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.     

Immunohistochemical expression of Bcl-2, Bcl-x, and Bax in follicular carcinomas of the thyroid.

Bcl-2, bcl-x, and bax proteins are involved in the regulation of apoptosis. There is limited data on the expression of these proteins in follicular carcinomas (FCs) of the thyroid. A retrospective clinicopathologic review with bcl-2, bcl-x, and bax immunostaining of 34 FCs and 7 follicular adenomas with incomplete capsular penetration (FAICP) was performed. The study included 41 patients (25 females; age range 16 to 84 y, mean 50.9 y). All patients underwent surgical resection. Seven FC patients developed recurrent disease: 1 patient was alive (14.2 y) and 6 patients died with metastatic disease (mean survival 5.9 y). All remaining patients were disease-free (mean follow-up 7.9 y). Only one FAICP recurred (patient alive at 11 y). The remaining patients were disease-free (mean follow-up 6.9 y). Normal thyroid tissue stained positively for bcl-2 and bcl-x, and did not stain with bax. Only 15 tumors (12 FC and 3 FAICP) stained positively for bcl-2. None of the recurrent tumors demonstrated evidence of bcl-2 staining. The majority of tumors stained positively for bax (83%, 29 FC and 5 FAICP) and for bcl-x (93%, 32 FC and 6 FAICP); there was no correlation of staining with outcome. The majority of follicular neoplasms were positive for bax and negative for bcl-2 by immunohistochemistry. Aberrant expression of apoptosis-associated proteins may play a role in the pathogenesis of FC of the thyroid. All recurrent and fatal tumors were negative for bcl-2. These data suggest that the loss of bcl-2 expression may correlate with poorer prognosis.     

Bcl-2, Bax and Bcl-x expression following kainic acid administration at convulsant doses in the rat.

Neuronal death was produced in the CA1 and CA3 areas of the hippocampus, amygdala, and piriform and entorhinal cortices after intraperitioneal administration of kainic acid at convulsant doses to adult rats. To assess the involvement of members of the Bcl-2 family in cell death or survival, immunohistochemistry, western and northern blotting to Bcl-2, Bcl-x and Bax, and in situ hybridization to Bax were examined at different time-points after kainic acid treatment. Members of the Bcl-2 family were expressed in the cytoplasm of pyramidal neurons in the hippocampus, and in a subset of neurons of the piriform and the entorhinal cortices, amygdala and neocortex in the normal adult brain. Dying neurons in the pyramidal cell layer of CA1 and CA3 areas, entorhinal and piriform cortices, and amygdala also expressed Bcl-2, Bax and Bcl-x following excitotoxicity, although many dying cells did not. In addition, a number of cells in the affected areas showed Bax immunoreactivity in their nuclei at 24-48 h following kainic acid administration, thus indicating Bax nuclear translocation in a subset of dying cells. Western blots disclosed no modifications in the intensity of the bands corresponding to Bcl-2, Bcl-x and Bax, between control and kainic acid-treated rats. No modifications in the intensity of the bcl-2 messenger RNA band on northern blots was observed in kainic acid-treated rats. However, a progressive increase in the intensity of the bax messenger RNA band was found in kainic acid-treated rats at 6 h, 12 h and 24 h following kainic acid administration. Interestingly, a slight increase in Bax immunoreactivity was observed in the cytoplasm of neurons of the dentate gyrus at 24-48 h, a feature which matches the increase of bax messenger RNA in the same area, as shown by in situ hybridization at 12-24 h following kainic acid injection. The present results suggest that cell death or survival does not correlate with modifications of Bcl-2, Bax and Bcl-x protein, and messenger RNA expression, but rather that kainic acid excitotoxicity is associated with Bax translocation to the nucleus in a subset of dying cells.     

