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.     

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.     

Concomitant progressive multifocal leucoencephalopathy and primary central nervous system lymphoma expressing JC virus oncogenic protein, large T antigen.

This report describes the concomitant occurrence of the JC virus (JCV) induced demyelinating disease progressive multifocal leucoencephalopathy (PML) and a primary central nervous system lymphoma (PCNS-L) in a patient with AIDS. Postmortem neuropathological examination revealed characteristic features of PML including multiple lesions of demyelination, enlarged oligodendrocytes with hyperchromatic nuclei (many containing eosinophilic intranuclear inclusions), and enlarged astrocytes with bizarre hyperchromatic nuclei. Immunohistochemical analysis demonstrated the expression of the JCV capsid protein VP-1 in the nuclei of infected oligodendrocytes and astrocytes. The PCNS-L lesion located in the basal ganglia was highly cellular, distributed perivascularly, and consisted of large atypical plasmacytoid lymphocytes. Immunohistochemical examination of this neoplasm identified it to be of B cell origin. Moreover, expression of the JCV oncogenic protein, T antigen, was detected in the nuclei of the neoplastic lymphocytes. This study provides the first evidence for a possible association between JCV and PCNS-L.     

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.     

Effects of JC virus infection on anti-apoptotic protein survivin in progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system resulting from the productive infection of oligodendrocytes by the opportunistic polyomavirus JC virus (JCV). Apoptosis is a host defense mechanism to dispose of damaged cells; however, certain viruses have the ability to deregulate apoptotic pathways to complete their life cycles. One such pathway involves survivin, a member of the inhibitor of apoptosis family, which is abundantly expressed during development in proliferating tissues but should be absent in normal, terminally differentiated cells. Immunohistochemistry performed in 20 cases of PML revealed the presence of survivin in JCV-infected oligodendrocytes and bizarre astrocytes within demyelinated plaques. Survivin up-regulation was also found in oligodendroglial and astrocytic cultures infected with JCV. Cell cycle analysis and DNA laddering demonstrated a significantly lower number of cells undergoing apoptosis on JCV infection compared with noninfected cultures; small interfering RNA inhibition of survivin resulted in a dramatic increase in apoptotic cells in JCV-infected cultures. This is the first report describing the activation of survivin by JCV infection in vitro and in PML clinical cases. These observations provide new insights into the anti-apoptotic mechanisms used by JCV to complete its lytic cycle and may suggest new therapeutic targets for PML.     

Alterations of DNA damage repair pathways resulting from JCV infection

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disorder of the CNS caused by infection of glial cells with the polyomavirus, JCV. Here we report that genomic stability and DNA repair are significantly dysregulated by JCV infection of human astrocytes. Metaphase spreads exhibited increased ploidy correlating with duration of infection. Increased micronuclei formation and phospho-Histone2AX expression also indicated DNA damage. Western blot analysis revealed perturbation in expression of some DNA repair proteins including a large elevation of Rad51. Immunohistochemistry on clinical samples of PML showed robust labeling for Rad51 in nuclei of bizarre astrocytes and inclusion body-bearing oligodendrocytes that are characteristic of JCV infection. Finally, in vitro end-joining DNA repair was altered in extracts prepared from JCV-infected human astrocytes. Alterations in DNA repair pathways may be important for the life cycle of JCV and the pathogenesis of PML.     

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.     

Immunohistochemical determination of in vivo distribution of Bax, a dominant inhibitor of Bcl-2.

