Biomedical subjects

J C Reed

Publications and source records attributed to J C Reed.

At least 217 records · Page 12Linked to original sources

PubMed1998

BMD188, A novel hydroxamic acid compound, demonstrates potent anti-prostate cancer effects in vitro and in vivo by inducing apoptosis: requirements for mitochondria, reactive oxygen species, and proteases.

A newly synthesized cyclic hydroxamic acid compound, BMD188 [cis-1-hydroxy-4-(1-naphthyl)-6-octylpiperidine-2-one], was found to induce the apoptotic death of cultured prostate cancer cells by activating caspase-3. Orally administered BMD188 significantly inhibited the primary growth of prostate cancer cells (Du145) orthotopically implanted into SCID mice. Mechanistic studies indicated that BMD188 did not alter the protein levels of several Bcl-2 family members. In contrast, the BMD188 effect required three essential factors: reactive oxygen species (ROS), the mitochondrial respiratory chain function, and proteases. First, the apoptosis-inducing effect of BMD188 could be blocked by ROS scavengers such as Desferal. Second, both BMD188-induced PARP cleavage as well as PC3 cell apoptosis could be dramatically inhibited by several complex-specific mitochondrial respiration blockers. The involvement of mitochondria was also supported by the observations that BMD188 dramatically altered the mitochondrial distribution and morphology without affecting the cellular ATP levels. Finally, the apoptosis-inducing effect of BMD188 in PC3 cells could be significantly inhibited by serine protease inhibitors (TPCK and TLCK) as well as by caspase inhibitors (zVAD-fmk and DEVD-CHO). Collectively, the present study suggests that BMD188 and its analogs may find clinical applications in the treatment of prostate cancer patients by inducing apoptotic death of prostate cancer cells.

Animals↗

PubMed1998

Bcl-2, Bax and Bcl-x expression in neuronal apoptosis: a study of mutant weaver and lurcher mice.

We investigated the expression of the apoptosis modulating proteins Bcl-2, Bax and Bcl-x in the cerebellum of mutant lurcher and weaver mice. Lurcher Purkinje cells and weaver germinal (granule neuron progenitor) cells both die via apoptosis during the postnatal cerebellar development. No significant changes in the expression patterns were detected prior to the actual cell death process. Instead apoptotic lurcher Purkinje cells exhibited increased Bax and Bcl-x expression, while surviving cells had an expression pattern similar to that of healthy littermates. Increased Bax expression was also found in apoptotic weaver germinal cells, while no change of Bcl-x expression was detected. Bcl-2 was expressed at low levels in cerebellar neurons and no loss of Bcl-2 was evident. The observed expression patterns of Bcl-2, Bax and Bcl-x protein in apoptotic lurcher and weaver neurons support the hypothesis that the execution of neuronal apoptosis involves increased expression of Bax, which could represent a general mechanism in diverse neurodegenerative processes.

Animals↗

PubMed1998

p53-regulated apoptosis is differentiation dependent in ultraviolet B-irradiated mouse keratinocytes.

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (-/-) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53-/- mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m2 UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53-/- cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53-/- cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and is therefore of questionable significance in protection against skin cancer induction.

Animals↗

PubMed1998

Toso, a cell surface, specific regulator of Fas-induced apoptosis in T cells.

Fas is a surface receptor that can transmit signals for apoptosis. Using retroviral cDNA library-based functional cloning we identified a gene, toso, that blocks Fas-mediated apoptosis. Toso expression was confined to lymphoid cells and was enhanced after cell-specific activation processes in T cells. Toso appeared limited to inhibition of apoptosis mediated by members of the TNF receptor family and was capable of inhibiting T cell self-killing induced by TCR activation processes that up-regulate Fas ligand. We mapped the effect of Toso to inhibition of caspase-8 processing, the most upstream caspase activity in Fas-mediated signaling, potentially through activation of cFLIP. Toso therefore serves as a novel regulator of Fas-mediated apoptosis and may act as a regulator of cell fate in T cells and other hematopoietic lineages.

