In vivo patterns of Bcl-2 family protein expression in breast carcinomas in relation to apoptosis

The expression of the pro-apoptotic proteins (Bax, Bak) and anti-apoptotic proteins (Bcl-2, Bcl-X, Mcl-1) was studied by immunohistochemistry in 110 invasive ductal breast carcinomas. The results were correlated with tumour grade, expression of oestrogen receptor (ER) and p53 protein, and the apoptotic index by combined morphology, immunohistochemistry, and a terminal UTP nick end labelling (TUNEL) procedure. Overall, Bcl-2, Bcl-X, Mcl-1, Bax, Bak, ER, and p53 were detected in 62, 75, 68, 75, 60, 68 and 26 per cent of the cases respectively, but at different levels in each case. A high apoptotic index was correlated with high tumour grade (p < 0·001), overexpression of p53 (p < 0·001), Bak expression (p < 0·001), and low expression of Bcl-2 (p < 0·001) and ER (p < 0·001). No correlation was found between the apoptotic index and Bax, Bcl-X, and Mcl-1 immunostaining results. The expression of Bcl-2 and Bcl-X was correlated to that of ER. Overall, the results of this study strongly suggest that Bcl-2 and Bak expression is critical in regulating apoptosis in breast carcinomas. Copyright © 1999 John Wiley & Sons, Ltd.     

Prognostic value of Bax, Bcl-2, p53, and TUNEL staining in patients with radically resected ampullary carcinoma

BACKGROUND: There is a lack of data in the literature concerning the identification of potential prognostic factors in ampullary adenocarcinoma. AIMS: To examine the prognostic significance of Bax, Bcl-2, and p53 protein expression and the apoptotic index in a large cohort of uniformly treated patients with radically resected ampullary cancer. METHODS: All patients with a pathological diagnosis of ampullary cancer and radical resection were evaluated. Expression analysis for p53, Bax, and Bcl-2 was performed by immunohistochemistry. Apoptotic cells were identified by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). RESULTS: Thirty nine tumour specimens from patients with radically resected ampullary adenocarcinoma were studied. A positive significant correlation between Bax and p53 expression was found by rank correlation matrix (p < 0.001). A trend towards a positive correlation was found between the apoptotic index and p53 expression (p = 0.059). By univariate analysis, overall survival was influenced by Bax expression, p53 expression, and TUNEL staining (p = 0.001, p = 0.01, and p = 0.03, respectively). Bcl-2 expression did not influence overall survival in these patients (p = 0.55). By multivariate Cox regression analysis, the only immunohistochemical parameter that influenced overall survival was Bax expression (p = 0.020). CONCLUSIONS: These results provide evidence that apoptosis may be an important prognostic factor in patients with radically resected ampullary cancer. This study is the first to assess the clinical usefulness of Bax expression in radically resected ampullary cancer.     

Differential Regulation of Bax, Bcl-2, and Bcl-X Proteins in Focal Cortical Ischemia in the Rat

Focal ischemia in the parietal cortex of the rat results in massive neuronal death in the infarct zone and penumbra between 12 hours and 6 days after photothrombosis. To examine a possible role of Bcl-2 family proteins in this process of cell death, we investigated their expression by immunoblot assays and immunocytochemistry, and correlated expression patterns with TUNEL as well as morphological signs indicative of apoptosis. In the center of the lesion Bax immunostaining was increased in many degenerating neurons between 4 hours and 3 days after the induction of photothrombosis. At all time points examined, Bcl-2 and Bcl-X protein levels were markedly reduced in injured neurons as compared to the unlesioned side. At the border of the ischemic lesion, two areas were distinguished: 1 – 2 days after induction of photothrombosis, pyknotic cells located immediately adjacent to the lesion core displayed nuclear Bcl-X and Bax immunoreactivity. In contrast, large, morphologically intact neurons located more towards the healthy brain parenchyma displayed an increase in cytoplasmic Bcl-2 and Bcl-X proteins. Double staining for each of the Bcl-2 family proteins and TUNEL revealed that DNA strand breaks and nuclear fragmentation seen in cells located in the lesion core were often associated with increased levels of Bax, but not with elevated Bcl-2 or Bcl-X protein levels, suggesting a role for Bax in the induction of apoptotic death in these cells. The upregulation of Bcl-2 and Bcl-X expression in surviving neurons close to the penumbra might reflect an active survival mechanism that protects these neurons from cell death following a sublethal insult.     

