Expression of Bcl-2 family proteins in psoriasis

Aim

To elucidate the mechanisms involved in apoptosis of psoriatic keratinocytes by examining the expression of pro-apoptotic (Bak, Bax) and anti-apoptotic (Bcl-2, Bcl-X) Bcl-2 family of proteins, as well as the expression of p53 and Ki-67 proteins in normal skin, and uninvolved and involved psoriatic skin.

Methods

A total of 90 skin samples (30 cases of involved and uninvolved psoriatic skin and normal skin) were examined immunohistochemicaly to determine the protein expression of p53, Ki-67, Bcl-2, Bcl-X, Bax, and Bak. The results were quantified and expressed as a percentage of positive keratinocytes.

Results

There was a significant increase in Ki-67 (17.05 vs 3.65; P<0.001), Bcl-X (40.21 vs 13.97; P<0.001), Bak (89.46 vs 73.36; P<0.001), and Bax (50.00 vs 29.25; P<0.001) expression and a decrease in Bcl-2 (3.23 vs 6.25; P=0.008) expression in involved psoriatic skin, as well as an increase in Bcl-X (25.13 vs 13.97; P<0.001) expression in uninvolved psoriatic skin, when compared to normal skin. Samples with higher percentage of Ki-67 positive cells showed a higher percentage of p53 positive cells (correlation coefficient r = 0.75 in involved psoriatic samples, P<0.001; r = 0.88 in uninvolved psoriatic samples, P<0.001; and r = 0.85 in normal skin samples, P<0.001). Samples with higher percentage of p53 positive cells expressed pro-apoptotic Bak and Bax in higher percentage of cells; the correlation coefficients were r = 0.74 and r = 0.68 in involved psoriatic samples (P<0.001 for both), r = 0.75 and r = 0.69 in uninvolved psoriatic samples (P<0.001, for both), and r = 0.87 and r = 0.70 in normal skin samples (P<0.001, for both).

Conclusion

Increased expression of Bcl-X protein was associated with psoriatic epidermal hyperplasia. Strong Bax and Bak expression in involved psoriatic skin are probably inhibitory mechanisms counteracting intensive proliferation.Psoriasis is a chronic, relapsing, inflammatory, and hyperproliferative skin disorder characterized by well-circumscribed erythematosquamous lesions (1). Clinically, psoriasis is characterized by significant epidermal keratinocyte hyperplasia with proliferation, keratinocyte maturation, and turn-over rates as important mechanisms in its pathogenesis (2,3).In normal human skin, keratinocytes in the superficial layer of the epidermis undergo apoptosis and regulate proliferation of cells in the basal layer (4). As opposed to normal skin, keratinocytes derived from psoriatic plaques were shown to be resistant to apoptosis (5). Numerous TUNEL-positive keratinocytes were also positive for proliferating nuclear antigen and Ki-67, which are indicative of proliferating cells (5). Inappropriate regulation of apoptosis was proposed as a possible explanation for epidermal thickening in hyperproliferative inflammatory skin disorders (6).The list of molecular mediators influencing apoptosis is rapidly expanding with Bcl-2 and its homologous proteins, emerging as one of the most important regulators of programmed cell death and playing a crucial role in the balance between cell survival and cell death. Protein p53 is a well described tumor suppressor that plays a central role in the initiation of apoptosis and cell cycle control (2,7,8).The mechanisms involved in the psoriatic plaque formation are not completely elucidated. In the era of biological therapy, better understanding of different apoptotic cell cycle regulatory mechanisms involved in this process would enable a development of novel specific therapeutic approaches for treating psoriatic patients. In this study, we examined the expression and distribution of the pro-apoptotic (Bak, Bax) and anti-apoptotic (Bcl-2, Bcl-X) Bcl-2 family proteins, as well as the expression of p53 protein in psoriatic epidermis and normal human skin. High TUNEL-positive rate in psoriatic keratinocytes was linked to high proliferation rate, so staining of Ki-67 was also performed.     

