Subcallosal striations: early findings of multiple sclerosis on sagittal, thin-section, fast FLAIR MR images

Since Bcl-2 protects a variety of cell types from programmed cell death, whereas Bax promotes apoptosis, the present study examines Bcl-2 and Bax proteins, and bcl-2 and bax mRNA expression in the developing cerebellum of the rat following methylazoxymethanol (MAM) acetate administration by using immunohistochemistry, Western blotting and Northern blotting. Bcl-2 expression in the developing cerebellum is observed in proliferating and differentiating cells, whereas Bax expression is higher in differentiating cells than in proliferating cells during development. Administration of MAM (0.05 microliter/g, i.p.) at postnatal day 3 produces apoptotic cell death, as detected by the characteristic morphology and positivity with the method of in situ end-labeling of nuclear DNA fragmentation of dying cells, in the external granule cell layer of the cerebellum. Dying cells are not stained with Bcl-2 and Bax antibodies. Furthermore, no modification in the intensity of Bcl-2 and Bax protein bands and in the intensity of Bcl-2 and bax mRNA bands on Western and Northern blots, respectively, were observed between control and treated rats. These data indicate that MAM-induced apoptosis is not associated with modifications in the expression of Bcl-2 and Bax.     

Prognostic significance of Bax protein expression in diffuse aggressive non-Hodgkin's lymphoma

Bax is a proapoptotic member of the Bcl-2 protein family. The incidence and prognostic significance of Bax protein expression in diffuse non-Hodgkin's lymphomas with a large cell component (DLCL) was determined by an immunohistochemical method by using paraffin-embedded tumors from a cohort of patients treated uniformly with combination chemotherapy (n = 139). All patients were between 16 and 70 years of age and had advanced stage disease of diffuse large cell type (diffuse mixed, diffuse large cell, immunoblastic, or anaplastic large cell). Paraffin sections from diagnostic biopsies were successfully immunostained for Bax in 113 cases. Of these, 7 (6%) tumors were scored as Bax immunonegative (< 1% Bax-stained tumor cells), 42 (37%) as low (1% to 10%), 9 (8%) as low-intermediate (11% to 30%), 25 (22%) as high-intermediate (31% to 70%), and 30 specimens (27%) as high for Bax expression (> 70%). Of the 7 Bax-immunonegative lymphomas, all also scored low (< or = 10% immunostained tumor cells) for Bcl-2 expression, whereas 78 of the 106 (74%) Bax-immunopositive tumors had low Bcl-2 expression. By itself, Bax expression was not of prognostic significance in univariate analysis, although there was a clear trend for patients with Bax-immunonegative lymphomas (n = 7) to relapse sooner and to die faster than patients whose tumors contained Bax-immunopositive malignant cells (n = 106; 8-year overall survival 29% versus 55%; P = .06). When combined with Bcl-2 immunostaining data, Bax provided additional prognostic information. Among patients with Bcl-2 low-expressing DLCLs, for example, Bax immunonegativity was associated with lower 8-year relapse-free survival (RFS; 29% v 61%; P < .01) and lower 8-year overall survival (OS; 29% v 63%; P < .05), suggesting that absence of Bax protein connotes a more aggressive phenotype when Bcl-2 protein is also not expressed at high levels. In contrast, low Bax expression was associated with improved 8-year disease-free survival (52% v 16%; P < .02), RFS (47% v 11%; P < .02), and OS (64% v 11%; P < .01) in patients whose tumors expressed Bcl-2 at high levels, suggesting that the combination of high levels of Bax and Bcl-2 expression is more deleterious than high levels of Bcl-2 expression alone. Bax expression failed to provide additional prognostic information beyond Bcl-2 expression in multivariate analysis that included the clinical International Prognostic Index factors (age, stage, lactate dehydrogenase, performance status, and number of extranodal sites) and immunophenotype. Taken together, the results suggest that Bax expression is not a major prognostic marker in DLCL. However, the interactions of the Bcl-2 and Bax expression data with respect to clinical outcome may shed new insights into the biological significance of Bcl-2/Bax protein heterodimerization.     