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.     

Blocking neutrophil influx reduces DNA damage in hyperoxia-exposed newborn rat lung

Hyperoxia-induced neutrophil infux in neonatal rats may contribute to impaired lung development through oxidative DNA damage. To determine whether blocking neutrophil influx prevents DNA damage, we treated newborn rats with 95% O2 beginning at birth, and at 3 and 4 d with nonimmune immunoglobulin G (IgG) (control) or anti-cytokine-induced neutrophil chemoattractant (CINC). At 8 d, lungs were inflation-fixed. Random sections were labeled using terminal transferase nick end-labeling (TUNEL), and DNA oxidation was measured using anti-8-OH-2'-deoxyguanosine (OHdG). To determine whether hyperoxia-induced TUNEL represented apoptosis, we labeled sections with anti-Bax (proapoptotic) and anti-Bcl-2 (antiapoptotic). We labled additional sections with anti-M30, directed against an epitope formed by caspase 6 digestion of cytokeratin 18 during apoptosis. Hyperoxia induced marked increases in TUNEL and OHdG signal in lung parenchymal cells, which was substantially prevented by treatment with anti-CINC. The large effects of hyperoxia on TUNEL were not accompanied by substantial effects on Bax, Bcl-2, or M30. We conclude that neutrophil influx during hyperoxia damages DNA by nicking and oxidation, and that blocking neutrophil influx can prevent this. Effects of 95% O2 on TUNEL are not primarily due to apoptosis in this model. Neutrophil-mediated oxidative DNA damage may contribute to abnormal lung development in newborns subjected to significant oxidative stress.     

Differential expression of cell death regulators in response to thapsigargin and adriamycin in Bcl-2 transfected DU145 prostatic cancer cells

Functional overexpression of Bcl-2 has been reported to confer an anti-apoptotic potential in a variety of cell types. The role of Bcl-2 in epithelial cell-cycle control and in interactions with other cell-cycle regulators is not clearly understood. Its expression has been correlated with the hormono- and chemo-resistant phenotype in advanced prostate cancer. The aim of this study was to investigate the mechanisms through which Bcl-2 mediates increased cytotoxic chemoresistance by assessing alterations in the expression of cell death regulatory molecules. The DU145 human prostatic adenocarcinoma cell line was stably transfected with a Bcl-2 encoding expression plasmid. Two Bcl-2 transfectants, DKC9 and DKC11, were expanded for further study. The effects of Bcl-2 expression on cellular proliferation, cell death (+/- adriamycin or thapsigargin), and expression of cell-cycle/death regulators (p53, PCNA, Bax, Bak, Bcl-X(L)) were evaluated. Compared with controls, Bcl-2 transfectants showed no difference in the rate of proliferation, a decrease in p53 (approximately two-fold), an increase in Bax (approximately two-fold) and PCNA (approximately three-fold), and no change in the levels of Bcl-X(L) and Bak proteins. DKC9 and DKC11 also exhibited a significantly increased chemoresistance to adriamycin (0.0025-5 microM) and thapsigargin (0.0025-5 microM) compared with controls. In the presence of thapsigargin or adriamycin, levels of Bcl-2 and its heterodimeric partner Bax were elevated approximately two-fold with no change in Bak in Bcl-2 transfectants in contrast to controls, where Bak was increased (two-fold). This is the first study to demonstrate that Bcl-2 transfection modulates the expression of mutant p53, Bax, and PCNA in prostate cancer cells. Moreover, Bcl-2 overexpression conferred a significant cytotoxic chemoresistance and altered the balance of expression of death promoters (from Bak, a dominant death promoter in controls, to Bax) in response to thapsigargin and adriamycin.     