Role of Bcl-2 family proteins in apoptosis: apoptosomes or mitochondria?

Apoptosis is an essential physiological process for the selective elimination of cells, which is involved in a variety of biological events. The Bcl-2 family is the best characterized protein family involved in the regulation of apoptotic cell death, consisting of anti-apoptotic and pro-apoptotic members. The anti-apoptotic members of this family, such as Bcl-2 and Bcl-x_L, prevent apoptosis either by sequestering proforms of death-driving cysteine proteases called caspases (a complex called the apoptosome) or by preventing the release of mitochondrial apoptogenic factors such as cytochrome c and AIF (apoptosis-inducing factor) into the cytoplasm. After entering the cytoplasm, cytochrome c and AIF directly activate caspases that cleave a set of cellular proteins to cause apoptotic changes. In contrast, pro-apoptotic members of this family, such as Bax and Bak, trigger the release of caspases from death antagonists via heterodimerization and also by inducing the release of mitochondrial apoptogenic factors into the cytoplasm via acting on mitochondrial permeability transition pore, thereby leading to caspase activation. Thus, the Bcl-2 family of proteins acts as a critical life–death decision point within the common pathway of apoptosis.     

Apoptosis in nontumorous and neoplastic human pituitaries: expression of the Bcl-2 family of proteins

Analyses of apoptosis and of the apoptosis regulatory proteins Bcl-2, Bax, Bcl-X, and Bad were done in 95 nontumorous and neoplastic pituitary tissues by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL), immunohistochemistry, and Western blotting. The apoptotic index was relatively low in all groups but was at least fourfold higher in pituitary carcinomas compared with any other groups. Pituitaries from pregnant and postpartum women had a fivefold higher apoptotic index compared with matched controls from nonpregnant females. Preoperative treatment of adenomas with octreotide or dopamine agonists did not change the apoptotic index significantly. The lowest levels of Bcl-2, Bax, and Bcl-X expression were in pituitary carcinomas as detected by immunostaining. An immortalized human pituitary adenoma cell line, HP75, developed in our laboratory using a replication-defective recombinant human adenovirus with an early large T-antigen, had a much higher level of apoptosis than nontumorous and neoplastic pituitaries. Treatment with transforming growth factor (TGF)-beta1 and protein kinase C (PKC) inhibitors increased apoptosis in this cell line. Analysis of the Bcl-2 family of proteins after treatment with TGF-beta1 and PKC inhibitors showed a 20% to 30% decrease in Bcl-X in the treated groups compared with controls. These results, which represent the first study of apoptosis in pituitaries from pregnant and postpartum cases and in pituitary carcinomas, indicate that 1) the apoptotic rate is low in nontumorous and neoplastic pituitary tissues but is relatively higher in pituitary carcinomas, 2) there are alterations in the expression of the Bcl-2 family of proteins in pituitary neoplasms with a decrease in Bcl-2 expression in pituitary carcinomas that may contribute to pituitary tumor pathogenesis and/or proliferation, and 3) cultured pituitary tumor cells respond to TGF-beta1 and PKC inhibitors by undergoing apoptotic cell death.     

Immunohistochemical analysis of Bcl-2 family proteins in adenocarcinomas of the stomach.

