the mitochondrial apoptosis pathway and of proapoptotic Bcl-2-related proteins for melanoma progression.

Objective: To investigate infiltrating cells in the liver of children with type 1 autoimmune hepatitis (AH-1)., Methods: liver biopsies from 24 untreated AH-1 patients (14 children, 10 adults), five patients with hepatitis C virus related chronic hepatitis (HCV), and 10 control liver specimens (CL) were processed for immunohistochemical cell characterisation., Results: Two different cell distribution patterns were detected in the liver of patients with AH-1: (1) CD4(+) and CD20(+) cells were found in the central areas of the portal tracts (portal distribution); (2) CD8(+) cells were observed at the periphery of the portal space (periportal distribution). Some cell subsets, like CD56, CD57, Fas-L, and Bak, showed a non-defined distribution pattern. The presence of two well defined patterns of cell distribution was not observed in HCV and CL (CD4(+), CD20(+), and CD8(+) cells were uniformly distributed in the portal space). In AH-1 and CL, the NK markers CD56 and CD57 were found scattered throughout the liver parenchyma. However, in HCV biopsies, CD56(+) cells were also clearly increased in both the portal and the periportal areas. Biopsies of AH-1 and HCV patients showed a uniform distribution of Fas-L and Bak in the portal and periportal areas, with Bak staining also detected in the hepatic parenchyma., Conclusions: Despite clinical and genetic differences, there was a similar distribution of liver infiltrating mononuclear cells in children and adults with AH-1. These results raise the possibility of reclassifying cryptogenic chronic hepatitis by immunohistochemical analysis of infiltrating liver cells.

A large number of methods devoted to the identification of apoptotic cells and the analysis of the morphological, biochemical, and molecular changes that take place during this universal biological process have been developed. Apoptotic cells are recognized on the basis of their reduced DNA content and morphological changes that include nuclear condensation and which can be detected by flow cytometry (sub-G1 DNA content), Trypan Blue, or Hoechst staining. Changes in plasma membrane composition and function are detected by the appearance of phosphatidylserine on the plasma membrane, which reacts with Annexin V-fluorochrome conjugates. Combined with propidium iodide (PI) staining, this method can distinguish between the early and late apoptotic events. The best-recognized biochemical hallmarks of apoptosis are the activation of cysteine proteases (caspases), condensation of chromatin, and fragmentation of genomic DNA into nucleosomal fragments. Recognized by a variety of assays, activated caspases cleave many cellular proteins and the resulting fragments may serve as apoptosis markers. Finally, the mitochondria and the Bcl-2 family proteins play an important role in this process that can be recognized by translocation of apoptogenic factors, such as Bax and cytochrome c, in and out of mitochondria.

Bcl-2 family proteins play an important role in the growth and biological behavior of tumors. This study aimed to determine Bcl-2 family proteins in laryngeal carcinoma and to examine their relationship with spontaneous apoptosis. The material studied was from 39 patients with laryngeal carcinoma. It was found that the expression of both Bak and Bax was lower in tumor tissues than in nontumor tissues. However, there was no difference in the expression of Bcl-2 between tumor and nontumor tissues. The frequency of spontaneous apoptosis was lower in tumor tissues than in nontumor tissues but was not significantly related to the expression of Bak, Bax, or Bcl-2. Bak was decreased in moderately differentiated tumors compared to well-differentiated tumors. In contrast to Bak, the expression of Bcl-2 was increased in moderately differentiated tumors compared to well-differentiated tumors. These results indicate that the reduction in Bak may be associated with an increase in tumor grade and dedifferentiation in laryngeal carcinomas. The lack of correlation between apoptosis and the expression of Bcl-2 family proteins suggests that spontaneous apoptosis in laryngeal carcinoma is a complex process and that molecules other than Bak, Bax, and Bcl-2 participate in it.