Cytotoxic chemotherapy upregulates pro-apoptotic Bax and Bak in the small intestine of rats and humans

AIMS: Small intestinal crypt cells rapidly undergo apoptosis in response to cytotoxic drug treatment that results in gastrointestinal toxicity. The Bcl-2 family have been implicated in both positive and negative regulation of intestinal cell apoptosis. The aim of this study was to examine the effect of cytotoxic treatment on Bcl-2 protein expression in patients and rats with tumours. METHODS: Four pro- and four anti-apoptotic members of the Bcl-2 family, caspase-3 and p53 were examined in small intestinal crypts before and after treatment in rats and humans. Immunohistochemistry identified changes in protein expression over time, while relative RT-PCR was used to investigate mRNA expression in rat small intestine. RESULTS: Cytotoxic treatment increased p53 and caspase-3 which coincided with elevated levels of apoptosis. Bax and Bak protein and mRNA expression also significantly increased at 6 hours following treatment in rats. Bax and Bak protein increased at day 1 after treatment in humans. Anti-apoptotic Mcl-1 protein decreased within 24hours. Other Bcl-2 family members showed only modest changes. CONCLUSION: Increased expression of Bax and Bak but not other Bcl-2 family members is associated with apoptosis in small intestinal crypts and may amplify the sensitivity and susceptibility of crypt cells to chemotherapy-induced enteropathy.     

Increased expression of apoptosis-associated proteins in puromycin aminonucleoside nephrosis

Increased apoptosis has been reported in acute puromycin aminonucleoside nephrosis (PAN). The aim of this study was to investigate if increased apoptosis is related to increased expression of apoptosis-associated proteins (AAP) in this model of nephrosis. Sprague-Dawley rats were made nephrotic by intraperitoneal injection of one dose of puromycin aminonucleoside. Renal tissues were obtained at 1, 2 and 7 weeks after injection and apoptosis was investigated by TUNEL and by electron microscopy. Fas, Fas ligand, p53, Bax and Bcl-2 expressions were analyzed by the respective monoclonal and polyclonal antibodies, using indirect immunofluorescence. In the glomerulus of nephrotic animals, increased apoptosis was accompanied with increased expression of p53, Fas and Bax. In the interstitium, high expression of apoptosis, Fas, Fas-L and Bax were observed and in tubules increased apoptosis was accompanied with increased expression of p53, Fas and Fas-L. Bcl-2 was increased in interstitium and tubules during PAN. The incidence of apoptosis during PAN was correlated with the expression of AAP in glomerulus (p53), interstitium (Fas, Fas-L and Bax) and tubules (Fas, Fas-L, p53 and Bcl-2). There was correlation between Fas and Fas-L expression in interstitium and tubules. About 4% of glomerular and 25% of tubular p53 positive cells were apoptotic cells. The data suggest that increased local expression of AAP could contribute to renal apoptosis in the glomerular, interstitial and tubular compartments during this experimental model of nephrosis.     

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.     

叶酸拮抗同型半胱氨酸诱导的内皮细胞凋亡的作用机制

目的： 明确同型半胱氨酸（Hcy）对内皮细胞凋亡的影响以及叶酸的拮抗作用，阐明Bax和Bcl-2在同型半胱氨酸诱导内皮细胞凋亡及叶酸拮抗中的作用。方法: 用不同浓度的Hcy处理内皮细胞后，应用末端转移标记技术（TUNEL）以及Annexin V/PI染色加流式细胞术了解细胞凋亡状态，免疫组化方法检测Bax、Bcl-2的表达。结果: Hcy能促进细胞凋亡，叶酸具有拮抗作用。Hcy能促进细胞Bax、Bcl-2的表达，上调Bax/Bcl-2比值，叶酸能减少细胞表达Bax及Bcl-2，下调Bax/Bcl-2比值。结论: Bax、Bcl-2参与了Hcy诱导内皮细胞凋亡以及叶酸拮抗作用的过程。     