The apoptosis-regulating proteins Bcl-2, Bax, Bcl-X, Bak, and Mcl-1 were examined by immunohistochemical methods in 48 archival specimens of adenocarcinoma of the stomach, and the results were correlated with tumor histology (intestinal versus diffuse pattern) and clinical stage (early- versus late-stage disease, ie, stages I and II versus stage III). Tumor cells containing immunostaining for the anti-apoptotic proteins Bcl-2, Bcl-X, and Mcl-1 were present in 26 (54%), 41 (85%), and 36 (75%) of the 48 cases evaluated, respectively, whereas immunopositivity for the pro-apoptotic proteins Bax and Bak was found in 44 (92%) and 42 (88%) specimens Comparisons of these immunostaining results with tumor histology revealed statistically significant differences for Bax (P = 0.03), Bcl-X (P = 0.003), and Mcl-1 (P = 0.005), which were all more frequently immunopositive for tumors with an intestinal than a diffuse histological pattern (chi 2 analysis). In addition, the percentage of immunopositive tumor cells was significantly higher for Bcl-X (62 +/- 6% versus 45 +/- 6%, mean +/- SE, P = 0.01) and for Mcl-1 (48 +/- 6% versus 30 +/- 6%; P = 0.04) in tumors with intestinal versus diffuse histology (unpaired t-test). In contrast, the percentage of Bcl-2-immunopositive tumor cells was higher in tumors with diffuse histology compared with intestinal (32 +/- 5% versus 12 +/- 5%; P = 0.01), whereas the percentages of Bax- and Bak-immunopositive tumor cells were not significantly different between these two histological types. In 34 specimens, residual normal gastric epithelial cells (foveolar cells) were present for direct comparisons of immunointensity with tumor cells. The immunointensity for the Bcl-2, Bcl-X, and Mcl-1 proteins was stronger in tumor cells compared with normal foveolar cells in 7 (21%), 15 (44%), and 8 (2.1%) of 34 cases, respectively, whereas the immunointensity of the proapoptotic proteins Bax and Bak was reduced compared with normal cells in 8 (24%) and 24 (71%) cases. Immunointensity, however, did not correlate with histology. clinical stage was not significantly associated with the presence or absence of immunopositive tumor cells, the percentage of immunopositive cells, or immunointensity. Taken together, these results establish for the first time that several Bcl-2 family proteins are expressed in gastric adenocarcinomas and suggest that the repertoire of these proteins may differ depending on the histological type. The findings therefore support the notion that the intestinal and diffuse types of gastric cancer arise at least in part through different mechanisms.     

Bodyguards and assassins: Bcl-2 family proteins and apoptosis control in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) is the most common B-cell malignancy in the Western world and exists as subtypes with very different clinical courses. CLL is generally described as a disease of failed apoptosis. Apoptosis resistance may stem from a combination of microenvironmental survival signals as well as from intrinsic alterations in the apoptotic machinery within the CLL cell. The molecular mechanism involved in controlling apoptosis in CLL is complex and is influenced by many factors, including Bcl-2 family proteins, which are critical regulators of cell death. Here we review the significance of apoptosis dysregulation in CLL, focusing on the role of Bcl-2 and related Bcl-2 family proteins, such as Bax and Mcl-1. The differential properties of the newly described subsets of CLL are also highlighted.     

Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis

We investigated the frequency of spontaneous apoptosis and expression of the Bcl-2 family of proteins during normal spermatogenesis in man. Testicular tissue with both normal morphology and DNA content was obtained from necro-donors and fixed in Bouin's solution. A TdT-mediated dUTP end-labelling method (TUNEL) was used for the detection of apoptotic cells. Expression of apoptosis regulatory Bcl-2 family proteins and of p53 and p21(Waf1) was assessed by immunohistochemistry. Germ cell apoptosis was detected in all testes and was mainly seen in primary spermatocytes and spermatids and in a few spermatogonia. Bcl-2 and Bak were preferentially expressed in the compartments of spermatocytes and differentiating spermatids, while Bcl-x was preferentially expressed in spermatogonia. Bax showed a preferential expression in nuclei of round spermatids, whereas Bad was only seen in the acrosome region of various stages of spermatids. Mcl-1 staining was weak without a particular pattern, whereas expression of Bcl-w, p53 and p21(Waf1) proteins was not detected by immunohistochemistry. The results show that spontaneous apoptosis occurs in all male germ cell compartments in humans. Bcl-2 family proteins are distributed preferentially within distinct germ cell compartments suggesting a specific role for these proteins in the processes of differentiation and maturation during human spermatogenesis.     