Apoptosis proteins may play a role in prognosis and therapy response; however, they have not been fully investigated in gastric cancer. We aimed to assess the expression of proteins in the Bcl-2 family. Immunohistochemistry was employed to examine the expression of the antiapoptotic proteins Bcl-2 and Bcl-XL and the proapoptotic proteins Bad, Bak, Bax, Bid, Bim, and p53 in 21 cases of gastric cancer. Immunopositivity was observed in 12/21 (57%) cases for p53, 16/21 (76%) cases for Bcl-XL, and 5/21 (23%) cases for Bcl-2. For the proapoptotic members of the Bcl family, loss of protein expression was observed: Bid (14/21 cases; 66%), Bad (13/21 cases; 61%), Bax (12/21 cases; 57%), Bak (9/21 cases; 42%), and Bim (4/21 cases; 19%). This study identified apoptosis proteins that exhibit heterogeneous expression between primary gastric carcinomas.

Background: Escherichia coli-derived Shiga toxin (Stx), the cause of the enteropathic hemolytic uremic syndrome, is a potent inducer of apoptotic cell death. The present study was performed to examine the hypothesis that Stx initiates apoptosis by activating the mitochondrial pathway involving mitochondrial-associated, pro-apoptotic Bcl-2 family proteins Bax and Bak., Materials and Methods: To determine if Stx2-mediated apoptosis is dependent on Bax or Bak, a gene-silencing approach was employed using sequence-specific small interfering (si)RNA duplexes. Silencing of Bax and Bak protein expression in human renal proximal tubular epithelial (HK-2) cells and its effect on Shiga toxicity was assessed by immunofluorescence microscopy and Western blotting., Results: Transfection of HK-2 cells, shown to be exquisitely sensitive to Stx, with siRNA duplexes successfully diminished Bak, but not Bax protein expression. In order to determine if silencing of pro-apoptotic gene expression affects Stx-induced apoptosis, HK-2 cells were transfected with Bak-specific or control siRNA, exposed to lethal concentrations of Stx2 and assessed for cleavage of poly(ADPribose) polymerase-1 (PARP) as a marker of apoptosis, using Western blot technology. We observed that siRNA-induced reduction of Bak expression levels correlated with decreased PARP cleavage., Conclusion: Results suggest that Stx-induced cell death involves pro-apoptotic Bak and that silencing of Bak gene expression affords partial protection against Stx-mediated apoptosis.

Follicular lymphoma (FL) is the second most common non-Hodgkin's lymphoma and generally is incurable. Reliable prognostic markers to differentiate patients who progress rapidly from those who survive for years with indolent disease have not been established. Most cases overexpress Bcl-2, but the pathogenesis of FL remains incompletely understood. To determine whether a proteomic approach could help overcome these obstacles, we procured lymphoid follicles from 20 cases of FL and 15 cases of benign follicular hyperplasia (FH) using laser capture microdissection. Lysates were spotted on reverse-phase protein microarrays and probed with 21 antibodies to proteins in the intrinsic apoptotic pathway, including those specific for posttranslational modifications such as phosphorylation. A panel of three antibodies [phospho-Akt(Ser473), Bcl-2, and cleaved poly(ADP-ribose) polymerase] segregated most cases of FL from FH. Phospho-Akt(Ser473) and Bcl-2 were significantly increased in FL (P = 0.001 and P < 0.0001, respectively). Additionally, the Bcl-2/Bak ratio completely segregated FL from FH. High ratios of Bcl-2/Bak and Bcl-2/Bax were associated with early death from disease with differences in median survival times of 7.3 years (P = 0.0085) and 3.8 years (P = 0.018), respectively. Using protein microarrays, we identified candidate proteins that may signify clinically relevant molecular events in FL. This approach showed significant changes at the posttranslational level, including Akt phosphorylation, and suggested new prognostic markers, including the Bcl-2/Bak and Bcl-2/Bax ratios. Proteomic end points should be incorporated in larger, multicenter trials to validate the clinical utility of these protein microarray findings.