Ubiquitin immunostaining and inclusion body myositis: Study of 30 patients with inclusion body myositis

Distinction of inclusion body myositis (IBM) from other forms of inflammatory myopathy is significant from prognostic and therapeutic standpoints. This study retrospectively examines ubiquitin expression by paraffin immunohistochemistry in muscle biopsy material from 30 patients with IBM. Patients included 19 men and 11 women (ages 29 to 80 years; mean, 64 years). All biopsies were characterized by endomysial chronic inflammation, muscle fiber degeneration and regeneration, rimmed vacuoles, and angular atrophic esterase-positive muscle fibers. Ragged red fibers were identified in biopsies of five patients and a partial cytochrome C-oxidase deficiency by enzyme histochemistry in biopsies of 10 patients. Evidence of intranuclear or cytoplasmic tubulofilamentous structures confirming a diagnosis of IBM was observed in all 30 cases. Paracrystalline mitochondrial inclusions were noted in five patients. Discrete myocyte intranuclear ubiquitin-positive inclusions were noted in 14 patients (47%). Discrete intracytoplasmic ubiquitin-positive inclusions were noted in 24 (80%) patients. Positive staining of rimmed vacuoles by ubiquitin was observed in 25 (83%) patients. Diffuse staining of scattered muscle fibers was observed in 21 (70%) patients. In a control group including patients with polymyositis (n = 3), dermatomyositis (n = 3), necrotizing vasculitis (n = 1), and granulomatous myositis (n = 1), discrete intranuclear or cytoplasmic ubiquitin-positive inclusions were not observed. Rimmed vacuoles were not seen either by light microscopy or ubiquitin immunostaining in any of the eight cases. Occasional myofibers from all eight cases showed diffuse, positive muscle fiber staining. Although not present in all cases, evidence of ubiquitinpositive myocytic intranuclear or cytoplasmic inclusions or positive-staining rimmed vacuoles in the setting of an inflammatory myopathy may be suggestive of a diagnosis of inclusion body myositis. Use of ubiquitin immunohistochemistry may be useful in cases in which frozen tissue or tissue processed for electron microscopy is not available, and IBM is suspected. Light or electron microscopic evidence of mitochondrial abnormalities were noted in a significant subset of patients (13 of 30; 43%) of patients with IBM.     

Bcl-2, Bax, and Bcl-x expression in the CA1 area of the hippocampus following transient forebrain ischemia in the adult gerbil

Delayed neuronal death was produced in the CA1 area of the hippocampus following 5 min of forebrain ischemia in adult gerbils. Immunohistochemistry and Western blotting to Bcl-2, Bax, and Bcl-x was examined in control (age-matched, non-operated and sham-operated) and ischemic gerbils. Bcl-2 immunoreactivity was low in CA1 neurons, but Bax was highly expressed in CA1 neurons of control gerbils. Moderate Bcl-x immunoreactivity was observed in control CA1 neurons. Strong Bcl-2 and Bcl-x immunoreactivity was found in CA1 neurons following ischemia. Bcl-2, Bax, and Bcl-x were localized in dying cells, thus suggesting that expression of Bcl-2 was not sufficient to prevent nerve cells from dying. Although the Bcl-x antibody does not discriminate between Bcl-xL and Bcl-xS content in tissue sections, Western blots disclosed a marked increase in the intensity of the band corresponding to Bcl-xS, but not of the band corresponding to Bcl-xL in ischemic hippocampi, thus indicating that the increase in Bcl-xS is associated with delayed cell death following transient forebrain ischemia in the adult gerbil. Received: 24 June 1997 / Accepted: 29 January 1998     

Apoptosis and expression of Bax, Bcl-x, and Bcl-2 apoptotic regulatory proteins in colorectal carcinomas, and association with p53 genotype/phenotype.