Adaptor Proteins, Signal Transducing↗

PubMed1998

The Mitochondrial F0F1-ATPase proton pump is required for function of the proapoptotic protein Bax in yeast and mammalian cells.

The proapoptotic mammalian protein Bax associates with mitochondrial membranes and confers a lethal phenotype when expressed in yeast. By generating Bax-resistant mutant yeast and using classical complementation cloning methods, subunits of the mitochondrial F0F1-ATPase proton pump were determined to be critical for Bax-mediated killing in S. cerevisiae. A pharmacological inhibitor of the proton pump, oligomycin, also partially abrogated the cytotoxic actions of Bax in yeast. In mammalian cells, oligomycin also inhibited Bax-induced apoptosis and activation of cell death proteases. The findings imply that an intact F0F1-ATPase in the inner membrane of mitochondria is necessary for optimal function of Bax in both yeast and mammalian cells.

Animals↗

PubMed1998

Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast.

The mammalian proapoptotic protein Bax confers a lethal phenotype when expressed in yeast. By exploiting this phenotype, we have identified a novel human Bax inhibitor, BI-1. BI-1 is an evolutionarily conserved integral membrane protein containing multiple membrane-spanning segments and is predominantly localized to intracellular membranes, similar to Bcl-2 family proteins. Moreover, BI-1 can interact with Bcl-2 and Bcl-XL but Bax or Bak, as demonstrated by in vivo cross-linking and coimmunoprecipitation studies. When overexpressed in mammalian cells, BI-1 suppressed apoptosis included by Bax, etoposide, staurosporine, and growth factor deprivation, but not by Fas (CD95). Conversely, BI-1 antisense induced apoptosis. BI-1 thus represents a new type of regulator of cell death pathways controlled by Bcl-2 and Bax.

Animals↗

PubMed1998

Expression of multiple apoptosis-regulatory genes in human breast cancer cell lines and primary tumors.

The expression of several apoptosis-regulating genes was evaluated in 9 human breast cancer cell lines, 2 immortalized human mammary epithelial lines, 1 normal breast tissue biopsy, and 3 primary breast tumors, using a multiple antigen detection (MAD) immunoblotting method. The anti-apoptotic proteins Bcl-2, Bcl-X(L), Mcl-1, and BAG-1 were present at immunodetectable levels in 7, 10, 10, and 9 of the 11 lines. Comparing these 11 cell lines among themselves revealed that steady-state levels of Bcl-2, Bcl-X(L), Mcl-1, and BAG-1 were present at relatively higher levels in 4, 6, 5, and 5 of the lines, respectively. In contrast, the pro-apoptotic proteins Bax and Bak were detected in all 11 cell lines, and were present at relatively higher levels in 10 and 5 of the 11 lines, respectively. The Interleukin-1beta converting enzyme (ICE) homolog CPP32 (Caspase-3) was expressed in 10/11 breast cell lines. High levels of p53 protein, indicative of mutant p53, were found in 8 of the 11 lines and correlated inversely with Bax expression (p = 0.01). Bcl-2 and BAG-1 protein levels were positively correlated (p = 0.03). Immunoblot analysis of primary adenocarcinomas revealed expression of the anti-apoptotic proteins Bcl-2, Bcl-X(L), Mcl-1, and BAG-1, as well as the pro-apoptotic proteins Bax, Bak, and CPP32, in at least 2 of the 3 tumors examined. Immunohistochemical analysis was also performed for all of these proteins using 20 paraffin-embedded breast cancer biopsy specimens that all contained residual normal mammary epithelium in combination with both invasive cancer and carcinoma in situ. All of these apoptosis-regulating proteins were detected in primary breast cancers, though the percentage of immunopositive tumor cells varied widely in some cases. Comparisons of the intensity of immunostaining in normal mammary epithelium and invasive carcinoma suggested that Bcl-2 immunointensity tends to be lower in cancers than normal breast epithelium (p = 0.03), whereas CPP32 immunointensity was generally higher in invasive cancers (p < 0.0001). Taken together, the results demonstrate expression of multiple apoptosis-modulating proteins in breast cancer cell lines and primary tumors, suggesting complexity in the regulation of apoptosis in these neoplasms of mammary epithelial origin.