Modulation of Bcl-2 family proteins in primary endothelial cells during apoptosis

We studied the expression of Bcl-2 family proteins during cytokine- and verotoxin (VT)-induced apoptosis in primary human umbilical vein endothelial cells (HUVECs). Our experiments demonstrated that high initial expression of Bcl-2 protein was significantly downregulated in HUVECs treated with IFN-gamma whereas TNF-alpha gave a less pronounced decrease in Bcl-2 level. Treatment with the combination of cytokines was more efficient in downregulating Bcl-2 protein. HUVECs pretreated with cytokines and incubated with VT gave a further significant decrease in Bcl-2 level. Simultaneous measurement of Bcl-xl level did not reveal any significant changes. Bax protein was upregulated in HUVECs stimulated with TNF-alpha alone or in combination with IFN-gamma. However, addition of VT did not give any further increase in Bax level suggesting that Bax upregulation is more important for cytokine- rather than VT-mediated apoptosis. Total endothelial cell growth factor deprivation gave a significant increase in apoptosis accompanied by a decrease of Bcl-2 in apoptotic cells while Bcl-xl and Bax levels were unaffected. Our data indicate that anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax are reciprocally regulated during apoptosis, whilst Bcl-xl is essentially unaffected. This implies that Bcl-2/Bax ratio rather than Bcl-xl controls apoptosis in primary endothelial cells.     

The role of Bcl-2 family members in the progression of cutaneous melanoma

The overwhelming problem of cutaneous melanoma is chemoresistance. Subversion of the biochemical changes that lead to chemoresistance intersects the apoptosis pathways. The mitochondrion has been a focal point of this intersection for the development of therapeutic strategies aimed at reducing the progression of melanoma. The Bcl-2 family of apoptotic regulators is arguably the most pivotal component to this mitochondrial response. The shear number of studies conducted on the relationship between melanoma and Bcl-2 members prompted us to evaluate the literature available and discern some rational utility of the data. We have found that there are striking inconsistencies for the expression of Bcl-2 family proteins with melanoma progression, particularly for Bcl-2. Roughly one-third of the data suggests an increase in Bcl-2 expression with advancing melanoma, while another third suggests a decrease. Furthermore, the remaining third found on the whole, a detectable level of Bcl-2 in all tissues of melanocytic origin. These discrepancies are difficult to rectify in light of the apparent success of recent clinical trials utilizing Bcl-2 antisense strategies. The general consensus in the literature is that pro-apoptotic Bax is decreased with melanoma progression while anti-apoptotic Bcl-x_L and Mcl-1 appear to increase with progression. We suggest that the biochemical techniques being used for analysis present too great of a heterogeneity, which could be mitigated with more standard procedures and reagents. Finally the utility of ‘multi-specific’ antisense tactics could be a more effective way of targeting advanced melanoma disease. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhanced T cell apoptosis in common variable immunodeficiency: negative role of the fas/fasligand system and of the Bcl-2 family proteins and possible role of TNF-RS

CVI is a primary immunodeficiency characterized by a failure of B cell differentiation associated with an array of T cell defects, such as enhanced T cell apoptosis. In this study we investigated the mechanisms underlying CVI enhanced T cell death. We analysed both the expression of Fas using flow cytometry techniques and the expression of FasL mRNA using RT-PCR in CVI T cells. We could not find any significant differences between CVI and normal subjects with regard to Fas expression, although there was a subgroup of CVI patients with very high Fas expression which was accompanied by an up-regulation of FasL mRNA. However, attempts to induce Fas-mediated apoptosis in these high Fas expressing cells, as evaluated by propidium iodide staining and APO2.7 staining, were unsuccessful. We also investigated intracellular levels of Bcl-2, bcl-xl and bax in CD4(+) and CD8(+) CVI T cells, as well as the bax/Bcl-2 ratio, using flow cytometry techniques but could not detect any differences between CVI and normal subjects. Finally we analysed TNF-RI and TNF-RII mRNA expression in CD4(+) and CD8(+) CVI T cells using semiquantitative RT-PCR and found a significant increase in expression of both TNF-Rs in CD4(+) T cells from CVI patients. Our data suggest that the increased expression of both TNF-Rs on T cells may be one of the mechanisms responsible for the accelerated T cell apoptosis in CVI.     

