Biomedical subjects

J C Reed

Publications and source records attributed to J C Reed.

At least 307 records · Page 17Linked to original sources

PubMed1997

Expression of pro- and anti-apoptosis gene products in brains from paediatric patients with HIV-1 encephalitis.

We have previously demonstrated the presence of DNA fragmentation in neurons, macrophages and microglia consistent with apoptosis, but not in reactive astrocytes in brain tissue from paediatric patients with HIV-1 encephalitis (HIVE). To further understand the underlying mechanism(s) for these findings as they relate to gene-directed neural cell death, we studied the in-situ expression of the Bcl-2 family of proteins, including the pro-apoptosis gene product Bax, the anti-apoptosis gene product Bcl-2, and Bcl-x. We demonstrate significantly elevated numbers of Bax-positive microglia and macrophages immunoreactive in basal ganglia and cerebral cortex of children who had HIVE, in comparison to HIV-1 infected children without encephalitis or children who were seronegative for HIV-1. In contrast, patients with HIVE, but not HIV-1 without encephalitis, or seronegative controls, had increased expression of Bcl-2 and Bcl-x in reactive astrocytes in cortex and basal ganglia. In vitro studies using Western blot analysis demonstrated an up-regulation in the levels of Bax, and phosphorylated (i.e. inactive) Bcl-2 in HIV-1 infected macrophages, and in LPS-activated macrophages, relative to levels in virus-negative unstimulated macrophages. These results suggest that productive HIV-1 infection, or cellular activation, renders macrophages more vulnerable to apoptosis. Taken together, these findings suggest that brain-resident macrophages and microglia in patients with HIV-1 encephalitis are more prone to undergo apoptosis and that astrocytes in contrast may be resistant to apoptosis. This may represent a mechanism to limit microglial activation and the spread of productive HIV-1 infection in the CNS of children with HIV-1 encephalitis.

Adolescent↗

PubMed1997

Hypericin-induced apoptosis of human malignant glioma cells is light-dependent, independent of bcl-2 expression, and does not require wild-type p53.

Hypericin and tamoxifen are experimental agents for the adjuvant chemotherapy of malignant glioma. We report that hypericin and tamoxifen induce apoptosis of 7 human malignant glioma cell lines in a concentration- and time-dependent manner. Illumination is essential for the cytotoxicity of hypericin but not tamoxifen. Apoptosis is unaffected by inhibitors of RNA and protein synthesis or free radical scavengers, does not require wild-type p53 activity, and occurs in glioma cells expressing high levels of bcl-2. There is no correlation between hypericin and tamoxifen-induced cytotoxicity and inhibition of protein kinase C (PKC). Ectopic expression of a murine bcl-2 transgene provides modest protection from tamoxifen but does not affect hypericin toxicity. Hypericin and tamoxifen do not modulate glioma cell killing induced by tumor necrosis factor-alpha (TNF-alpha) or CD95 ligand. Both drugs augment the acute cytotoxicity of various cancer chemotherapy drugs but fail to shift their EC50 values in modified colony formation assays. These data do not provide further supportive evidence how to enhance the limited efficacy of tamoxifen treatment for human malignant glioma. However, hypericin is a promising agent for the treatment of malignant glioma if local photodynamic activation of hypericin in the glioma tissue can be achieved.

Anthracenes↗

PubMed1997

Bcl-2 family proteins: regulators of apoptosis and chemoresistance in hematologic malignancies.