Apoptosis↗

PubMed1998

A sequential treatment regimen with melatonin and all-trans retinoic acid induces apoptosis in MCF-7 tumour cells.

Neoplastic events are marked by uncontrolled cell proliferation. One major focus of cancer research has been to identify treatments that reduce or inhibit cell growth. Over the years, various compounds, both naturally occurring and chemically synthesized, have been used to inhibit neoplastic cell proliferation. Two such oncostatic agents, melatonin and retinoic acid, have been shown to suppress the growth of hormone-responsive breast cancer. Currently, separate clinical protocols exist for the administration of retinoids and melatonin as adjuvant therapies for cancer. Using the oestrogen receptor (ER)-positive MCF-7 human breast tumour cell line, our laboratory has studied the effects of a sequential treatment regimen of melatonin followed by all-trans retinoic acid (atRA) on breast tumour cell proliferation in vitro. Incubation of hormonally responsive MCF-7 and T47D cells with melatonin (10(-9) M) followed 24 h later by atRA (10(-9) M) resulted in the complete cessation of cell growth as well as a reduction in the number of cells to below the initial plating density. This cytocidal effect is in contrast to the growth-suppressive effects seen with either hormone alone. This regimen of melatonin followed by atRA induced cytocidal effects on MCF-7 cells by activating pathways leading to apoptosis (programmed cell death) as evidenced by decreased ER and Bcl-2 and increased Bax and transforming growth factor beta 1 (TGF-beta1) expression. Apoptosis was reflected morphologically by an increase in the number of lysosomal bodies and perinuclear chromatin condensation, cytoplasmic blebbing and the presence of apoptotic bodies. The apoptotic effect of this sequential treatment with melatonin and atRA appears to be both cell and regimen specific as (a) ER-negative MDA-MB-231 and BT-20 breast tumour cells were unaffected, and (b) the simultaneous administration of melatonin and atRA was not associated with apoptosis in any of the breast cancer cell lines studied. Taken together, the results suggest that use of an appropriate regimen of melatonin and atRA should be considered for preclinical and clinical evaluation against ER-positive human breast cancer.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1998

A multivariate analysis of tumour biological factors predicting response to cytotoxic treatment in advanced breast cancer.

The study was designed to identify factors that could predict response to chemotherapy in breast cancer. A total of 173 patients with measurable or evaluable metastatic breast cancer were enrolled in a randomized trial between November 1987 and January 1991 to receive a monthly dose of 5-fluorouracil (500 mg m(-2)), epirubicin (60 mg m(-2)) and cyclophosphamide (500 mg m(-2)) either administered in four weekly doses or in an every-4-week dose as first-line cytotoxic treatment. In 103 evaluable patients we performed a multivariate analysis of the tumour biological factors, i.e. histological grade, oestrogen receptor (ER), progesterone receptor (PR), S-phase fraction (SPF), ploidy, p53, c-erbB-2, Bcl-2 and Bax expression, which showed significance in the univariate analysis according to treatment response, time to progression (TTP) or overall survival (OS). In the univariate analysis only SPF, grade and the proapoptotic protein Bax correlated with the response to cytotoxic treatment. In the multivariate analysis SPF had the strongest correlation, followed by grade and Bax. In the univariate analysis grade, PR, Bax and Bcl-2 correlated significantly with TTP, whereas in the multivariate analysis only PR showed a statistically significant correlation. In the univariate analysis PR and Bax correlated with OS and both retained its significance in the multivariate analysis. The factors that correlated significantly with the response to cytotoxic treatment in the univariate analysis, i.e. grade, SPF and Bax, seemed to predict independently the response to treatment in the multivariate analysis also. TTP and OS could be predicted partly by the same factors, although the association was quite weak. More studies and new tumour biological factors are needed to identify the group of breast cancer patients who get the most benefit from chemotherapy.

Analysis of Variance↗

PubMed1998

Analysis of apoptosis and expression of bcl-2 gene family members in the human and baboon ovary.

