Effects of acute ethanol exposure on regulatory mechanisms of Bcl-2-associated apoptosis promoter, bad, in neonatal rat cerebellum: differential effects during vulnerable and resistant developmental periods

BACKGROUND: Prenatal alcohol exposure produces anatomical and behavioral abnormalities associated with fetal alcohol syndrome (FAS). Animal FAS models have demonstrated temporal windows of vulnerability in the developing cerebellum, with substantial ethanol (EtOH)-mediated apoptotic activation during these periods. In rodents, the cerebellum is most sensitive to EtOH on postnatal days 4 to 6 (P4 to P6). At slightly later ages (P7 and later), this region is less vulnerable to EtOH. The present study investigated EtOH effects on mechanisms related to activities of Bad, a proapoptotic member of the Bcl-2 gene family, to further characterize processes underlying these disparate EtOH sensitivities. In healthy cells, Bad is retained in the cytosol by association with 14-3-3, a primarily cytosolic protein. Bad promotes apoptosis by disassociating from 14-3-3 and sequestering Bcl-xL through heterodimerization. This dimerization prevents the neutralizing association of Bcl-xL with Bax, freeing Bax to perform in a prodeath manner. Caspase-dependent cleavage of Bad to a 15-kDa fragment increases its proapoptogenic capacity. METHODS: Two hours following EtOH exposure of P4 and P7 animals via inhalation, we determined how exposure affects intracellular localization and proteolytic cleavage of Bad and expression of cerebellar 14-3-3, using subcellular fractionation and Western blot techniques. Ethanol effects on interactions between Bad and 14-3-3 or Bcl-xL at the more vulnerable and less vulnerable ages were determined using an enzyme-linked immunosorbent assay-based technique to detect native protein-protein interactions. RESULTS: At P4, EtOH increased mitochondrial localization of Bad, expression of a 15-kDa fragment recognized by Bad antibody, and formation of Bad:Bcl-xL complexes. At that more vulnerable age, EtOH also decreased formation of Bad:14-3-3 complexes. At P7, EtOH increased Bad:14-3-3 complexes and reduced Bad:Bcl-xL complexes. Cytosolic 14-3-3 remained unchanged by EtOH at P4 and P7. CONCLUSIONS: Ethanol-induced alterations of Bad-related mechanisms at P4 favor a prodeath response. EtOH does not influence these same mechanisms in a manner that promotes cell death at P7. Divergent Bad-related responses at these 2 developmental ages likely contribute to their differential EtOH vulnerability.     

The role of neurotrophic factors,apoptosis-related proteins,and endogenous antioxidants in the differential temporal vulnerability of neonatal cerebellum to ethanol

BACKGROUND: Ethanol produces abnormalities in the developing nervous system, with certain regions being vulnerable during well-defined periods. Neonatal rodent cerebellum is particularly susceptible to ethanol during the early postnatal period [on postnatal days 4-5 (P4-5)], while this region is resistant to ethanol at a slightly later time (P7-9). We assessed basal levels of several substances which may be involved in differential temporal ethanol vulnerability in neonatal cerebellum, and analyzed alterations in these substances after early ethanol exposure. METHODS: Assessments were made of neurotrophic factors nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4; apoptosis-related proteins Bcl-2, Bcl-xl, Bax, Bcl-xs, Bad, phosphorylated-Bad, phosphorylated-Akt, and phosphorylated-c-Jun N-terminal kinase; and the antioxidants superoxide dismutase, glutathione reductase, and catalase. These analyses quantified basal levels (in controls), and sequential changes following acute ethanol exposure at the vulnerable and resistant cerebellar periods (P4, P7). RESULTS: Comparisons of basal levels of the molecules assessed between P4 and P7 revealed higher levels of total proapoptotic Bad at p4, higher levels of the protective pAkt kinase at P7, and lower levels of proapoptotic pJNK at P7. Other basal levels did not differ. While ethanol-mediated alterations were found at both ages favoring both apoptosis and survival, the apoptosis-promoting changes produced on P4 exceeded those on P7, and most occurred within the first 2 hr after exposure, a critical survival/death period. The number of alterations favoring survival were similar at the two ages, but at P7 most occurred within the first 2 hr after exposure, possibly acting in a protective manner. CONCLUSIONS: Differential temporal vulnerability to ethanol in the neonatal cerebellum appears to be paralleled by cellular alterations in neurotrophic factors, apoptosis-regulatory proteins, and/or antioxidant activities which generally favor apoptosis at the most sensitive age and survival at the resistant age.     

Expression of Apo-1/Fas (CD95), Bcl-2, Bax and Bcl-x in myeloma cell lines: relationship between responsiveness to anti-Fas mab and p53 functional status

The down-regulation of apoptosis may be an essential mechanism for tumour cell expansion in slowly proliferating tumours such as multiple myeloma. We studied eight myeloma cell lines for the presence of Bcl-2, which inhibits apoptosis, of Bax, which counteracts Bcl-2, of Bcl-x_L and Bcl-x_S, which act in an anti- and pro-apoptotic fashion, respectively, and of Apo-1/Fas, which induces programmed cell death, when activated by the Apo-1/Fas ligand or the relevant monoclonal antibody (mab). All cell lines constitutively expressed homogenous amounts of Bcl-2, but displayed different amounts of Bax and Bcl-x proteins. The Apo-1/Fas antigen could be detected in seven out of eight myeloma lines, but expression levels varied considerably. The relative expression levels of Apo-1/Fas correlated with that of Bax, but not with that of Bcl-2 or Bcl-x subtypes. Furthermore, the effectiveness of the Apo-1/Fas mab was associated with the relative expression levels of the Apo-1/Fas and with that of the Bax antigen, but not with that of the Bcl-2 and Bcl-x antigens. We further showed that wild-type p53 function is not required for Apo-1/Fas-induced apoptosis, nor is it necessary for the expression of Bax or Apo-1/Fas antigens in myeloma.   In conclusion, our results suggest a p53-independent co-regulation of Apo-1/Fas and Bax, as well as a role for Bax in Apo-1/Fas-induced apoptosis in myeloma.