Pro- and anti-apoptotic members of the Bcl-2 family in skeletal muscle: a distinct role for Bcl-2 in later stages of myogenesis.

Apoptotic myonuclei appear during myogenesis and in diseased muscles. To investigate cell death regulation in skeletal muscle, we examined how members of the Bcl-2 family of apoptosis regulators are expressed and function in the C2C12 muscle cell line and in primary muscle cells at different stages of development. Both anti-apoptotic (Bcl-W, Bcl-X(L)) and pro-apoptotic (Bad, Bak, Bax) members of the Bcl-2 family were expressed in developing skeletal muscle in vivo. Each was also expressed in embryonic (E11-12), fetal (E15-16), and neonatal muscle stem cells, myoblasts, and myotubes in vitro. In contrast, Bcl-2 expression was limited to a small group of mononucleate, desmin-positive, myogenin-negative muscle cells that were seen in fetal and neonatal, but not embryonic, muscle cell cultures. The cell surface protein Sca-1, which is associated with muscle and blood stem cells, was found on approximately 1/2 of these Bcl-2-positive cells. Loss of Bcl-2 did not affect expression of other family members, because neonatal muscles of wild-type and Bcl-2-null mice had similar amounts of Bcl-X(L), Bcl-W, Bad, Bak, and Bax mRNAs. Loss of Bcl-2 did have functional consequences; however, because neonatal muscles of Bcl-2-null mice had only approximately 2/3 as many fast muscle fibers as muscles in wild-type mice. Thus, Bcl-2 function is required for particular stages of fetal and postnatal myogenesis.     

Bcl-2 family proteins and mechanisms of the acceleration and inhibition of tumor progression in vivo

Earlier the authors demonstrated that the process of tumor progression in vivo may be inhibited or accelerated depending on the conditions of tumor growth (accelerated by tumor cell dissemination or delayed in locally growing tumors). It was also shown that tumor progression is inhibited in case of bcl-2 gene transduction in tumor cells. In this study, the research into mechanisms of the acceleration or inhibition of tumor progression and the role that Bcl-2 family proteins may play in these phenomena was continued. The results of the study demonstrated the following 1) immediate in vivo activation of endogenous proapoptotic Bax protein in disseminated tumor cells, not protected by Bcl-2 against apoptosis, and its correlation with accelerated tumor progression; 2) complete suppression of in vivo Bax activation in tumor cells protected by Bcl-expression, and inhibited tumor progression; 3) alternative character of Bcl-2 and Bax expression under the conditions of accelerated and inhibited tumor progression. Thus, the data presented support the hypothesis that the rates of tumor progression in vivo are regulated depending on the initial anti- and proapoptotic programs of tumor cells.     

Expression of Bcl-2 family proteins during chemotherapeutic agents-induced apoptosis in the hepatoblastoma HepG2 cell line

This study demonstrates that two anticancer drugs, taxol and doxorubicin (Dox), can kill human hepatoblastoma HepG2 cells in a dose-dependent manner via the induction of apoptosis. Characteristic events, including externalization of phosphatidylserine, cytoplasmic shrinkage, chromatin condensation and DNA degradation, were observed in a large majority of the drug-treated cells. DNA fragmentation showed that a ladder of DNA fragments of approximately 200 bp multiples was observed in taxol-treated, but not in Dox-treated, cells. In addition, the expression patterns of Bcl-2 family members during taxol or Dox treatment were investigated. Results from Western blot analysis indicated that HepG2 cells did not express either the death repressor Bcl-2, or the death promoters Bcl-XS and Bax. However, during the apoptotic process one death repressor, Bcl-XL, and two death promoters, Bak and Bad, were expressed. The expression levels of Bcl-XL and Bak remained unchanged, whereas the level of Bad was down-regulated. As the ratio between death repressors and death promoters in the Bcl-2 family will determine the sensitivity of cells to apoptotic stimuli, the findings suggest that the changed expression patterns of Bcl-2 family proteins caused by anticancer drugs in liver cancer cells may be involved in chemoresistance.     