人硫氧还蛋白对肺缺血再灌注损伤时细胞凋亡及Bcl-2/Bax的影响

目的:探讨细胞凋亡与肺缺血再灌注损伤的关系以及人硫氧还蛋白对凋亡及其相关基因的影响。方法:健康清洁级Wistar大鼠84只,随机分为对照组、肺缺血再灌注1h、3h、5h组和人硫氧还蛋白干预1h、3h和5h组。复制肺缺血再灌注损伤模型。采用电子显微镜和原位缺口末端标记法观测肺组织细胞凋亡的变化和凋亡指数,免疫组化技术检测肺组织细胞Bcl-2、Bax及凋亡信号调节激酶1(ASK1)蛋白表达的变化。结果:肺缺血再灌注组肺组织细胞凋亡指数、ASK1、Bcl-2和Bax蛋白表达均显著高于对照组(均P0.01),超微结构呈严重损伤性改变。人硫氧还蛋白干预组ASK1、Bax的表达显著下降(均P0.01),Bcl-2的表达及Bcl-2/Bax比值显著上调(P0.05或P0.01),肺组织细胞凋亡指数也显著低于缺血再灌注组(P0.01)。肺组织细胞凋亡指数分别与ASK1、Bax蛋白之间均呈显著正相关(分别r=0.775、r=0.814;均P0.01);与Bcl-2/Bax蛋白呈显著负相关关系(r=-0.275,P0.05)。结论:Bcl-2/Bax比值下调启动的肺组织细胞凋亡可能参与了肺缺血再灌注损伤的发生。人硫氧还蛋白可能通过下调ASK1的表达,提高Bcl-2/Bax的比值减少肺组织细胞凋亡,从而减轻肺缺血再灌注损伤。     

Regulation of Airway and Alveolar Epithelial Cell Apoptosis by p53-Induced Plasminogen Activator Inhibitor-1 during Cigarette Smoke Exposure Injury

Increased expression of tumor suppressor protein p53 and of plasminogen activator inhibitor (PAI)-1 is associated with cigarette smoke (CS) exposure-induced lung epithelial injury. p53 induces PAI-1 through mRNA stabilization in lung epithelial cells. However, it is unclear how this process affects lung epithelial damage. Here, we show that CS induces p53 and PAI-1 expression and apoptosis in cultured Beas2B and primary alveolar type (AT)II cells. CS exposure augmented binding of p53 protein with PAI-1 mRNA. Inhibition of p53 from binding to PAI-1 mRNA through expression of p53-binding 70 nt PAI-1 mRNA 3'UTR sequences suppressed CS-induced PAI-1 expression. Treatment of Beas2B cells with caveolin-1 scaffolding domain peptide (CSP) suppressed p53 expression and p53-PAI-1 mRNA interaction. These changes were associated with parallel inhibition of CS-induced PAI-1 expression and apoptosis in Beas2B cells. Wild-type mice exposed to passive CS likewise show augmented p53 and PAI-1 with parallel induction of ATII cell apoptosis, whereas mice deficient for p53 or PAI-1 expression resisted apoptosis of ATII cells. CSP suppressed CS-induced ATII cell apoptosis in wild-type mice and abrogated p53-PAI-1 mRNA interaction with parallel inhibition of p53 and PAI-1 expression. The protection against ATII cell apoptosis by CSP involves inhibition of passive CS-induced proapoptotic Bax and Bak expression and restoration of the prosurvival proteins Bcl-X(L). These observations demonstrate that inhibition of p53 binding to PAI-1 mRNA 3'UTR attenuates CS-induced ATII cell apoptosis. This presents a novel link between p53-mediated PAI-1 expression and CS-induced ATII cell apoptosis.     