AIMS: Spontaneous apoptosis and expression of the apoptotic regulatory proteins Bax, Bcl-x, and Bcl-2 were investigated in 50 colorectal carcinomas. The p53 genotypes/phenotypes and BAX genotypes were also determined, and possible associations of these with apoptosis and/or with expression of the different apoptotic regulatory proteins were studied. METHODS: Terminal deoxynucleotidyl transferase (TdT) mediated dUTP labelling of DNA fragments was used to detect apoptotic tumour cells in sections and peroxidase immunohistochemistry was used to assess protein expression. p53 genotype/phenotype was determined using constant denaturant gel electrophoresis/immunoblotting and bax genotype was determined using polymerase chain reaction based methods. RESULTS: The distribution of tumour apoptotic indices was bimodal with a natural cut off at 1.0% (range, 0.0-5.4%); the median fraction of apoptotic tumour cells was 0.8%. Tumour apoptosis was not associated significantly with tumour DNA ploidy status. Normal mucosal tissue had less than 0.1% apoptotic cells. Staining intensities for Bax, Bcl-x, and Bcl-2 were strong; that is, equivalent to or greater than positive normal mucosal cells, in 11 of 50, 20 of 49, and 20 of 48 carcinomas. Frameshift mutations in the bax gene were detected in three of 42 tumours analysed, all of which were DNA diploid, and Bax protein expression in these tumours was absent or very low. Bax, Bcl-x, and Bcl-2 protein expression were not correlated with tumour apoptosis or tumour DNA ploidy status. p53 was expressed in 34 of 50 tumours and p53 gene mutations were detected in 22 of 29 p53 positive tumours analysed. Apoptosis was significantly lower in a greater number of p53 positive tumours than p53 negative tumours. In addition, Bcl-2 protein expression was significantly higher in a greater number of p53 positive tumours compared with p53 negative tumours. Bax and Bcl-x protein expression were not significantly associated with p53 phenotype/genotype. CONCLUSIONS: The results indicate that acquisition of a p53 phenotype is associated with lower spontaneous apoptosis and higher expression of Bcl-2. The results also suggest that p53 is not a major determinant for Bax expression in colorectal carcinomas in vivo.     

Apoptosis and expression of Bax, Bcl-x, and Bcl-2 apoptotic regulatory proteins in colorectal carcinomas, and association with p53 genotype/phenotype.

AIMS: Spontaneous apoptosis and expression of the apoptotic regulatory proteins Bax, Bcl-x, and Bcl-2 were investigated in 50 colorectal carcinomas. The p53 genotypes/phenotypes and BAX genotypes were also determined, and possible associations of these with apoptosis and/or with expression of the different apoptotic regulatory proteins were studied. METHODS: Terminal deoxynucleotidyl transferase (TdT) mediated dUTP labelling of DNA fragments was used to detect apoptotic tumour cells in sections and peroxidase immunohistochemistry was used to assess protein expression. p53 genotype/phenotype was determined using constant denaturant gel electrophoresis/immunoblotting and bax genotype was determined using polymerase chain reaction based methods. RESULTS: The distribution of tumour apoptotic indices was bimodal with a natural cut off at 1.0% (range, 0.0-5.4%); the median fraction of apoptotic tumour cells was 0.8%. Tumour apoptosis was not associated significantly with tumour DNA ploidy status. Normal mucosal tissue had less than 0.1% apoptotic cells. Staining intensities for Bax, Bcl-x, and Bcl-2 were strong; that is, equivalent to or greater than positive normal mucosal cells, in 11 of 50, 20 of 49, and 20 of 48 carcinomas. Frameshift mutations in the bax gene were detected in three of 42 tumours analysed, all of which were DNA diploid, and Bax protein expression in these tumours was absent or very low. Bax, Bcl-x, and Bcl-2 protein expression were not correlated with tumour apoptosis or tumour DNA ploidy status. p53 was expressed in 34 of 50 tumours and p53 gene mutations were detected in 22 of 29 p53 positive tumours analysed. Apoptosis was significantly lower in a greater number of p53 positive tumours than p53 negative tumours. In addition, Bcl-2 protein expression was significantly higher in a greater number of p53 positive tumours compared with p53 negative tumours. Bax and Bcl-x protein expression were not significantly associated with p53 phenotype/genotype. CONCLUSIONS: The results indicate that acquisition of a p53 phenotype is associated with lower spontaneous apoptosis and higher expression of Bcl-2. The results also suggest that p53 is not a major determinant for Bax expression in colorectal carcinomas in vivo.     