Recent data support a role for apoptosis, under tight regulatory control by bcl-2, oxidative stress response, tumor suppressor, and CASP gene family members, in mediating granulosa cell demise during follicular atresia in the rodent and avian ovary. Herein we evaluated the occurrence of apoptosis in the human and baboon ovary relative to follicular health status, and analyzed expression of several cell death genes in these tissues. In situlocalization of DNA strand breaks in fixed human and baboon ovarian tissue sections indicated that apoptosis was essentially restricted to granulosa cells of atretic antral follicles. Biochemical analysis of DNA oligonucleosomes in individual follicles isolated from baboon ovaries during the ovulatory phase revealed the presence of apoptotic DNA fragments in subordinate but not dominant follicles, thus substantiating the in situ labeling studies. Messenger RNA transcripts encoded by the bax death susceptibility gene, the bcl-xlong survival gene, the bcl-xshort pro-apoptosis gene, the p53 tumor suppressor gene, and two members of the CASP gene family (CASP-2/Ich-1, CASP-3/CPP32), were detected by Northern blot analysis of total RNA prepared either from human ovaries or from Percoll-purified granulosa-lutein cells obtained from patients undergoing assisted reproductive technologies. Lastly, immunohistochemical localization of the BAX death-susceptibility protein in the human ovary revealed abundant expression in granulosa cells of early atretic follicles, whereas BAX protein was extremely low or non-detectable in healthy or grossly-atretic follicles. We conclude that apoptosis occurs during, and is probably responsible for, folicular atresia in the human and baboon ovary. Moreover, apoptosis in the human ovary is likely controlled by altered expression of the same cohort of cell death regulatory factors recently implicated as primary determinants of apoptosis induction or suppression in the rodent ovary.

Adult↗

PubMed1998

Developmental expression patterns of Bcl-2, Bcl-x, Bax, and Bak in teeth.

The ontogenic profile of expression of four members of the Bcl-2 family (Bcl-2, Bcl-x, Bax and Bak) was examined in the mouse by immunohistochemistry using paraffin sections. All four members were expressed in changing patterns during critical stages of tooth morphogenesis. Expression was detected in epithelial cell populations including the dental lamina, internal dental epithelium (IDE; differentiating ameloblasts), stratum intermedium and stellate reticulum cells, as well as in the condensed dental mesenchyme. The temporo-spatial localization of the various members of the Bcl-2 family in dental epithelium and mesenchyme showed striking overlapping areas but often their expression patterns differed. In general, contemporaneous co-expression of the Bcl-2 and Bax proteins, and of the Bcl-x and Bak proteins was noted in various types of cells during the developmental process, with the intensity of Bcl-2>Bax and of Bak>Bcl-x. Expression was pronounced at sites where interaction between surface ectoderm and induced mesenchyme takes place, and at the enamel knot, which is regarded as organization/regulating center for tooth development. Around birth, after the structural maturation was accomplished, the expression was down-regulated. The absence of elevated expression of each of these four members of the Bcl-2 family after birth in the teeth suggests that these proteins are relevant during the accomplishment of the basic architecture but not once the structure of the tooth is established.

Animals↗

PubMed1998

Bcl-2 family proteins as ion-channels.

The Bcl-2 protein family function(s) as important regulators of cellular decisions to heed or ignore death signals. The three-dimensional structure of the Bcl-2 homolog, Bcl-XL, bears a strong resemblance to some pore-forming bacterial toxins. This similarity suggested that the Bcl-2 family proteins may also possess channel-forming capability. This review summarizes the recent initial studies on the in vitro channel activity of Bcl-2, Bcl-XL and Bax and offers some speculation as to the physiological role that these channels may play in the cell death pathway.

Apoptosis↗

PubMed1998

Differential p53 phosphorylation and activation of apoptosis-promoting genes Bax and Fas/APO-1 by irradiation and ara-C treatment.