Nearly all anticancer drugs presently available to clinicians kill tumor cells by activating an endogenous biochemical pathway for cell suicide, known as programmed cell death or apoptosis. However, many malignant cells develop defects in the regulation of genes that control apoptosis, rendering them resistant to the induction of apoptosis by a wide variety of stimuli, including chemotherapeutic drugs and radiotherapy. The BCL-2 family of proto-oncogenes are critical regulators of apoptosis whose expression frequently becomes altered in human cancers, including some of the most common types of lymphomas and leukemias. The first member of this gene family to be identified was BCL-2, by virtue of its involvement in t(14;18) chromosomal translocations commonly found in B-cell non-Hodgkin's lymphoma (NHL). Subsequently, however, overexpression of Bcl-2 has been reported in a wide variety of cancers, without associated chromosomal translocations. A variety of experiments have provided conclusive evidence that elevations in Bcl-2 expression cause resistance to chemotherapeutic drugs, while decreases in Bcl-2 expression promote apoptotic responses to anticancer drugs. Information about the biochemical mechanisms of action of the Bcl-2 protein and other members of the Bcl-2 family is beginning to suggest strategies for overcoming the cytoprotective effects of Bcl-2 overexpression in lymphomas, leukemias, and other types of cancer.

Apoptosis↗

PubMed1997

Analysis of Bax and Bcl-2 expression in p53-immunopositive breast cancers.

Bax and Bcl-2 are proteins that regulate programmed cell death and apoptosis. The expression of these proteins can be regulated, at least in part, by the tumor suppressor p53, but the effects of p53 are highly tissue specific. In an effort to better understand the relation between p53 and the in vivo control of the expression of Bax and Bcl-2 in adenocarcinomas of the breast, we evaluated by immunohistochemistry the expression of Bcl-2 and Bax in 149 invasive ductal carcinomas, 135 of which were chosen because of their p53 immunopositivity. The percentages of Bcl-2-immunopositive tumor cells were significantly lower in the p53-positive (median 20%) subsets as compared to the p53-negative (median 85%) subsets (P = 0. 004). Comparisons of the percentages of p53-immunopositive tumor cells with the percentages of Bcl-2- and Bax-positive cells (as continuous variables) revealed a significant inverse correlation between Bcl-2 and p53 (r = -0.41, P < 0.001) but not between Bax and p53. In the p53-positive subset, the percentages of Bax- and Bcl-2-immunopositive tumor cells were correlated positively (r = 0. 27, P = 0.002), suggesting that the expression of these genes may be co-regulated to some extent in these breast cancers. Higher percentages of Bcl-2-positive tumor cells were also associated with estrogen receptor positivity (P = 0.03), low histological tumor grade (P = 0.03), and low T stage (P = 0.02), whereas Bax immunostaining was associated only with c-erbB-2 immunopositivity (P = 0.02). Although the number of cases was small and treatment was non-uniform, preliminary correlations with clinical outcome data suggest that the prognostic significance of Bcl-2 may be enhanced by inclusion of Bax data in patients with p53-immunopositive adenocarcinoma of the breast, at least for patients with node-negative disease. Taken together, these data suggest that, despite the ability of p53 to bind directly to the Bax gene promoter, the regulation of Bax in human breast cancers does not necessarily correlate with p53 status, implying that regulation of this pro-apoptotic gene in these tumors is complex and probably influenced by several factors.

Breast Neoplasms↗

PubMed1996

Interleukin-2 (IL-2) upregulates BAG-1 gene expression through serine-rich region within IL-2 receptor beta c chain.

BAG-1 is a Bci-2-binding protein which functions in protection from apoptotic cell death. Here we provide evidence for interleukin-2 (IL-2)-mediated upregulation of BAG-1 expression. In hematopoietic cell line BAF-B03 F7 cells, gene transfer mediated expression of the IL-2R beta c chain is sufficient to confer proliferation and cell survival responses to IL-2. In these IL-2R beta c-expressing cells, BAG-1 mRNA was dramatically induced by IL-2. The IL-2-mediated induction of BAG-1 expression required the activation of tyrosine kinase(s) and was sensitive to rapamycin as the induction of bcl-2 expression was. Analysis of the transfectants which express mutant IL-2R beta c chains or mutant Janus family protein tyrosine kinase Jak3 lacking the kinase domain showed that the IL-2-mediated BAG-1 gene expression required the serinerich region within the IL-2R beta c chain, but Jak3 activation was dispensable. The signaling pathway for BAG-1 gene expression thus highly resembles that for bcl-2 gene expression, strongly suggesting that their induction shares the same signaling pathway. In addition, deletion of the serine-rich region led to loss of IL-2-mediated protection from apoptotic cell death. Taken together, these studies demonstrate that the serine-rich region of the IL-2R beta c chain mediates the coordinated expression of bcl-2 and BAG-1 genes, thereby contributing to suppression of apoptosis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

CD40-induced growth inhibition in epithelial cells is mimicked by Epstein-Barr Virus-encoded LMP1: involvement of TRAF3 as a common mediator.