Immunohistochemical expression of Bax and Bcl-2 in penile carcinoma

There is a complex interplay between the pro-apoptotic Bax and anti-apoptotic Bcl-2 family of proteins and the tumor suppressor gene p53. The pathogenic role of Bax and Bcl-2 protein expression in penile carcinomas has not previously been investigated. We examined Bax and Bcl-2 expression in verrucous (VC) and squamous cell carcinoma (SCC) of the penis. Herein we also present a concise review of p53, Bcl-2/Bax ratios, and their relationship to apoptosis. Fourteen cases of penile carcinoma, including 7 VC and 7 well-differentiated SCC, were analyzed for Bax and Bcl-2 expression by immunohistochemical analysis of paraffin embedded archived tissues. The number of positively staining tumor cells was enumerated per 100 tumor cells within non-overlapping high power fields. The Bax immunoreactivity was similar in VC (19+/-3%) and well-differentiated SCC (15+/-4%) (p = 0.69). The expression of Bcl-2 protein was significantly higher in well-differentiated SCC (69+/-12%) compared to VC (36+/-14%) (p = 0.04). The mean Bcl-2/Bax ratio was significantly lower in VC (1.89) compared to well-differentiated SCC (4.6) (p = 0.05). These findings indicate that penile VC and SCC are immunophenotypically distinct. Bax expression is comparable in verrucous and low-grade squamous cell carcinomas, but Bcl-2 expression of Bcl-2 is significantly higher in the squamous cell carcinomas.     

Discordant expression of Bcl-x and Bcl-2 by keratinocytes in vitro and psoriatic keratinocytes in vivo.

Apoptosis is a required event in maintaining kinetic homeostasis within continually renewing tissues such as skin. However, no systematic study of the apoptotic process in epidermal keratinocytes of the skin has been performed. In this report, we examined the expression of proteins associated with promoting (Fas) or preventing (Bcl-2, Bcl-x, CD40) apoptosis in the normal, psoriatic, and malignant keratinocyte. Immunohistochemical staining and flow cytometry analysis revealed that normal cultured keratinocytes express low levels of Fas, CD40, and Bcl-x that was enhanced by cytokines including gamma-interferon (IFN-gamma) and a phorbol ester tumor promoter, TPA. Only faint Bcl-2 staining was detected in cultured keratinocytes exposed to IFN-gamma and TPA compared with the prominent expression of Bcl-x. Biopsies of normal skin, psoriatic plaques, and basal cell carcinomas were examined to extend the in vitro observations. Immunohistochemical staining revealed that while keratinocytes in normal epithelium express low to absent levels of Fas and Bcl-x, psoriatic keratinocytes expressed significantly higher levels of Fas and Bcl-x. In contrast, malignant keratinocytes in basal cell carcinomas expressed high levels of Bcl-2, but minimal Bcl-x, and no Fas. Immunoblot analysis revealed that the long form of Bcl-x (Bcl-xI), which prevents apoptosis in lymphocytes, is expressed by cultured keratinocytes and psoriatic plaque keratinocytes. We conclude that normal cytokine-activated keratinocytes can express an apoptotic (Fas) and an anti-apoptotic protein (Bcl-x). The overexpression of Bcl-x in psoriasis, or Bcl-2 in basal cell carcinomas, may contribute to the longevity of these cells by blocking the normal apoptotic process involved in the terminal differentiation program of epidermal keratinocytes.     

Elevated expression of Bcl-2 and Bcl-x by intestinal intraepithelial lymphocytes: resistance to apoptosis by glucocorticoids and irradiation

Administration of glucocorticoids or exposure to ionizing radiation in vivo results in a rapid cell death of thymocytes. We report that murine small intestinal intraepithelial lymphocytes (IEL) are resistant to both steroid- and radiation-induced deletion. This is due to resistance to apoptosis, as evidenced by the absence of detectable apoptotic IEL nuclei in situ after in vivo glucocorticoid treatment. IEL express normal levels of glucocorticoid receptors and these receptors bind [3H]dexamethasone to equivalent levels as other lymphocyte populations. Thus, their survival is due to post-receptor signaling mechanisms. Many IEL express high levels of Bcl-2 and that of these Bcl-2high IEL are largely TCR gamma delta +. Those IEL that do express high levels of Bcl- 2 are CD8 alpha + beta - CD4-. In addition, IEL express Bcl-x, another protein shown to be involved in the protection of cells from apoptotic signals. IEL represent the first lymphocyte population in vivo shown to have high levels of expression of both molecules, that otherwise occur only in activated lymphocytes in vitro. These data suggest that the Bcl- 2+Bcl-x+ IEL are activated cells and not an effete population of cells necessarily destined to die. Also, the high levels of Bcl-2 and Bcl-x in this in vivo activated population supports the in vitro correlate of protection from activation-induced cell death.      