Lack of mutation in the WAF1/CIP1 gene during bovine leukemia virus-induced leukemogenesis

As a cyclin-dependent kinases (Cdks) inhibitor (CDI), the protein p21WAF1/CIP1 is able to interfere with cell cycle progression. Its expression is upregulated by wild-type p53, and the p21WAF1/CIP1 protein appears to be a potent effector of the p53-dependent cell cycle regulatory pathway. We have previously reported that p53 mutations frequently occur during bovine leukemia virus (BLV)-induced leukemogenesis in cattle but not in sheep. Therefore, we have investigated the involvement of p21WAF1/CIP1 mutations in the tumorigenic process associated with BLV. We first cloned the bovine and ovine WAF1 genes and determined the complete nucleotide sequences of their second coding exons. These sequences share respectively 79% and 80% homology with those of the human counterpart exon. In order to screen for mutations that could be associated with BLV-induced pathogenicity, we performed single strand conformation polymorphism (SSCP) assays on the WAF1 genes from BLV-induced tumors. No WAF1 mutations were detected in any of the ten BLV-induced bovine tumor samples. Among eleven sheep tumors and three ovine cell lines, only one sample revealed a single mutation in the WAF1 coding sequence, but this mutation was silent at the translational level. We concluded that mutations of the WAF1 gene are not involved in the development of the tumors during BLV-induced leukemogenesis.     

Choledochoduodenostomy for palliation in unresectable pancreatic cancer

Bcl-2 and bax are cellular proteins that are important in the regulation of apoptosis. Overexpression of bcl-2 protein is associated with prolonged cell survival, whereas overexpression of bax correlates with increased apoptosis after injury. It has been suggested that the ratio of bcl-2 and bax determines a cell's susceptibility to apoptosis. We studied bcl-2 and bax expression by immunohistochemical methods in 46 cases of B-cel non-Hodgkin's lymphoma characterized by the Revised European-American Lymphoma (REAL) classification to determine whether expression of these two proteins correlated with the histological subtype or the predicted clinical behavior (indolent v aggressive). For each case, both the percentage of cells staining as well as the intensity of staining of bcl-2 and bax were recorded, and a bcl-2-bax protein ratio (BBPR) was calculated. Bax staining was identified in 100% of the lymphomas studied. In contrast, bcl-2 staining was seen in only 67%. Bcl-2 expression correlated with the subtype of lymphoma with positive staining in 100% of small lymphocytic lymphomas, 80% of follicle center lymphomas, 38% of diffuse large cell lymphomas, 33% of high-grade B-cell Burkitt's-like lymphomas, 0% of Burkitt's lymphomas, and 0% of B-cell lymphoblastic lymphomas. The BBPR of indolent lymphomas (mean, 1.8) was significantly greater than the BBPR of aggressive lymphomas (mean, 0.6) (P < or = .002). This suggests that bax and bcl-2 expression may be linked to biological behavior in non-Hodgkin's B-cell lymphomas.     

Progression to persistent lymphocytosis and tumor development in bovine leukemia virus (BLV)-infected cattle correlates with impaired proliferation of CD4+ T cells in response to gag- and env-encoded BLV proteins

The mechanism of leukemogenesis and persistent lymphocytosis (PL; benign expansion of B lymphocytes) in cattle infected with bovine leukemia virus (BLV; a retrovirus closely related to human T-cell leukemia virus type 1) is unknown; however, the immune system likely plays an important role in controlling the outcome of infection. In this study, we compared T-cell competence in serologically positive alymphocytotic (AL) animals with T-cell functions in animals with progressive stages of infection, PL and tumor bearing (TB). Dramatic differences were observed in lymphocyte proliferation to recombinant proteins encoded by BLV gag (p12, p15, and p24) and env (gp30, and gp51) genes in different disease stages. Lymphocytes from AL cattle recognized an average of three of five recombinant proteins per animal. Expansion of antigen pulsed lymphocytes in interleukin-2 increased protein recognition to almost five per animal. In contrast, lymphocytes from PL and TB animals failed to recognize any BLV recombinant proteins. Short-term T-cell cultures from the PL group expanded in interleukin-2, as well as the PL and TB cells cultured in indomethacin (3 to 6 microg/ml), increased the average of recognized proteins per animal to one. Cells proliferating to BLV antigens were CD4+ T lymphocytes, as shown by cell depletion studies. The positive effect of indomethacin suggests involvement of prostaglandin E2 as a negative regulatory factor in the later stages of disease. Thus, for the first time, advancing stages of BLV infection were correlated with decreased T-cell competence, providing deeper insight into pathogenesis of retroviral infections.     