CD40, a member of the tumour necrosis factor receptor family, is expressed on the surface of B lymphocytes where its ligation provides a potent survival signal. CD40 is also expressed in basal epithelial cells and in a number of different carcinomas where its function remains unknown. We observed that contrary to the studies in normal B cells, CD40 ligation in carcinoma cell lines and in normal primary epithelial cells resulted in growth inhibition and enhanced susceptibility to apoptosis induced by anti-neoplastic drugs, TNF-alpha, Fas and ceramide. This effect was also observed in CD40-transfected Rat-1 fibroblasts. The expression of Bcl-2 did not affect growth inhibition induced by CD40 ligation in epithelial cells but the Epstein - Barr Virus-encoded latent membrane protein 1 (LMP1) blocked the effect. Whilst transient expression of LMP-1 resulted in the inhibition of epithelial cell growth, this effect was not observed with a LMP1 mutant lacking the binding domain for TRAF3, a protein which may mediate signal transduction by interacting with the cytoplasmic domains of both CD40 and LMP1. Transient expression of TRAF3 also inhibited epithelial cell growth, whilst expression of a dominant-negative TRAF3 partially blocked the inhibitory effect of CD40 ligation and of transient LMP1 expression. These results suggest that CD40 regulates epithelial cell growth in a manner mimicked by LMP1 and implicate TRAF3 as a common mediator in the transduction of the growth inhibitory signals generated via the CD40 and LMP1 pathways.

Animals↗

PubMed1996

Bcl-2 targets the protein kinase Raf-1 to mitochondria.

A green fluorescent protein (GFP)-Raf-1 fusion protein was used to show that Bcl-2 can target this kinase to mitochondria. Active Raf-1 fused with targeting sequences from an outer mitochondrial membrane protein protected cells from apoptosis and resulted in phosphorylation of BAD, a proapoptotic Bcl-2 homolog. Plasma membrane-targeted Raf-1 did not protect from apoptosis and resulted in phosphorylation of ERK-1 and ERK-2. Untargeted active Raf-1 improved Bcl-2-mediated resistance to apoptosis, whereas a kinase-inactive Raf-1 mutant abrogated apoptosis suppression by Bcl-2. Bcl-2 can therefore target Raf-1 to mitochondrial membranes, allowing this kinase to phosphorylate BAD or possibly other protein substrates involved in apoptosis regulation.

Apoptosis↗

PubMed1996

Expression pattern of candidate cell death effector proteins Bax, Bcl-2, Bcl-X, and c-Jun in sensory and motor neurons following sciatic nerve transection in the rat.