Bax, Bcl-2, and NF-kappaB expression in sanguinarine induced bimodal cell death

The apoptosis related proteins Bax, Bcl-2, and NF-kappaB were analyzed in sanguinarine induced apoptosis and blister cell death (BCD) of K562 erythroleukemia cells and in sanguinarine treated high Bcl-2 expressing JM1 pre-B lymphoblastic cells, utilizing immunofluorescence-flow cytometry. Sanguinarine induced apoptosis of K562 cells was found to have increased Bax expression and decreased NF-kappaB, whereas BCD showed a decrease in Bax expression and an increase in NF-kappaB. In contrast, high Bcl-2 expressing JM1 cells, when exposed to the same concentrations (and duration) of sanguinarine that induced PCD and BCD in K562 cells, failed to show the respective morphologies while showing a concomitant increase in Bcl-2. Results from studies with K562 cells suggest that Bax is pro-apoptotic and also that NF-kappaB activation may be associated with BCD. Results from studies with JM1 cells suggest that Bcl-2 is anti-apoptotic and anti-BCD. Results from JM1 cells strengthen the assumption in the literature of the central role Bcl-2 plays in chemoresistance by assuming an anti-PCD role. These results also suggest that, in JM1 cells, Bcl-2 may further complicate chemoresistance by being anti-BCD in nature, in addition to its anti-PCD role.     

小鼠海马发育中Bcl-2和Bax表达的体视学观察

目的探讨B淋巴细胞瘤-2基因(Bcl-2)和促凋亡基因Bax(Bax)在C57/BL6小鼠海马发育过程中的表达变化。方法取胚龄(E)18、20 d和生后(P)1、3、7、14、21、28 d以及2、3、6、15、18个月的C57/BL6小鼠海马,每组8只,应用免疫组织化学技术及体视学方法检测Bcl-2和Bax蛋白的表达。结果 E18 d~P21 d,在齿状回(DG)的颗粒层、海马阿蒙角(CA)以及CA各区(CA1~CA4)的锥体层,Bcl-2和Bax阳性细胞及其体密度均呈现出先逐渐增加再逐渐降低的趋势,除DG区Bax阳性细胞及其体密度在P14 d达到最高外,其他均在P7 d达到最高(P0.01)。P28 d后均趋于稳定(P0.05)。CA锥体层及DG颗粒层Bcl-2/Bax的体密度比值在P1d显著降低(P0.01),之后趋于稳定(P0.05)。结论小鼠海马Bcl-2和Bax的表达在胚胎发育晚期和生后发育早期较多,而在成年及老年期的表达稳定在较低水平,它们可能参与了海马的塑形过程。     

Mitochondrial DNA variants in inclusion body myositis characterized by deep sequencing