Rural attachments for students in the health professions: are they worthwhile?

Bcl-2 family proteins are principal regulators of cell death during normal development as well as in many disease states. Differentiated cerebellar granule neurons are protected from apoptosis by depolarizing concentrations of potassium. Further, these cells acquire resistance to glutamate-mediated excitotoxicity when pre-exposed to subtoxic concentrations of the glutamate receptor agonist, N-methyl-D-aspartate. Here, we report that the expression of bcl-2, bcl-xL, bcl-xS, bax and bad mRNA as well as of Bcl-2, Bax, Bcl-XL, Bcl-XS and Bag-1 proteins is not modulated in these two paradigms of neuronal cell death. However, mitochondrial release of cytochrome c, which is thought to be controlled by Bcl-2 family proteins, is detected 5 h after switching the neurons to low potassium conditions. Thus, there appears to be regulation of Bcl-2 family protein bioactivity in the absence of altered protein expression during potassium deprivation-induced apoptosis of cerebellar granule neurons.     

Upregulation of bax protein levels in neurons following cerebral ischemia

The patterns of expression of the bcl-2, bax, and bci-X genes were examined immunohistochemically in neurons of the adult rat brain before and after 10 min of global ischemia induced by transient cardiac arrest. High levels of the cell death promoting protein Bax and concomitant low levels of the apoptosis-blocking protein Bcl-2 were found in some populations of neurons that are particularly sensitive to cell death induced by transient global ischemia, such as the CA1 sector of the hippocampus and the Purkinje cells of the cerebellum. Moreover, within 0.5 to 3 hr after an ischemic episode, immunostaining for Bax was markedly increased within neurons with morphological features of degeneration in many regions of the brain. Use of a two-color staining method for simultaneous analysis of Bax protein and in situ detection of DNA-strand breaks revealed high levels of Bax immunoreactivity in many neurons undergoing apoptosis. Postischemic elevations in Bax protein levels in the hippocampus, cortex, and cerebellum were also demonstrated by immunoblotting. At early times after transient ischemia, regulation of Bcl-2 and Bcl-x protein levels varied among neuronal subpopulations, but from 3 hr on, those neurons with morphological evidence of degeneration uniformly contained reduced levels of Bci-2 and particularly Bci-X immunoreactivity. The findings suggest that differential expression of some members of the bcl-2 gene family may play an important role in determining the relative sensitivity of neuronal subpopulations to ischemia and that postischemic alterations in the expression of bax, bcl-2, and bcl-x may contribute to the delayed neuronal cell death that occurs during the repurfusion phase after a transient ischemic episode.     

Induction of Bax-alpha precedes apoptosis in a human B lymphoma cell line: potential role of the bcl-2 gene family in surface IgM-mediated apoptosis

The members of the bcl-2 gene family are major regulators of programmed cell death, but their role in sIg-triggered apoptosis remains unclear. Using sensitive and resistant variants of the human B cell line BL-41, we studied the expression of the bcl-2 gene family during surface IgM-mediated apoptosis. We found constitutive Bcl-2 and Bcl-x expression, which remained unaltered after sIg cross-linking, in both resistant and sensitive cells. This and other experiments suggest that constitutive expression of Bcl-2 or Bcl-x alone is not sufficient to protect from activation-induced cell death in B cells. We therefore investigated Bax-alpha, the death-promoting splice variant of Bax, and found strong induction of both mRNA and protein upon sIg stimulation in sensitive cells. However, resistant subclones showed only weak expression, which was not inducible by sIg cross-linking. We provide evidence that up-regulation of Bax-alpha and the resulting imbalance of Bcl-2/Bax might be a major regulator of sIg-mediated apoptosis. Additionally, we found strong constitutive expression of Bcl-xs, the death promoting variant of Bcl-x, in sensitive cells, whereas resistant cells showed only weak Bcl-xs expression. Thus, we observed a much stronger expression of the death-promoting proteins Bax-alpha (inducible) and Bcl-xs (constitutive) in sensitive cells than in resistant cells. We therefore propose a potential role of the novel bcl-2 gene family members bcl-x and bax in surface IgM-triggered apoptosis.     