球囊损伤血管内皮后平滑肌细胞凋亡及相关基因表达的变化

目的:探讨球囊损伤血管内皮后平滑肌细胞(SMC)凋亡的机制。方法:采用末端脱氧核苷酸转移酶介导的三磷酸脱氧尿嘧啶缺口末端标记法(TUNEL)和免疫组织化学技术检测球囊损伤内皮后血管平滑肌细胞(VSMC)凋亡及Bax/Bcl-2蛋白的变化。结果:球囊损伤内皮后第3d,血管中层出现凋亡的SMC;损伤后第7d,内膜和中层SMC凋亡率最高,凋亡的SMC主要分布在内膜层;以后逐渐降低,至损伤后第28d,仅内膜层有少量凋亡的SMC。Irbesartan显著增加SMC凋亡(P＜0.01)。球囊损伤内皮后第3d,血管中层Bax/Bcl-2表达显著高于假手术组(P＜0.01);损伤后第7d血管中Bax表达最高,是假手术组的3倍,以后表达减少。球囊损伤内皮后Bcl-2表达逐渐增多,至第28d表达最高。Bax/Bcl-2也在损伤后第7d达最高,至第28d降至基础水平以下。Irbesartan使Bax表达增高、Bcl-2表达降低、Bax/Bcl-2升高。结论:Bax/Bcl-2参与了球囊损伤内皮后VSMC凋亡的调节。     

生育年龄妇女早期卵泡细胞凋亡和Bcl-2/BAX蛋白表达

目的:研究生育年龄妇女早期卵泡的细胞凋亡和Bcl-2/BAX蛋白表达。方法:12例卵巢组织标本来自进行妇科手术的生育年龄妇女(年龄23-38岁),并经过组织病理学检查证实无明显形态异常。利用末端标记法(TUNEL)和免疫组织化学方法研究早期卵泡(包括始基卵泡、间期和初级卵泡)细胞凋亡和Bcl-2/BAX蛋白表达。结果:早期卵泡内18.75%卵细胞表现TUNEL阳性,但是卵泡内未见TUNEL阳性颗粒细胞。BAX在早期卵泡的卵细胞内表达,阳性表达率76.07%;相反未见Bcl-2在卵细胞表达。另外,早期卵泡内颗粒细胞也没有表达Bcl-2和BAX。结论:生育年龄妇女早期卵泡的卵细胞发生凋亡,促凋亡蛋白BAX在卵细胞凋亡过程中发挥调节作用,由此提示BAX介导的卵细胞凋亡可能是生育年龄妇女早期卵泡闭锁的机制。     

Similarities of prosurvival signals in Bcl-2-positive and Bcl-2-negative follicular lymphomas identified by reverse phase protein microarray

Overexpression of Bcl-2 protein has been known to play a role in the pathogenesis of follicular lymphoma (FL). However, 10-15% of FLs are negative for Bcl-2 by immunohistochemistry, raising the possibility that another gene product(s) may provide prosurvival signal(s). We used reverse phase protein microarray to analyze lysates of follicle center cells isolated by laser capture microdissection from: Bcl-2+ FL, Bcl-2- FL and reactive follicular hyperplasia (FH) (nine cases each group). TUNEL assay confirmed similar and reduced levels of apoptosis in Bcl-2+ FL and Bcl-2- FL, indicating the likelihood of Bcl-2-independent inhibition of apoptosis. Arrays were quantitatively analyzed with antibodies to proteins involved in the apoptotic pathway. As expected, Bcl-2 levels were up to eight-fold higher in Bcl-2+ FL than in FH and Bcl-2- FL. However, there was no difference in levels of Mcl-1 and survivin among these three groups. Bcl-X(L) showed a trend for increased expression in Bcl-2- FL as compared with Bcl-2+ FL, although the differences did not reach statistical significance (P>0.1). The increase in Bcl-X(L) may provide an alternative antiapoptotic signal in FL negative for Bcl-2 protein. Interestingly, Bax expression was higher in FL (Bcl-2+ or -) than in FH (P=0.001). Notably, phospho-Akt (Ser-473) was increased in FL (Bcl-2+ or -) (P<0.03) with increased phospho-Bad (Ser-136), as compared with levels in FH. The activation of the Akt/Bad pathway provides further evidence of prosurvival signals in FL, independent of Bcl-2 alone. These data suggest that nodal FL represents a single disease with a final common biochemical pathway.     