Muscle pathology in inclusion body myositis (IBM) typically includes inflammatory cell infiltration, muscle fibers with rimmed vacuoles and cytochrome c oxidase (COX)‐deficient fibers. Previous studies have revealed clonal expansion of large mitochondrial DNA (mtDNA) deletions in the COX‐deficient muscle fibers. Technical limitations have prevented complete investigations of the mtDNA deletions and other mtDNA variants. Detailed characterization by deep sequencing of mtDNA in muscle samples from 21 IBM patients and 10 age‐matched controls was performed after whole genome sequencing with a mean depth of mtDNA coverage of 46,000x. Multiple large mtDNA deletions and duplications were identified in all IBM and control muscle samples. In general, the IBM muscles demonstrated a larger number of deletions and duplications with a mean heteroplasmy level of 10% (range 1%‐35%) compared to controls (1%, range 0.2%‐3%). There was also a small increase in the number of somatic single nucleotide variants in IBM muscle. More than 200 rearrangements were recurrent in at least two or more IBM muscles while 26 were found in both IBM and control muscles. The deletions and duplications, with a high recurrence rate, were mainly observed in three mtDNA regions, m.534‐4429, m.6330‐13993, and m.8636‐16072, where some were flanked by repetitive sequences. The mtDNA copy number in IBM muscle was reduced to 42% of controls. Immunohistochemical and western blot analyses of IBM muscle revealed combined complex I and complex IV deficiency affecting the COX‐deficient fibers. In conclusion, deep sequencing and quantitation of mtDNA variants revealed that IBM muscles had markedly increased levels of large deletions and duplications, and there were also indications of increased somatic single nucleotide variants and reduced mtDNA copy numbers compared to age‐matched controls. The distribution and type of variants were similar in IBM muscle and controls indicating an accelerated aging process in IBM muscle, possibly associated with chronic inflammation.     

Gain and loss of extracellular molecules in sporadic inclusion body myositis and polymyositis--a proteomics-based study

Sporadic inclusion body myositis (sIBM) contains non-necrotic myofibers that are surrounded and/or invaded by inflammatory cells. In this study we aimed to identify selective molecules that are present at this site. Myofibers of four biopsies of sIBM that were surrounded and/or invaded by inflammatory cells were microdissected, pooled and profiled by proteomic studies using mass spectrometry. Normal skeletal muscle tissue served as control. Based on the table of proteins that were detected in sIBM only, we selected nine extracellular matrix molecules and validated the results performing immunofluorescence. Seven out of nine proteins that were detected in sIBM by mass spectrometry showed different immunohistochemical results in myositis and normal controls. Of these, the small leucine-rich repeat proteins proline arginine-rich end leucine-rich repeat protein (PRELP) and biglycan were deposited precisely at myofibers surrounded and/or invaded by inflammatory cells both in sIBM and polymyositis. The basement membrane (BM) molecules merosin, perlecan, nidogen-2 and collagen IV were variably destroyed or increased at these sites. P component, which ensheathed all myofibers in normal controls, was absent from invaded myofibers. Similar to BM remodeling, the specific deposition of PRELP and biglycan may represent a mechanism to defend against immune attack. Loss of P component may affect the anchorage of the myofiber in the endomysium.     

DCC基因在大肠癌中的表达及与Bcl-2, Bax关系

目的：探讨DCC(deleted in colorectal carcinoma)基因在大肠癌组织中的表达情况及与Bcl-2，Bax蛋白之间的关系。 方法：采用免疫组织化学法结合图像分析技术观察74例大肠癌组织中DCC，Bcl-2，Bax蛋白的表达情况。 结果：大肠癌组织中，DCC蛋白失表达率为 54.1% (40/74)，其表达在不同的肿瘤分化程度 (P＝0.002)及Dukes分期中有差异 (P＝0.042)； Bcl-2蛋白表达阳性率为60.8% (45/74)，其表达与不同的大肠癌临床Dukes分期 (P＝0.032)及是否有淋巴结转移 (P<0.001)中有差异；Bax蛋白表达阳性率为48.6% (36/74)，在不同的大肠癌组织学类型、分化程度、Dukes分期及淋巴结是否转移中均无差异 (P>0.05)。大肠癌中DCC蛋白表达与Bcl-2表达水平呈负相关（r＝－0.201, P＝0.043），与Bax表达水平呈正相关（r＝0.296, P＝0.005）。 结论：大肠癌中DCC表达缺失频率较高，且可能通过上调Bcl-2蛋白表达和下调Bax蛋白表达阻止细胞凋亡，从而促进大肠癌发生。     