Numerous studies have demonstrated a prolonged expression of c-Jun transcription factor in neurons following axotomy, and it has been hypothesized that c-Jun may be causally involved in neuroregeneration in vivo. By contrast, there is growing evidence from in vitro studies that induction of c-Jun may be necessary for neuronal cell death induced by growth factor starvation. It has been demonstrated that protein levels of cell death repressor Bcl-2 and cell death promotor Bax determine the threshold for neuronal cell death and that their expression is dynamically modulated at the onset of neurodegeneration. In the present study, we investigated by double-immunolabeling methods activation of c-Jun transcription factor and expression of members of the Bcl-2 family of cell death effector proteins in axotomized neurons. Six days after transection of the sciatic nerve in young rats, when axotomized neurons start to degenerate, strong nuclear Jun immunostaining in spinal cord motoneurons was associated with intense cytoplasmic Bax labeling and signs of neuronal atrophy. Bcl-2 and Bcl-X proteins were present only at moderate to low levels. In situ end-labeling by terminal transferase revealed nuclear DNA fragmentation in scattered motoneurons of the ipsilateral ventral horn (1 or 2 labeled nuclei per section). In the L5 dorsal root ganglia (DRG) levels of Bax, Bcl-2, and Bcl-X proteins were highly variable. High levels of Bax immunoreactivity together with intense Jun immunofluorescence were frequently observed in small-diameter sensory neurons. RT-PCR analysis revealed expression of exclusively the anti-apoptotic bcl-xL mRNA isoform in rat DRG which decreased significantly following sciatic nerve transection. These findings indicate that the high susceptibility of central neurons and small-sized DRG neurons to axotomy-induced cell death might be related to their low ratio of cell death repressor Bcl-2 and Bcl-XL to cell death promotor Bax expression. It should be noted, however, that numerous strongly Jun-positive DRG neurons contained low levels of Bax or high levels of Bcl-2 and Bcl-X immunoreactivity. Thus, high levels of c-Jun protein in axotomized neurons do not necessarily suggest a destination to die, and other factors may determine the outcome of axotomy.

Animals↗

PubMed1996

Minimal region of loss at 13q14 in B-cell chronic lymphocytic leukemia.

Allelic loss at nonrandom chromosomal sites is thought to mark the position of tumor suppressor genes involved in the pathogenesis and progression of human malignancies. Solid tumors in particular have been found to harbor multiple genetic changes resulting in loss of function mutations. Tumor suppressor genes have also been found to be involved in the progression of lymphoid tumors. Previous reports have suggested the involvement of a tumor suppressor gene located on the long arm of chromosome 13, between the retinoblastoma (RB) and D13S25 loci, in the pathogenesis and or progression of more than 40% of B-cell chronic lymphocytic leukemia (B-CLL), a common lymphoid malignancy whose molecular etiology remains largely unknown. In the present study, we report the construction and characterization of a YAC contig spanning a region of approximately 3 cM between the RB gene and the D13S31 locus. We also screened 60 paired normal/tumor B-CLL samples for allelic loss on chromosome 13 with nine microsatellite markers located between RB and D13S25. This analysis has allowed us to narrow the smallest region of loss to a segment of 550 kb located between the 206XF12 and D13S25 markers.

Alleles↗

PubMed1996

Up-regulation of Bcl-xL expression protects CD40-activated human B cells from Fas-mediated apoptosis.

CD40--CD40L interactions between resting B cells and activated T cells are essential for germinal center formation. It has been shown that CD40L can induce both Fas expression and susceptibility to Fas-mediated killing in B cells, while anti-Ig can partially rescue B cells from Fas-mediated killing. However, the intracellular mechanism for this phenomenon is not known. We examined the expression of Fas and bcl-2 family gene products, such as Bcl-2, Bcl-x, Bax, and Mcl-1, in human tonsillar B cells. The activation of naive B cells by CD40L induced transient expression of Bcl-xL. As the Bcl-xL level decreased in CD40-activated B cells, the cells became susceptible to apoptosis by anti-Fas antibodies. Though anti-Ig did not change the Fas expression, it protected CD40-activated B cells from Fas-mediated killing by up-regulating Bcl-xL expression. The addition of anti-Ig did not significantly change Bcl-2, Bax, and Mcl-1 levels compared to those of B cells activated by CD40L alone.

Apoptosis↗

PubMed1996

Altered expression of Bcl-2, Bcl-X, Bax, and c-Fos colocalizes with DNA fragmentation and ischemic cell damage following middle cerebral artery occlusion in rats.