In this study, we examined the effects of radiation and ara-C on induction of apoptosis and on the apoptosis-promoting genes p53, Bax and Fas/APO-1, in BV173 human leukemia cells, which harbor the wild-type p53 gene. It has been reported that p53 upregulates Fas/APO-1 and Bax expression. Both irradiation and ara-C treatment resulted in apoptosis and induction of p53 proteins within hours. The Bax gene was activated in irradiated and ara-C-treated BV173 cells, but Fas/APO-1 was induced only in irradiated BV173 cells. Radiation and ara-C treatment did not induce Bax or Fas/APO-1 protein expression in p53-null HL60 cells. Radiation weakly induced Fas/APO-1 expression in KBM-7 cells, which harbor a partially defective p53 gene. Both HL60 and KBM-7 cells are more resistant to radiation- and ara-C-induced apoptosis than BV173 cells. These results suggest that functional p53 is necessary for the activation of Bax and Fas/APO-1 expression. However, elevated p53 protein is not sufficient to activate Fas/APO-1 gene expression in ara-C-treated cells. Using two-dimensional gel electrophoresis, we found that the p53 proteins in irradiated and ara-C-treated BV173 cells have different isoelectric points; they converged to a single isoelectric point after in vitro treatment with phosphatase. These results suggest that different genotoxic treatments cause different phosphorylations of p53, which may account for the different levels of activation of Fas/APO-1 expression.

Apoptosis↗

PubMed1998

Investigation of glucocorticoid-induced apoptotic pathway: processing of caspase-6 but not caspase-3.

Glucocorticoids (GCs) are essential therapeutic reagents for the treatment of lymphomas and leukemias. GCs cause cell death in certain types of lymphoid cells mediated by the process known as apoptosis. This cell death is completely inhibited by Bcl-2. Here we report that Bcl-2 and benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), a broad spectrum caspase inhibitor, prevent loss of mitochondrial membrane potential (delta psi m) and the production of reactive oxygen species (ROS) caused by GC, while acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), an inhibitor of the caspase-3 family proteases, does not. This suggests that the inhibition by Bcl-2 and activation of some initiator caspases are upstream events of mitochondrial damage, whereas the activation of caspase-3 family proteases occurs downstream of mitochondrial changes. We also demonstrate that caspase-6 but not caspase-3 is cleaved and activated during GC-mediated apoptosis and that poly(ADP-ribose) polymerase (PARP), a substrate of caspases, also undergoes proteolysis. In addition, we provide the evidence that DNA fragmentation is markedly inhibited by Ac-DEVD-CHO, while cell death, assessed by the damage of the plasma membrane, is marginally inhibited or merely delayed.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Prolonged cell survival enhances peritoneal dissemination of gastric cancer cells.

Bcl-2 and a Bcl-2-binding protein BAG-1 function in protection from apoptosis induced by a variety of stimuli. Deregulated expression of Bcl-2 leads to inhibition of apoptosis and is correlated with development of various cancers. Here, we provide evidence that prolonged cell survival introduced by overproduction of Bcl-2 or BAG-1 strongly enhances peritoneal dissemination of human gastric cancer MKN74 cells. Gene transfer-mediated overexpression of Bcl-2 or BAG-1 led to prolonged cell survival of MKN74 cells against serum-starved apoptosis and anoikis. When the viable transfectants were inoculated into the intraperitoneal cavity of BALB/c nude mice, the Bcl-2-expressing MKN74 cells and the BAG-1-expressing MKN74 cells exhibited strongly enhanced peritoneal dissemination in BALB/c nude mice and whole disseminated tumor weights were increased by 4-fold and 3.3-fold, respectively, compared with the control transfectants. The enhanced peritoneal dissemination of MKN74-Bcl-2 and MKN74-BAG-1 transfectants correlated well with resistance to cell death induced by serum-starvation and anoikis. However, the overexpression of Bcl-2 or BAG-1 caused no significant difference among the transfectants in cell growth rates, either in vitro or in vivo. Taken together, these studies demonstrate that resistance to apoptosis is a crucial factor for development of peritoneal dissemination of human gastric cancer cells.

Adenocarcinoma↗

PubMed1998

Decreased levels of p26-Bcl-2, but not p30 phosphorylated Bcl-2, precede TGFbeta1-induced apoptosis in colorectal adenoma cells.