Regulation of cell survival during B lymphopoiesis: apoptosis and Bcl-2/Bax content of precursor B cells in bone marrow of mice with altered expression of IL-7 and recombinase-activating gene-2

B cell development in mouse bone marrow depends critically upon IL-7. To examine the possible in vivo trophic role of IL-7, we have quantitated apoptosis and Bcl-2 family proteins in populations of phenotypically defined B lineage cells in IL-7-deficient and IL-7-overexpressing mice. Using immunofluorescence labeling, multiparameter flow cytometry, and a short-term culture assay, we show that the apoptotic rates of precursor B cells, but not of more mature B cells, are enhanced by IL-7 gene deletion, associated with increased intracellular content of Bax and decreased Bcl-2, while, conversely, an IL-7 transgene suppresses precursor B cell apoptosis and produces low Bax and high Bcl-2 levels. During normal B cell development, high Bax/Bcl-2 ratios characterize cells undergoing greatest apoptotic cell death. Pro-B cells in RAG-2-/- mice, all destined to abort, show elevated Bax levels and Bax/Bcl-2 ratios. By comparison with the elevated rate of pro-B cell apoptosis in RAG-2-/- mice, provisional estimates have been made for the fraction of pro-B cells undergoing apoptosis in normal mice (70%), IL-7-/- mice (85%), and IL-7 transgenic mice (35%). The results demonstrate that IL-7 strongly promotes in vivo cell survival and maintains antiapoptotic Bcl-2/Bax ratios during the development of precursor B cells in mouse bone marrow.     

Involvement of Bcl-2 family and caspase-3-like protease in NO-mediated neuronal apoptosis

To clarify mechanisms of neuronal death in the postischemic brain, we examined whether astrocytes exposed to hypoxia/reoxygenation exert a neurotoxic effect, using a coculture system. Neurons cocultured with astrocytes subjected to hypoxia/reoxygenation underwent apoptotic cell death, the effect enhanced by a combination of interleukin-1beta with hypoxia. The synergistic neurotoxic activity of hypoxia and interleukin-1beta was dependent on de novo expression of inducible nitric oxide synthase (iNOS) and on nitric oxide (NO) production in astrocytes. Further analysis to determine the neurotoxic mechanism revealed decreased Bcl-2 and increased Bax expression together with caspase-3 activation in cortical neurons cocultured with NO-producing astrocytes. Inhibition of NO production in astrocytes by N(G)-monomethyl-L-arginine, an inhibitor of NOS, significantly inhibited neuronal death together with changes in Bcl-2 and Bax protein levels and in caspase-3-like activity. Moreover, treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by an NO donor, sodium nitroprusside. These data suggest that NO produced by astrocytes after hypoxic insult induces apoptotic death of neurons through mechanisms involving the caspase-3 activation after down-regulation of Bcl-2 and up-regulation of Bax protein levels.     

Conditions for the consolidation of fractures of the femoral diaphysis after locked intramedullary flexible osteosynthesis

In the previous study, we have shown that propentofylline (PPF) could induce the cellular differentiation and apoptosis-related growth regression in the human glioma cell lines. Its biological functions were partly due to the increasing endogeneous NGF and its high affinity receptor, trk A productions. Although little has been known about the precise machinary regulating the propentofylline induced apoptosis. Recently, we have found that propentofylline could modulate some apoptosis related genes products in the glioma cell lines, i.e. NGF, trk A mRNA levels and Fas protein expressions were increased, whereas bcl-2 mRNA level was decreased. In the present study, we examined the apoptotic signal cascade, especially focusing on the expressing pattern of Bcl-2/Bax gene products. In the normal human astrocyte cells, Bax-beta was markedly expressed, whereas Bcl-2 and Bax-alpha proteins and mRNA were weakly or even nondetectable. Accordingly, Bax beta might be a dominant variant in the normal glial cells, which could have the appropriate balance of proapoptotic (Bax beta) and anti-apoptotic proteins (Bcl-2). In the glioma cells, we showed the over-expressions of Bcl-2 and Bax alpha compared with the normal counterparts. According to Bax dominant theory, Bax, not Bcl-2 may have a major role in regulating apoptosis by means of homodimerization. In might be implied that in the glioma cells, excessive expressions of Bcl-2 and Bax alpha would favor the formation of the Bax alpha/Bax beta heterodimer or the Bax beta/Bcl-2 heterodimer rather than the Bax beta/Bax beta homodimer, which might be presumed to be functional proteins. And finally the increasing relative ratio of Bax alpha/ Bax beta or Bax beta/Bcl-2 to Bax beta/Bax beta could allow the tumor cells to survive. Thus over-expression of the bcl-2 and bax alpha gene renders the glioma cells resistant to apoptosis. In the present study, PPF could promote Bax beta over-expression and Bcl-2 retardative expression in the glioma cells, whereas had no effect on Bax alpha expression. Therefore, PPF might promote apoptotic cell death through the mechanism that restore the glioma cells to the appropriate balance of proapoptotic and anti-apoptotic proteins like as normal astrocytes. Our results indicated that propentofylline might have a potential role as apoptotic modulators in the human glioma cell lines, not only its protective activities against neuronal ischemic damages.     