Analysis of multiple mitochondrial DNA deletions in inclusion body myositis

Inclusion body myositis (IBM) is a sporadic progressive myopathy, which is morphologically characterized by inflammatory cell infiltrates and rimmed vacuoles in muscle fibers. Mitochondrial changes are regularly present with ragged-red fibers showing deficiency of cytochrome c oxidase. In these muscle fiber segments, there is accumulation of mitochondria with mitochondrial DNA (mtDNA) deletions. There are different deletions in different muscle fibers. In this study, we have sequenced for the first time the multiple mtDNA deletions in muscle from four patients with IBM. The deletion breakpoints were sequenced from cloned polymerase chain reaction (PCR)-amplified mtDNA fragments. The sequencing was performed directly from the bacterial colonies used for cloning. Of 122 analyzed clones, 33 different deletions were identified. The majority of these have not previously been described. There was a marked predominance of deletion breakpoints in certain regions of mtDNA. These predominant breakpoint regions are similar to those described in other conditions with multiple deletions, such as autosomal dominant progressive external ophthalmoplegia (adPEO) and normal aging, but different from those described in diseases due to single deletions such as Kearns-Sayre syndrome and sporadic PEO. These findings indicate that common factors are involved in the development of multiple mtDNA deletions in IBM, adPEO, and aging. Hum Mutat 10:381–386, 1997. © 1997 Wiley-Liss, Inc.     

Influence of prostaglandin A2 on Bax, Bcl-2 and PCNA expression in MCF-7 cells

The effects of 20 microg/mL exogenous prostaglandin A(2) (PGA(2)) were determined on Bax, Bcl-2 and proliferating cell nuclear antigen (PCNA) expression levels in MCF-7 cells. Flow cytometric analysis indicated a pronounced increase in the S phase and a decrease in the G(1) phase, whereas a significant increase in the DNA content preceding the G(0)/G(1) peak was also observed after 48 h of exposure to PGA(2). Confirmation of apoptosis was determined after 12 h, 36 h and 48 h of PGA(2) exposure employing the mitosensor reagent that detects potential changes in the mitochondrial membrane. Twenty-eight percent of PGA(2)-exposed cells were in apoptosis when compared to the 7.1% vehicle-treated cells after 48 h. PGA(2) exposure led to statistically significant increase (1.25-fold) over vehicle-treated controls in Bax expression levels. Decreases in Bcl-2 (0.79-fold), as well as PCNA (0.69-fold) expression levels over vehicle-treated controls were observed. The Bax/Bcl-2 ratio for PGA(2)-exposed cells was 2.7. The present study suggests that an accumulation in the S phase, a decrease in expression levels of PCNA, as well as an altered ratio in favor of Bax, could lead to the induction of apoptosis in these cells.     

Bcl-2 and Bcl-x: regulatory switches for lymphoid death and survival

The survival and death of lymphoid cells is under the control of a genetic program. Cell death is activated at different stages of development and serves to remove unnecessary and autoreactive lymphocytes, as well as to limit the immune response. The survival of cells is regulated by a set of genes that act as repressors of the cell death mechanism. Of these, bcl-2 and bcl-x exhibit a striking pattern of regulation during lymphoid maturation and can inhibit several forms of apoptotic cell death. Here, Gabriel Núñez and colleagues review recent developments in the field, particularly focusing on the role of the Bcl-2 and Bcl-x proteins in regulating lymphoid death and survival.     

Bax/Bcl-2 expression levels of 2-methoxyestradiol-exposed esophageal cancer cells

2-Methoxyestradiol (2ME), an endogenous metabolite of 17beta-estradiol, has been reported to play an active role in the induction of apoptosis in both proliferating endothelial and cancer cells. Since it has been indicated that an increased ratio of pro-apoptotic Bax protein to anti-apoptotic Bcl-2 protein expression can be associated with apoptosis, and since the exact action mechanism of 2ME is still not clearly defined and appears to vary according to cell type, the influence of 1 microM 2ME was investigated on Bax and Bcl-2 expression levels in squamous esophageal carcinoma cells. 2ME exposure led to statistically significant decreases (0.69 over DMSO controls) in Bcl-2 expression levels. In contrast, no statistically significant effects were observed on Bax expression levels after exposure to 2ME. The Bax/Bcl-2 ratio for 2ME-exposed cells was 1.45, normalised against Bcl-2 levels. Although the exact mechanisms of apoptosis induction in squamous esophageal cancer cells require further investigation, the present study suggests that this altered ratio in favor of Bax could lead to the induction of apoptosis in these cells.