Expression of Bcl-2, but not Bax, correlates with estrogen receptor status and tumor proliferation in invasive breast carcinoma

Bcl-2 and Bax have been demonstrated to be associated with apoptosis in breast carcinoma, and the ratio between Bax and Bcl-2 seems to be an important determinant of cellular sensitivity to induction of apoptosis. However, little information is available on the relationship between Bcl-2, Bax and the proliferative activity of breast carcinoma. The purpose of this study was to investigate the significance of apoptosis-related genes bcl-2 and Bax and their correlation with expression of p53, tumor proliferation defined by MIB-1 expression and estrogen receptor status. Immunohistochemistry was performed to determine Bcl-2, Bax, p53, estrogen receptor (ER) and MIB-1 expression in paraffin-embedded tissues of 177 invasive breast cancers. Expression of the anti-apoptotic protein Bcl-2 was not correlated with the pro-apoptotic Bax. Bcl-2 immunostaining displayed a negative correlation with increasing histologic grade, p53 and MIB-1 (P < 0.0001, P < 0.05 and P < 0.0001, respectively) and a positive correlation with rising ER immunostaining (r = 0.305, P < 0.0001). Conversely, expression of the apoptosis-promoting protein Bax did not correlate with increasing histologic grade, p53, MIB-1 or ER status. Neither Bcl-2 expression nor Bax expression correlated with age, menopausal status, tumor size, histologic type or axillary lymph node status. These results imply that Bcl-2 is associated with good prognostic markers and the regulation of Bax is complex and does not necessarily correlate with mutant p53 status in breast cancers.     

Expression of Bax, Bcl-2, and P53 in progressive multifocal leukoencephalopathy.

It has been shown in vitro that JC viral protein can form a complex with wild-type p53 protein, which is a key regulator of both cell proliferation and cell death. Cellular factors, Bax and Bcl-2, are two essential downstream elements involved in p53-dependent apoptosis. To determine whether association of JC virus with p53 protein affects the expression of Bax and Bcl-2 in viral-infected cells in progressive multifocal leukoencephalopathy (PML), we studied the expression of Bax, Bcl-2, and p53 in 14 cases from 13 PML patients by using paraffin immunohistochemistry. Seven of 13 patients were known to be HIV positive. Overexpression of p53 was found in viral-infected oligodendrocytes and some astrocytes in all 14 cases. Intense immunostaining of Bax was strongly expressed in viral-infected oligodendrocytes and astrocytes. Bax immunostaining was also found in macrophages in the demyelinating lesions. Bcl-2 was not detected in viral-infected glial cells. The expression pattern of Bax positive/Bcl-2 negative in viral-infected glial cells suggests that the oligodendrocyte may be undergoing apoptosis which may in turn contribute to the demyelinating process in PML. The coexpression of p53 and Bax in the infected glial cells suggests that p53 detected by immunohistochemistry may still maintain its wild-type function.     

The expression of antiapoptotic proteins Bcl-2, Bcl-XL, and Mcl-1 in benign, dysplastic, and malignant biliary epithelium

AIM: Cholangiocarcinoma can be cured by surgery, but only in a minority of cases. The activation of apoptosis is a major mode of action of chemotherapy and radiotherapy, which have limited benefit in the treatment of cholangiocarcinoma. The antiapoptotic members of the Bcl-2 protein family (Bcl-2, Bcl-X(L), and Mcl-1) are important inhibitors of apoptosis, but have not been investigated extensively in cholangiocarcinoma. METHODS: The expression of Bcl-2, Bcl-X(L), and Mcl-1 was investigated in normal biliary epithelium (17), biliary dysplasia (three), and invasive cholangiocarcinoma (51), in addition to three human cholangiocarcinoma cell lines, by immunohistochemistry and immunofluorescence. RESULTS: The expression of Bcl-2 was not detected in normal or malignant biliary tissue. In contrast, granular cytoplasmic Bcl-X(L) and Mcl-1 staining was found in 60-100% of cells in all normal, dysplastic, and malignant specimens, including the human cell lines examined in this study. CONCLUSION: These findings indicate that Mcl-1 and Bcl-X(L), but not Bcl-2, are involved in the survival of normal and neoplastic cells in the biliary tree. By prolonging survival through blocking apoptosis, these proteins might be reducing the efficacy of cytotoxic anticancer treatments in cholangiocarcinoma.