The protein encoded by the bcl-2 gene is a regulator of programmed cell death and apoptosis. The cell survival-promoting activity of this protein is opposed by Bax, a homologous protein that forms heterodimers with Bcl-2 and accelerates rates of cell death. In this report, the in vivo patterns of bax gene expression were immunohistochemically assessed in the mouse, with a polyclonal antibody raised against a synthetic peptide corresponding to a unique region in the murine Bax protein. Direct comparisons were made with Bcl-2 by using anti-peptide antisera specific for the mouse Bcl-2 protein. The expression of bax was more widespread than bcl-2. For example, Bax immunoreactivity was present in the hepatocytes of the liver, the exocrine pancreas, and the renal tubule epithelial cells whereas Bcl-2 was absent from these tissues. Both the Bax and Bcl-2 proteins were present in several epithelia examined, including the small intestines, colon, breast, prostate, respiratory tract, and skin. The most intense Bax immunostaining was seen in cells located in the base of the crypts of the small intestinal mucosa, consistent with reports of high rates of spontaneous and inducible apoptosis in this region. Bcl-2 immunostaining was completely absent from these cells but was present in the absorptive epithelial cells of the small intestine. In contrast, Bax immunostaining in the colon tended to be stronger in the surface epithelial cells that had advanced up the crypts towards the lumen and that are destined for programmed cell death, whereas Bcl-2 immunoreactivity generally was stronger in the base of the colonic crypts. Similarly, bax expression in the gastric pits of the stomach occurred in a gradient such that higher levels of Bax immunostaining were found in the upper layers of gastric glands than in the lower regions. In addition, strong Bax immunostaining was detected in the androgen-dependent secretory epithelial cells of the prostate, whereas Bcl-2 was limited to the androgen-independent basal cells. Like Bcl-2, Bax was found in the thymic medulla but not the cortex, despite the propensity for immature cortical thymocytes to undergo apoptosis. Unlike Bcl-2, however, Bax immunostaining tended to be more intense in the germinal center lymphocytes of lymph nodes than in the interfollicular lymphocytes, consistent with the high rate of apoptotic cell death in the former.(ABSTRACT TRUNCATED AT 400 WORDS)     

脉冲电场联合野生型p53基因诱导人宫颈癌Hela细胞凋亡

背景：在前期研究基础上,设想将基因电转染作为脉冲电场治疗恶性肿瘤的辅助方法。 目的：探讨脉冲电场联合抑癌基因野生型p53基因诱导人宫颈癌hela细胞发生凋亡及相互作用。 方法：采用空质粒及含有野生型p53的质粒转染Hela细胞得到Hela-vector、Hela-p53细胞,将Hela细胞、Hela-vector和Hela-p53细胞分别施加固定脉宽100 μs、频率1 Hz、脉冲数8个、电场强度为1 500 V/cm的脉冲电场处理。 结果与结论：相同参数脉冲电场作用于各组细胞12 h后,MTT结果显示Hela-p53组细胞吸光度明显低于Hela组(P < 0.05);流式细胞仪及RT-PCR检测结果显示Hela-p53组的早期凋亡率和p53 mRNA表达较Hela组明显增加;Western bolt及激光共聚焦显微镜结果显示与Hela组比较,Hela-p53组细胞Bax蛋白表达明显上升,而Bcl-2蛋白则明显下降,Casepase-3相对荧光强度明显增强。表明野生型p53基因对宫颈癌Hela细胞生长有明显的抑制作用,野生型p53基因可以提高脉冲电场诱导宫颈癌Hela细胞凋亡的作用。     

二甲双胍对U937 细胞增殖、周期及凋亡的影响

目的：探究二甲双胍（metformin）对U937细胞增殖、周期及凋亡的影响。 方法：以U937细胞为研究对象,予不同浓度的二甲双胍处理,分别在24、48和72 h收集细胞。CCK-8法检测细胞增殖情况,流式检测细胞凋亡及细胞周期,并使用Western blot方法检测促凋亡蛋白Bax、抑凋亡蛋白Bcl-2、p-AMPK、p53的表达情况。结果：CCK8结果显示二甲双胍抑制U937的增殖,且呈时间-剂量依赖性。流式结果显示二甲双胍处理后细胞周期停滞在G0/G1期,G0/G1期细胞比例的增加呈时间-剂量依赖性。二甲双胍可诱导细胞凋亡,且凋亡率呈剂量依赖性;二甲双胍浓度为20 mmol/L时,凋亡率呈时间依赖性。Western blot结果显示二甲双胍处理后,p-AMPK、p53、Bax的表达上调,而Bcl-2的表达下调。 结论：二甲双胍能抑制U937细胞的增殖,阻滞细胞周期在G0/G1期,诱导细胞凋亡;其机制可能与其上调胞内促凋亡蛋白Bax的表达、下调抑凋亡蛋白Bcl-2的表达、激活AMPK/p53通路有关。     

Immunoreactivity of p53, Mdm2, p21(WAF1/CIP1) Bcl-2, and Bax in soft tissue sarcomas: correlation with histologic grade.

