Individual foci from the transformation assay, as in Fig., were cloned and grown into cell lines.
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Generation of Stable BMK Cell Lines from bax −/−, bak −/−, and bax −/− bak −/−Mice In order to study molecular events of apoptosis affected by Bax and Bak expression, stable BMK cell lines, transformed with E1A plus p53DD, were derived from the wild-type (W), bax −/− (X), bak −/−(K), and bax −/− bak −/− (D) mice. Thus, the loss of Bax and Bak function may not be equivalent to the gain of an anti-apoptotic Bcl-2 function. Post-transfection each condition was plated into three 6-cm tissue culture dishes. The BMK cells were resuspended in 550 μl of DMEM plus 5% FBS, divided into two portions of 250 μl each, and electroporated in the presence of 10 μg of linearized pCMVE1A plasmid DNA and 110 μg of salmon sperm carrier DNA or 10 μg of linearized pCMVE1A plasmid DNA and 10 μg of linearized p53DD plasmid DNA and 100 μg of salmon sperm carrier DNA. Following the incubation, 5 ml of DMEM plus 5% FBS was added and mixed by pipetting, and clumps were allowed to settle for 5 min, and the BMK were collected from the supernatant by centrifugation at 400 × g for 5 min. Each pair of kidneys was washed with PBS, placed into 10 ml of PBS containing 2.5 mg/ml dispase II (Roche Molecular Biochemicals) and 2.5 μg/ml collagenase A (Roche Molecular Biochemicals), mechanically disrupted, and then stirred at 37 ☌ for 30 min. Kidneys from each pup were removed and processed separately under sterile conditions. Preparation of BMK cells from primary kidneys from murine pups was based on the BRK preparation protocol with the following modifications. Transformation Assay Three matings were performed as follows: mice were bred by crossing mice that were bax +/− bak −/− with bax +/− bak −/− bax +/− bak +/− with bax +/− bak −/− and bax +/− bak +/+ with bax +/− bak +/+. These findings indicate that Bax and Bak function redundantly to release cytochrome c from the mitochondria to implement apoptosis in response to death receptor signaling, but both are dispensable for p53-mediated suppression of oncogenic transformation. The absence of either Bax or Bak did not affect the release of cytochrome c from mitochondria to the cytosol in response to TNF-α however, the loss of both dramatically prevented the release of cytochrome cand caspase-9 activation. The loss of either Bax or Bak did not abrogate TNF-α-induced apoptosis however, the loss of both Bax and Bak conferred resistance to apoptosis. Stable E1A plus p53DD-transformed BMK cell lines were derived from the foci from bax −/−, bak −/−, and bax −/− bak −/− mice in the BMK cell transformation assay and were characterized for their ability to undergo apoptosis by death receptor signaling pathways.
Thus, merely an increase in the amount of Bax or Bak may not result in cell death.
Evidence suggests that Bax and Bak undergo changes in protein conformation that have been linked to their pro-apoptotic function (, ). Although there is up-regulation of bax, bak, puma, and noxa by p53, transcriptional up-regulation of at least Bax is not sufficient for p53-mediated apoptosis, because a mutant of p53 that up-regulates bax is not able to induce apoptosis.
This caspase activation initiates a caspase cascade that is required for p53-dependent apoptosis (, ) and results in DNA fragmentation, cleavage of cellular proteins such as poly(ADP-ribose) polymerase and nuclear lamins, and cell death by apoptosis. In many cases, this event is pivotal in the regulation of apoptosis, because cytochrome c in the cytosol complexes with APAF-1 and, in turn, promotes caspase-9 activation. Cytochrome c release from the mitochondria occurs in many apoptotic signaling pathways including those implemented by p53 and TNF-α. Evidence suggests that Bax and Bak function is required for the release of cytochrome c from the mitochondria to the cytosol during apoptosis (, ).
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