Permanent occlusion of the middle cerebral artery in rats was used to assess the effects of focal ischemia on the expression of members of the bcl-2 family which have been implicated in the regulation of programmed cell death. Intraluminal occlusion of one middle cerebral artery for 6 h resulted in histologically detectable brain damage within the ipsilateral caudate putamen, basolateral cortex and parts of the thalamus. In the infarcted basolateral cortex and thalamus fragmentation of DNA was detected in many nuclei using in-situ end-labeling of DNA breaks by terminal transferase, whereas only scattered labeled nuclei were visible in the infarcted caudate putamen. Immunohistochemical analysis revealed activation of c-Fos in the infarcted cortex and thalamus and in the non-infarcted cingulate cortex as has been shown by others. A decrease in immunoreactivity for Bcl-2, and Bcl-X and an increase in immunostaining for Bax was observed exclusively in neurons within the ischemic cortex and thalamus. Within the infarcted caudate putamen, however, protein levels of all bcl-2 family members declined and c-Fos remained absent. By reverse transcription and polymerase chain reaction it was demonstrated that levels of bcl-2 mRNA markedly decreased in the ipsilateral hemisphere, whereas the amount of bax mRNA was elevated. These findings suggest that a shift in the ratio of cell death repressor Bcl-2 to cell death effector Bax and a concomitant activation of c-Fos may contribute to neuronal apoptosis in the infarcted thalamus and cortex.

Animals↗

PubMed1996

Programmed myocyte cell death affects the viable myocardium after infarction in rats.

To determine whether apoptotic and necrotic myocyte cell death occur acutely and chronically after infarction, the formation of DNA strand breaks and the localization of myosin monoclonal antibody labeling were analyzed in the surviving myocardium from 20 min to 1 month. DNA strand breaks in myocyte nuclei were detected as early as 3 h following coronary artery occlusion and were still present at 1 month. This cellular process was characterized biochemically by internucleosomal DNA fragmentation which produced DNA laddering on agarose gel electrophoresis. Quantitatively, 155 myocyte nuclei per 10(6) cells exhibited DNA strand breaks in the portion adjacent to the infarcted tissue at 3-12 h. This parameter increased to 704 at 1-2 days and subsequently decreased to 364 at 7 days, 188 at 14 days, and 204 at 1 month. In the remote myocardium, the number of myocyte nuclei with DNA strand breaks was 84 per 10(6) at 3-12 h and remained essentially constant up to 1 month. Programmed myocyte cell death was accompanied by a decrease in the expression of bcl-2 and an increase in the expression of bax. The changes in the expression of these genes were present at 1 and 7 days after coronary artery occlusion. In conclusion, the mechanical load produced by myocardial infarction and ventricular failure may affect the regulation of bcl-2 and bax in the viable myocytes, triggering programmed cell death and the remodeling of the ventricular wall.

Animals↗

PubMed1996

Cloning of cDNAs encoding the human BAG1 protein and localization of the human BAG1 gene to chromosome 9p12.

cDNAs encoding the human homolog of BAG1, a Bcl-2-binding protein with anti-apoptotic function, were cloned. DNA sequence analysis of human BAG1 cDNAs predicts a protein with an additional 55 amino acids at its NH2-terminus compared to the mouse protein. Immunoblot assays using monoclonal antibodies raised against bacterially produced h-BAG1 protein confirmed the larger size of the human protein (approximately 34 kDa) compared to mouse. PCR analysis of DNA from human x rodent somatic cell hybrids using human BAG1-specific primers localized the gene to human chromosome 9. Cosmid clones of h-BAG1 were obtained and used for fluorescence in situ hybridization analysis of normal metaphase chromosomes, thus localizing h-BAG1 to 9p12, a region associated with hereditary disorders that may involve developmental dysregulation of programmed cell death.

Amino Acid Sequence↗

PubMed1996

bcl-2 alters the antigen-driven selection of B cells in mukappa but not in mu-only Xid transgenic mice.