Tumor growth depends on 2 distinctive pathways: cell proliferation and apoptosis. The p53 pathway is an important regulator of the cell cycle as it triggers growth arrest or leads to apoptosis in response to cellular stress and therefore is commonly targeted during tumorigenesis. Apoptosis is also controlled by the Bcl-2 family, which includes proapoptotic and antiapoptotic proteins. The aim of this study was to investigate the expression of proteins that are involved in the p53 pathway and apoptosis in different types of soft tissue sarcomas and to correlate the expression of these proteins with the histologic grade of sarcoma cases. One hundred fifty-two cases of different types of soft tissue sarcomas were analyzed. The cases consisted of 54 low-grade, 40 intermediate-grade, and 58 high-grade sarcomas. Immunohistochemical stains for p21(WAF1/CIP1), p53, Mdm2, Bcl-2, and Bax proteins were carried out on tissue microarrays. Nuclear reactivity for p53 was detected in 49 cases (32.2%). Overexpression of Mdm2 was found in 18 cases (11.8%) and p21(WAF1/CIP1) immunostaining was seen in 28 tumors (18.4%). p53 and p21(WAF1/CIP1) expression correlated with the tumor grade (low grade, 5.6% and 3.7%; intermediate grade, 22.5% and 20%; high grade, 63.8% and 31%, respectively). Expression of Bax protein was a common finding in soft tissue sarcoma cases. It was detected in 141 cases (92.8%). Bcl-2 was identified in 59 tumors (38.8%) and was more prevalent in high-grade sarcomas (low grade, 25.9%; intermediate grade, 32.5%; high grade, 55.2%). It was concluded that alterations in the p53 pathway and genes that regulate apoptosis are common events in soft tissue sarcomas. The expression of p53, p21(WAF1/CIP1), and Bcl-2 is closely associated with the histologic grade of the tumor, and therefore these proteins may be used as prognostic markers.     

Oligodendroglial apoptosis occurs along degenerating axons and is associated with FAS and p75 expression following spinal cord injury in the rat

Apoptosis or programmed cell death has been reported after CNS trauma. However, the significance of this mechanism in the pathophysiology of spinal cord injury, in particular at the cervical level, requires further investigation. In the present study, we used the extradural clip compression model in the rat to examine the cellular distribution of apoptosis following cervical spinal cord injury, the relationship between glial apoptosis and post-traumatic axonal degeneration and the possible role of apo[apoptosis]-1, CD95 (FAS) and p75 in initiating post-traumatic glial apoptosis. In situ terminal-deoxy-transferase mediated dUTP nick end labeling revealed apoptotic cells, largely oligodendrocytes as identified by cell specific markers, in grey and white matter following spinal cord injury. Apoptotic cell death was confirmed using electron microscopy and by the demonstration of DNA laddering on agarose gel electrophoresis. Beta-amyloid precursor protein was used as a molecular marker of axonal degeneration on western blots and immunohistochemistry. Degeneration of axons was temporally and spatially co-localized with glial apoptosis. FAS and p75 protein expression was seen in astrocytes, oligodendrocytes and microglia, and was also seen in some apoptotic glia after cord injury. Both FAS and p75 increased in expression in a temporal course, which mirrored the development of cellular apoptosis. The downstream caspases 3 and 8, which are linked to FAS and p75, demonstrated activation at times of maximal apoptosis, while FLIP-L an inhibitor of caspase 8, decreased at times of maximal apoptosis. We conclude that axonal degeneration after traumatic spinal cord injury is associated with glial, in particular oligodendroglial, apoptosis. Activation of the FAS and p75 death receptor pathways may be involved in initiating this process.     