The expression of Bcl-2 family proteins (Bcl-2, Bcl-x, Bax, Bak and Bim) in human lymphocytes

Bcl-2 family proteins regulate programmed cell death, and may play an important role in the selection of lymphocytes. We investigated the expression of Bcl-2, Bcl-x, Bax, Bak and Bim in human lymphocytes using flow-cytometry. Bcl-2 was down-regulated in CD4(+)8(+) (DP) thymocytes and CD19(+)38(+) tonsillar lymphocytes (GC B cells). Among DP thymocytes, cells co-expressing CD69 up-regulated Bcl-2, suggesting that the role of Bcl-2 is promoting survival of positively selected DP cells. Unexpectedly, the expression level of Bcl-x was higher in DP cells than in Single Positive (SP) cells and in CD69(+) DP thymocytes it was lower than in CD69(+) DP thymocytes. Expression of Bim was low in DP thymocytes but high in a subset of GC B cells. Bim and Bax were expressed more highly in SP than in DP thymocytes. Among peripheral blood lymphocytes (PBL), CD8(+) T cells expressed an approximately ten-fold higher level of Bcl-x than CD4(+) T cells while both subsets expressed similar levels of Bcl-2. Bak expression was low and Bim expression was absent in PBL. These results suggest that not only Bcl-2 but other members of the Bcl-2 family are involved in T cell development in the thymus and affinity maturation of B cells in the germinal center.     

Inhibition of the 53BP2S-mediated apoptosis by nuclear factor kappaB and Bcl-2 family proteins

The p53 binding protein 2 (53BP2) has been identified independently as the interacting protein to p53, Bcl-2, and p65 subunit of nuclear factor kappaB (NF-kappaB). It was demonstrated that over-expression of 53BP2 (renamed as 53BP2S) induces apoptotic cell death. In this study we explored the effect of NF-kappaB activation elicited by a physiological NF-kappaB inducer, interleukin-1beta (IL-1beta), and anti-apoptotic Bcl-2 family proteins on the 53BP2S-mediated apoptosis. We found that both NF-kappaB activation and Bcl-2 family proteins could prevent the 53BP2S-mediated depression of mitochondrial transmembrane potential, activation of caspase-9, cleavage of poly ADP ribose polymerase (PARP), and cell death. These observations suggested that 53BP2S/Bbp and its directly or indirectly interacting proteins might play crucial roles in the regulation of apoptosis and contribute to carcinogenesis. It is also suggested that 53BP2S/Bbp induces apoptosis through the mitochondrial death pathway presumably by counteracting the actions of anti-apoptotic Bcl-2 family proteins. The regulatory network of the 53BP2S-mediated apoptosis cascade including its interacting proteins is discussed.     

Bcl-2家族蛋白参与调节CD3ε和5-氟尿嘧啶介导的T淋巴细胞凋亡

目的：研究Bcl-2家族成员在5-氟尿嘧啶(5-FU)和CD3ε介导的T淋巴细胞凋亡中的作用，阐述T淋巴细胞凋亡的分子机制，为相关肿瘤的治疗提供依据。方法：用CD3ε特异的单克隆抗体(200μg/ml)和5-FU(2．5μg/ml)单独或联合刺激JK、TJK和T3JK细胞，诱导细胞凋亡，用MTS比色法检测细胞死亡率，用Western blot检测Bid的表达和活化。用脂质体的方法转染细胞。结果：CD3ε特异的单克隆抗体和5-FU分别单独处理TJK细胞，都能诱导其凋亡并伴随Bid的活化，二者联合使用能显著增加TJK细胞凋亡和Bid的活化。Bcl-2过表达可明显降低TJK细胞对5-Fu的敏感性。结论：Bcl-2家族成员参与CD3ε和5-FU诱导的T淋巴细胞凋亡的调节，Bcl-2过表达能显著抑制T淋巴细胞对5-FU的敏感性。     