A point mutation in the pleckstrin homology domain of the mouse Bruton's tyrosine kinase (btk) gene results in an X-linked immune defect, Xid, characterized by immunologic unresponsiveness to polymeric carbohydrate Ags. In Xid mice, B cells specific for phosphocholine (PC) do not develop in peripheral lymphoid tissues because they either fail to be positively selected from the marrow or they are clonally deleted via an Ag-driven, receptor-mediated process. Overexpression of the bcl-2 gene allows PC-specific B cells to survive and mature in Xid mukappa anti-PC transgenic mice, but PC-specific B cells are not rescued by bcl-2 in Xid mu-only transgenic mice. The failure of bcl-2 to rescue PC-specific B cells, in mu-only transgenic mice suggests that either it does not correct the btk defect in the Ag-driven selection process that occurs in pre-B cells and/or in very immature B cells or that a btk-dependent proliferative phase is required for the selection and amplification of the PC-specific B cells in mu-only transgenic mice. The rescue of PC-specific B cells in mukappa transgenic mice indicates that bcl-2 can alter receptor-mediated B cell selection at late stages in B cell development. The rescued PC-specific B cells in Xid male mice do not exhibit an altered proliferation profile in response to B cell-stimulating agents compared with B cells from unmanipulated Xid mice; thus, they fail to respond to soluble anti-mu, or PC-dextran, but they proliferate in response to PC, anti-mu, or anti-id conjugated to Sepharose.

Animals↗

PubMed1996

Combination of a potent 20-epi-vitamin D3 analogue (KH 1060) with 9-cis-retinoic acid irreversibly inhibits clonal growth, decreases bcl-2 expression, and induces apoptosis in HL-60 leukemic cells.

All-trans retinoic acid (RA) is the first highly effective differentiation-inducing agent for remission induction in patients with acute promyelocytic leukemia. However, remissions are short-lived because the treatment fails to induce complete differentiation and fails to eradicate the malignant clone. To eliminate rapidly the malignant clone, in analogy with aggressive chemotherapy, the combination of potent differentiation- and apoptosis-inducing drugs working through different receptors and signal pathways may be useful. The active form of vitamin D3 (1,25-dihydroxyvitamin D3; 1,25(OH)2D3) inhibits proliferation and induces differentiation of myeloid leukemic cells. The 9-cis-RA, unlike all-trans-RA which binds only retinoic acid receptors, is a high affinity ligand for both retinoic acid receptors and retinoid X receptors. The aim of this study was to evaluate the therapeutic potential of combining a vitamin D(3) analogue, 20-epi-22-oxa-24a,26a,27a-tri-homo-1alpha,25(OH) 2D, (KH 1060), which belongs to the family of potent 20-epi-1,25(OH),D3 analogues, with 9-cis-RA by assessing their effects on the proliferation, differentiation, and apoptosis of the human leukemia cell line HL-60 in vitro. Our data show that KH 1060 alone is a very potent inhibitor of clonal proliferation of HL-60, but this effect is reversible, and that 9-cis-RA alone is a weak inhibitor of clonal proliferation of HL-60 cells. In contrast, the combination of KH 1060 and 9-cis-RA synergistically and irreversibly inhibited the clonal proliferation of HL-60 cells and induced apoptosis, as detected by morphological changes and DNA fragmentation. This combination also affected the expression of apoptosis-related genes. The bcl-2 protein became nearly undetectable, and expression of bax protein increased slightly (the bax:bcl-2 ratio was 14-fold higher than in untreated cells). Differentiation of treated HL-60 cells was assessed by their ability to produce superoxide, as measured by reduction of nitro blue tetrazolium, positive staining for alpha-naphthyl acetate esterase, phagocytosis, morphology, and analysis of membrane-bound differentiation markers with two-color immunofluorescence. Treatment with the combination of KH 1060 and 9-cis-RA was a potent inducer of differentiation of HL-60, with the cells developing a myelomonocytic phenotype. In summary, our data demonstrate that the combination of both KH 1060 and 9-cis-RA irreversibly and synergistically inhibited clonal growth, induced differentiation and apoptosis of HL-60 cells concomitantly with a very marked decreased expression of bcl-2, and increased the bax:bcl-2 ratio. This drug combination may have important therapeutic significance.

Antigens, Surface↗

PubMed1996

Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1.