大鼠额叶损伤后细胞凋亡及Bax与Bcl-2蛋白的表达变化

本研究目的为探讨大鼠额叶锐器损伤后细胞凋亡的变化规律及调控机制。通过电镜、TUNEL法及免疫组织化学染色方法检测细胞凋亡和Bax、Bcl2蛋白的表达变化,结果发现凋亡细胞在损伤后3h即可发现,24h达到高峰;伤后3hBax和Bcl2表达明显升高,Bax表达12h达到高峰,而Bcl2表达6h即达到高峰;伤后Bax/Bcl2比值上调,24h达到高峰,随后逐渐下降。结果表明大鼠额叶锐器伤后存在细胞凋亡,凋亡细胞数量的变化与伤后时程有关,Bax/Bcl2比值是决定细胞凋亡的重要因素。     

Induction of growth inhibition and apoptosis in pancreatic cancer cells by auristatin-PE and gemcitabine.

Pancreatic adenocarcinoma is the fifth leading cause of cancer related deaths in the United States. Treatment for this disease has largely been unsuccessful, which may partly be due to insufficient data regarding the molecular mechanisms of chemotherapeutic drugs currently being used as single agents or in combined modality regimens. In this study, we investigated the molecular mechanisms by which auristatin-PE, a newly developed experimental agent, and gemcitabine, a commercially available anti-cancer agent, exert their inhibitory effects on pancreatic cancer cell lines containing wild-type p53 (HPAC) and mutant p53 (PANC-1). Our results showed that auristatin-PE and gemcitabine inhibited cell growth and induced cell cycle arrest in G2/M and S phase, respectively. Auristatin-PE also induced apoptosis in both cell lines. Western blot analysis showed that auristatin-PE up-regulated the expression of wt-p53, p21WAF1 and Bax, and down-regulated Bcl-2 and cyclin B in HPAC cells, while only up-regulation of p21WAF1 and Bax was observed in PANC-1 cells. These results suggest that auristatin-PE may induce apoptosis and p21WAF1 expression through p53-dependent or independent pathways, and that up-regulation of p21WAF1 and Bax and down-regulation of Bcl-2 may be the molecular mechanism through which auristatin-PE inhibits cell growth and induces apoptosis. Furthermore, the up-regulation of p21WAF1 and down-regulation of cyclin B may contribute to the G2/M cell cycle arrest. Combination of auristatin-PE and gemcitabine showed significantly greater inhibition of cell growth and up-regulated expression of p21WAF1 and Bax. From these results, we conclude that the selection of therapeutic agents based on their molecular mechanism may improve therapeutic outcome, and that auristatin-PE may be more effective in the treatment of pancreatic cancer when given in combination with gemcitabine, rather than as a single agent.     

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.     

Bcl-2 family gene expression during severe hyperoxia induced lung injury

Exposure of the lung to severe hyperoxia induces terminal transferase dUTP end-labeling (TUNEL) indicative of DNA damage or apoptosis and increases expression of the tumor suppressor p53 and of members of the Bcl-2 gene family. Because cell survival and apoptosis are regulated, in part, by the relative abundance of proteins of the Bcl-2 family, we hypothesized that lung cells dying during exposure would show increased expression of pro-apoptotic members, such as Bax, whereas surviving cells would have increased expression of anti-apoptotic members, such as Bcl-X(L). The hypothesis is tested in the current study by determining which Bcl-2 genes are regulated by hyperoxia, with specific focus on correlating expression of Bax and Bcl-X(L) with morphologic evidence of apoptosis or necrosis. Adult mice exposed to greater than 95% oxygen concentrations for 48 to 88 hours had increased whole-lung mRNA levels of Bax and Bcl-X(L), no change in Bak, Bad, or Bcl-2, and decreased levels of Bcl-w and Bfl-1. In situ hybridization revealed that hyperoxia induced Bax and Bcl-X(L) mRNA in uniform and overlapping patterns of expression throughout terminal bronchioles and parenchyma, coinciding with TUNEL staining. Electron microscopy and DNA electrophoresis, however, suggested relatively little classical apoptosis. Unexpectedly, Western analysis demonstrated increased Bcl-X(L), but not Bax, protein in response to hyperoxia. Bax and Bfl-1 were not altered by hyperoxia in p53 null mice; however, oxygen toxicity was not lessened by p53 deficiency. These findings suggest that oxygen-induced lung injury does not depend on the relative expression of these Bcl-2 members.