The action of Bax and bcl-2 on T cell selection

T cell development and selection in the thymus are shaped by the induction of apoptosis. However, a direct role in T cell development and selection for any of the molecules known to regulate apoptosis has remained controversial. We have studied the effect of bax and bcl-2 transgenes in recombination activation gene 1-deficient (RAG-1(-/-)) mice transgenic for the major histocompatibility complex class I-restricted F5 T cell receptor. Overexpression of a bax transgene in the thymus seriously impairs the production of mature T cells, whereas bcl-2 overexpression greatly promotes it. The effect of bax and bcl-2 overexpression on antigen-induced negative selection was studied using fetal thymic organ cultures. This analysis showed that Bcl-2 strongly inhibits negative selection, whereas Bax does not affect it. Our data directly show that Bcl-2 family members have specific roles in T cell selection and also lend support to the hypothesis that Bax and Bcl-2 can antagonize each other's action in a certain apoptosis pathway while in another they can be functionally nonreciprocal.     

Bcl-2 is overexpressed and alters the threshold for apoptosis in a cholangiocarcinoma cell line

Cholangiocarcinoma is a malignant neoplasm originating from cholangiocytes. The mechanisms responsible for oncogenesis of cholangiocytes are unknown. Resistance to apoptosis, especially by altered expression of B-cell lymphoma/leukemia 2 (Bcl-2) family members, has been implicated as a mechanism contributing to malignant transformation. Thus, our aim was to test the hypothesis that altered expression of Bcl-2 family members by cholangiocarcinoma cells renders them resistant to apoptosis. We compared the apoptotic threshold and expression of the Bcl-2 protein family members, Bcl-2, Bcl-XL, and Bax, in two human cell lines: 1) nonmalignant human cholangiocytes immortalized by transfection with the simian virus 40 (SV 40) large T antigen; and 2) a malignant human cholangiocarcinoma cell line. Apoptosis was induced pharmacologically using beauvericin. Bcl-2, Bcl-x long, and Bax protein expression were evaluated by immunoblot analysis, and Bcl-2 expression was modulated using antisense technology. The cholangiocyte and malignant/nonmaligant phenotype of both cell lines was verified using both in vitro and in vivo approaches. Beauvericin induced apoptosis of nonmalignant cholangiocytes in a concentration- (0 to 25 micromol/L) and time- (0 to 6 hours) dependent manner. In contrast, malignant cholangiocytes were resistant to apoptosis. Although expression of Bcl-x long and Bax protein were similiar in the two cell lines, Bcl-2 protein expression was 15-fold greater in malignant than in nonmalignant cholangiocytes. An 18 mer bcl-2 antisense oligonucleotide reduced expression of Bcl-2 protein by 50% and increased the rate of beauvericin-induced apoptosis more than threefold in the malignant cells. Our results support the hypothesis that resistance to apoptosis by overexpression of Bcl-2 may be a feature of cholangiocarcinoma.     