The Bcl-2 protein blocks programmed cell death (apoptosis) through an unknown mechanism. Previously we identified a Bcl-2 interacting protein BAG-1 that enhances the anti-apoptotic effects of Bcl-2. Like BAG-1, the serine/threonine protein kinase Raf-1 also can functionally cooperate with Bcl-2 in suppressing apoptosis. Here we show that Raf-1 and BAG-1 specifically interact in vitro and in yeast two-hybrid assays. Raf-1 and BAG-1 can also be coimmunoprecipitated from mammalian cells and from insect cells infected with recombinant baculoviruses encoding these proteins. Furthermore, bacterially-produced BAG-1 protein can increase the kinase activity of Raf-1 in vitro. BAG-1 also activates this mammalian kinase in yeast. These observations suggest that the Bcl-2 binding protein BAG-1 joins Ras and 14-3-3 proteins as potential activators of the kinase Raf-1.

Animals↗

PubMed1996

30 KDa phosphorylated form of Bcl-2 protein in human colon.

Bcl-2 expression was studied in a human colon cell line (HT-29) and in human colonic biopsies by Western and Northern blotting. Polyclonal antibodies raised against the Bcl-2 protein detected the expected 26 KDa protein in human colon. However, although Bcl-2 mRNA was present, the 26 KDa Bcl-2 protein was absent in HT-29 cells. Instead, a 30 KDa protein band strongly reacting with anti-Bcl-2 antibodies was found in HT-29 cells, and also in human colon, tonsil, and some other tissues. Alkaline phosphatase shifted the 30 KDa protein to the 26 KDa position in a time-dependent manner. 32P-labeling of HT-29 cells showed that the 30 KDa protein was phosphorylated. A 27 KDa phosphorylated protein was also immunoprecipitated by anti-Bcl-2 antibody. Phosphopeptide mapping showed that the 27 KDa protein contained a minimum of 3 and the 30 KDa protein at least an additional four phosphorylation sites. Phosphoamino acid analysis revealed that both the 30 KDa and 27 KDa proteins were phosphorylated on serine residues. These findings strongly suggest that the 30 KDa protein is a phosphorylated form of Bcl-2, which is widely distributed in human tissues.

Binding Sites↗

PubMed1996

Immunohistochemical analysis of in vivo patterns of Bak expression, a proapoptotic member of the Bcl-2 protein family.

The in vivo patterns of bak gene expression were determined in human tissues using an immunohistochemical approach. Polyclonal antisera were raised in rabbits against a synthetic peptide corresponding to amino acids 14-36 of the human Bak protein, and were shown to be specific by immunoblot analysis of various human tissues and cell lines. Bak immunoreactivity was detected in a wide variety of cell types and was typically present within the cytosol in a punctuate pattern suggestive of association with intracellular organelles. Consistent with a proapoptotic role for the Bak protein, gradients of Bak protein production were observed in the complex epithelia of the nasopharynx, esophagus, colon, and bladder, with Bak immunointensity being highest in the upper layers and relatively low in the basal portions of these epithelia. Similarly, in the myeloid series of hematopoietic cells, Bak immunoreactivity was strongest in the terminally differentiated granulocytes, with only weak immunostaining occurring in most progenitor cells in the bone marrow. Among the other cell types and tissues with prominent Bak immunostaining were: (a) cardiomyocytes; (b) vascular and visceral smooth muscle cells; (c) basal cells of the prostate glands; (d) myoepithelial cells of the mammary glands; (e) distal convoluted tubules of the kidney; (f) epidermal keratinocytes; (g) enterocytes of the small intestine; (h) Sertoli and Leidig cells of the testes; (i) theca interna cells in the ovary; and (j) adrenal cortex (but not adrenal medulla). Nearly all neurons and glial cells of the central nervous system did not contain immunodetectable Bak protein, whereas sympathetic neurons as well as neurons in dorsal root ganglia and their axons were Bak immunopositive. Most circulating peripheral blood lymphocytes were negative for Bak immunostaining, whereas strong Bak immunoreactivity was found frequently in lymphocytes in the nodes and spleen. Overall, these patterns of bak expression are unique compared to other members of the bcl-2 gene family, and suggest that bak regulates cell death at specific stages of cell differentiation through tissue-specific control of its expression.

Antibodies, Monoclonal↗