Bcl-2 expression is confined to the base of the colonic crypt, whereas transforming growth factor beta (TGFbeta) is expressed in the upper crypt, as are the apoptotic death promoters, Bak and Bax. In colonic adenoma cells, TGFbeta induces a growth arrest. In some adenoma cell lines, this is accompanied by apoptosis and in others it is not. In this study, we used two human colonic adenoma cell lines: RG/C2, in which TGFbeta induces a G1 arrest without apoptosis, and BH/C1, in which TGFbeta induces both a G1 arrest and apoptosis. TGFbeta does not induce apoptosis in RG/C2 cells even if hydrocortisone and insulin are removed from the culture medium. In BH/C1 cells, TGFbeta induces apoptosis in the presence of insulin and hydrocortisone. Apoptosis induced by TGFbeta is preceded by a reduction in p26-Bcl-2 protein levels. There was no change in the levels of the p30 phosphorylated form of Bcl-2 or in levels of the proapoptotic proteins Bax or Bak. RG/C2 cells did not show decreased Bcl-2 levels in response to TGFbeta-induced growth inhibition. Therefore, TGFbeta regulates Bcl-2 expression in colonic adenoma cells which undergo apoptosis in response to TGFbeta, but not in those which are growth inhibited, but resistant to TGFbeta-induced apoptosis. TGFbeta may play an important role in the colonic epithelium, not only in the inhibition of cell proliferation, but also in the regulation of apoptosis.

Adenoma↗

PubMed1998

Immunohistochemical analysis of bcl-2, bax, mcl-1, and bcl-X expression in ovarian surface epithelial tumors.

Cell survival may be enhanced in tumors by the inhibition of apoptosis, which allows tumor promotors to exert their effects. The bcl-2 related genes have been shown to contribute to either the inhibition or induction of apoptosis in a variety of neoplasms; therefore, it was hypothesized that the expression of these genes might contribute to malignant transformation in ovarian surface epithelial tumors. The expression of bcl-2 family proteins was investigated in 28 ovarian surface epithelial tumors, including serous and mucinous benign, borderline, and malignant tumors by immunohistochemical staining with antibodies to bcl-2, bax, bcl-X, and mcl-1 proteins. Staining intensity was scored on a 1+ to 3+ scale and the benign, borderline, and malignant tumors were compared. Significantly less immunoreactive bcl-2 and bcl-X proteins were present in malignant serous tumors compared to their benign counterparts. No difference was seen in immunostaining for bax or mcl-1 when benign, borderline, or malignant serous tumors were compared. In the mucinous tumors, no differences were seen in immunostaining for any of the bcl-2 family proteins between tumor types. The loss of expression of the antiapoptotic proto-oncogenes bcl-2 or bcl-X in serous carcinomas compared to benign serous tumors, together with previous demonstrations that the presence of bcl-2 in ovarian surface epithelial cancers is a favorable prognostic indicator, suggests that bcl-2 and bcl-X have biological functions in the ovary other than inducing apoptosis, acting instead as tumor suppressor proteins.

Apoptosis↗

PubMed1998

Bcl-x(l) Bax interaction after transient global ischemia.

Five minutes of bilateral common carotid artery occlusion in the Mongolian gerbil results in a selective, delayed death of CA1 pyramidal neurons. Although Bcl-2 appears to protect a variety of cells from cell death, the precise role of this apoptosis-regulating protein is complicated. We used immunoblots to estimate levels of Bcl-2, Bcl-x(l), and Bax at various times after carotid occlusion. Rather than Bcl-2, Bcl-x(l) appears to be the predominant neuroprotective form of this family of proto-oncogenes in the gerbil hippocampus. After transient ischemia, Bcl-2 and Bcl-x(l) protein levels remained the same. However, Bax levels were dramatically increased at 6 hours after ischemia, compared with sham-operated animals, and were still elevated at 72 hours after ischemia. To monitor dimerization interactions among theses apoptosis-regulating molecules, immunoprecipitation studies were conducted. These studies demonstrated that Bcl-x(l) association with Bax increases after ischemia. Therefore, Bax may disrupt the more favorable Bcl-x(l) (Bcl-2) interactions necessary for normal neuronal functioning and thus promote transient ischemic death.

Animals↗