Apoptotic induction in transformed follicular lymphoma cells by Bcl-2 downregulation

The roles of Bcl-2 protein and the protein ratio of Bcl-2/Bax in regulating cell growth in various lymphoma cell lines were examined. A dose-dependent decrease in Bcl-2 protein expression was observed in the different lymphomas incubated with lipid-incorporated bcl-2 antisense oligonucleotides (L-bcl-2). Growth inhibition was observed in a transformed follicular lymphoma (FL) cell line, which has the t(14;18) translocation and Bcl-2 protein overexpression. One of the mechanisms by which L-bcl-2 growth inhibition is mediated in these transformed FL cells might be through apoptotic induction, because the treated cells had an increased apoptotic index and showed the typical DNA fragmentation. These studies indicate that Bcl-2 protein is critical in the growth regulation of transformed FL cells. L-bcl-2 did not induce growth inhibition in lymphoma cells not expressing Bcl-2 or Bax protein. Thus, the protein ratio of Bcl-2/Bax may also be important in regulating the growth of these lymphomas.     

Interaction of E1B 19K with Bax is required to block Bax-induced loss of mitochondrial membrane potential and apoptosis

The Bcl-2 homologous region 3 (BH3) is sufficient for interaction of pro-apoptotic with anti-apoptotic Bcl-2 family members, and functional antagonism may determine whether cell survival or death is the outcome of this protein-protein interaction. To address the biological role of BH3, two Bax-Bcl2 chimeras were generated in which 13 amino acids encompassing BH3 was swapped between anti-apoptotic Bcl-2 and pro-apoptotic Bax, thereby generating Bax with BH3 of Bcl-2 (Bax-BH3Bcl2), and Bcl-2 with BH3 of Bax (Bcl2-BH3Bax). Function and binding of the chimeras was then assessed utilizing the adenoviral Bcl-2 homologue, E1B 19K, which blocks apoptosis, and interacts with Bax, but not with Bcl-2. E1B 19K did not interact with Bax-BH3Bcl2 but did interact with Bcl2-BH3Bax. Bax-BH3Bcl2 retained pro-apoptotic function, while Bcl2-BH3Bax did not exhibit either pro- or anti-apoptotic activity. Thus, BH3 of Bcl-2 encodes binding specificity but not the apoptotic propensity. E1B 19K could not block Bax-BH3Bcl2-induced apoptosis, suggesting that E1B 19K may act to antagonize pro-apoptotic proteins rather than as an effector of survival. Furthermore, Bax expression disrupted the mitochondrial membrane potential, which could be rescued by E1B 19K expression. Thus, BH3 controls the binding specificity among Bcl-2 family members, and direct interaction between pro-apoptotic and anti-apoptotic proteins is a mechanism to regulate mitochondrial membrane potential and apoptosis.     

Bcl-2 but not Bax expression is associated with apoptosis in normal and transformed squamous epithelium

Aberrant regulation of apoptosis may contribute to tumorigenesis. Relative levels of apoptosis regulatory proteins, such as Bcl-2 and Bax as well as interactions of these proteins with other gene products, may contribute to the rate of apoptosis in neoplasia. We examined Bcl-2 expression in 104 squamous cell carcinomas of the head and neck, as well as histologically normal mucosa several centimeters away from the tumor, and in control normal mucosa from patients without cancer. Immunohistochemistry and immunoblotting demonstrated Bcl-2 expression in 30% (31 of 104) of squamous cell carcinoma, with an increase in Bcl-2 protein levels compared with control normal mucosa from noncancer patients. Bcl-2-positive tumors demonstrated a 5-fold decrease in the number of apoptotic cells compared with Bcl-2-negative tumors. Bcl-2 protein expression was associated with poorly differentiated tumor grade but was not correlated with Bax expression or patient survival. These findings demonstrate that Bcl-2 contributes to apoptosis in normal and transformed squamous epithelium.     

Bcl-2 and Bax proteins in Lewy bodies from patients with Parkinson's disease and Diffuse Lewy body disease

Double-labelling immunohistochemistry of Bcl-2 and Bax, and ubiquitin (as a marker of Lewy bodies) was examined in the brains of patients with Parkinson's disease and Diffuse Lewy body disease to learn about possible modifications of protein expression and the presence of Lewy bodies. Bcl-2 and Bax immunoreactivities were observed in Lewy body-bearing and non-Lewy body-bearing neurons in patients with parkinsonism. These results show that Bcl-2 and Bax are probably not implicated in Lewy body formation and that the presence of Lewy bodies does not have a direct impact on the expression of Bcl-2 and Bax proteins in individual neurons.