Immunoblot analysis of cellular expression of Bcl-2 family proteins, Bcl-2, Bax, Bcl-X and Mcl-1, in human peripheral blood and lymphoid tissues

The ability of Bcl-2 to inhibit apoptotic cell death is wellestablished. Several homologues of the bcl-2 gene, such as bax,bcl-x or mcl-1, have recently been identified. Like Bcl-2, bothBcl-XL and Mcl-1 appear to function as repressors of apoptoticcell death, whereas Bax facilitates it, indicating possibleinteractions among them in the control of cellular survival.To investigate the in vivo role of expression of bcl-2 genefamily products, immunoblot analysis using corresponding specificantisera was performed for peripheral blood cells and some lymphoidtissues in humans. We demonstrated that all Bcl-2 family proteinswere expressed at various levels in hematolymphoid cell subpopulationsisolated from peripheral blood, tonsil, spleen and thymus. Lymphoidexpression of Bcl-2 family proteins tended to increase followingactivation, but declined with time in culture. Loss of Bcl-2in cultured lymphoid cells was especially marked. Sole expressionof Bax, but not other members of the Bcl-2 family, was observedon neutrophils, seemingly reflecting their shortest life-spanamong blood leukocytes. The results support the notion thata balance of expression of Bcl-2 family proteins may regulatethe life and death of hematolymphoid cells at different stagesof cell differentiation and activation.     

Epstein-Barr virus-mediated protection against etoposide-induced apoptosis in BJA-B B cell lymphoma cells: role of Bcl-2 and caspase proteins

Summary. Epstein-Barr virus (EBV)-infected B cell lymphomas are resistant to apoptosis during cancer development and treatment with therapies. The molecular controls that determine why EBV infection causes apoptosis resistance need further definition. EBV-positive and EBV-negative BJA-B B cell lymphoma cell lines were used to compare the expression of selected apoptosis-regulating Bcl-2 and caspase proteins in EBV-related apoptosis resistance, after 8hr or 18–24hr etoposide treatment (80µM). Apoptosis was quantified using morphology and verified with Hoechst 33258 nuclear stain and electron microscopy. Fluorescence activated cell sorting (FACS) was used to analyse effects on cell cycle of the EBV infection as well as etoposide treatment. Anti-apoptotic Bcl-2 and Bcl-XL, pro-apoptotic Bax, caspase-3 and caspase-9 expression and activation were analysed using Western immunoblots and densitometry. EBV-positive cultures had significantly lower levels of apoptosis in untreated and etoposide-treated cultures in comparison with EBV-negative cultures (p<0.05). FACS analysis indicated a strong G2/M block in both cell sublines after etoposide treatment. Endogenous Bcl-2 was minimal in the EBV-negative cells in comparison with strong expression in EBV-positive cells. These levels did not alter with etoposide treatment. Bcl-XL was expressed endogenously in both cell lines and had reduced expression in EBV-negative cells after etoposide treatment. Bax showed no etoposide-induced alterations in expression. Pro-caspase-9 and -3 were seen in both EBV-positive and -negative cells. Etoposide induced cleavage of caspase-9 in both cell lines, with the EBV-positive cells having proportionally less cleavage product, in agreement with their lower levels of apoptosis. Caspase-3 cleavage occurred in the EBV-negative etoposide-treated cells but not in the EBV-positive cells. The results indicate that apoptosis resistance in EBV-infected B cell lymphomas is promoted by an inactive caspase-3 pathway and elevated expression of Bcl-2 that is not altered by etoposide drug treatment.