Cyclin-dependent kinases and P53 pathways are activated independently and mediate Bax activation in neurons after DNA damage - PubMed (original) (raw)
Cyclin-dependent kinases and P53 pathways are activated independently and mediate Bax activation in neurons after DNA damage
E J Morris et al. J Neurosci. 2001.
Abstract
DNA damage has been implicated as one important initiator of cell death in neuropathological conditions such as stroke. Accordingly, it is important to understand the signaling processes that control neuronal death induced by this stimulus. Previous evidence has shown that the death of embryonic cortical neurons treated with the DNA-damaging agent camptothecin is dependent on the tumor suppressor p53 and cyclin-dependent kinase (CDK) activity and that the inhibition of either pathway alone leads to enhanced and prolonged survival. We presently show that p53 and CDKs are activated independently on parallel pathways. An increase in p53 protein levels, nuclear localization, and DNA binding that result from DNA damage are not affected by the inhibition of CDK activity. Conversely, no decrease in retinoblastoma protein (pRb) phosphorylation was observed in p53-deficient neurons that were treated with camptothecin. However, either p53 deficiency or the inhibition of CDK activity alone inhibited Bax translocation, cytochrome c release, and caspase-3-like activation. Taken together, our results indicate that p53 and CDK are activated independently and then act in concert to control Bax-mediated apoptosis.
Figures
Fig. 1.
Determination of the commitment point for neuronal death by DNA damage and protection by CDK inhibitors. A, Determination of the commitment point for neuronal death by DNA damage. Cortical cultures were treated with camptothecin (campto; 10 μ
m
) at 0 hr and removed at the indicated times; neuronal survival was determined at 24 hr after treatment. B, Determination of the commitment point for protection by CDK inhibition. The CDK inhibitor flavopiridol (1 μ
m
) was added to cortical cultures treated with camptothecin at the indicated times; neuronal survival was determined at 24 hr after treatment. In each experiment the viability was assessed by nuclear counts. Each_point_ is the mean ± SEM of data from three cultures.
Fig. 2.
Induction of p53 expression and p53-specific DNA binding activity is not affected by cotreatment with the CDK inhibitor flavopiridol. Total cell lysates were prepared from wild-type cortical cultures treated with camptothecin (campto; 10 μ
m
) alone or cotreated with flavopiridol (1 μ
m
) and analyzed for p53 expression by Western blot analyses (A, B) or DNA binding by EMSA (C), as indicated in Materials and Methods. Densitometric analysis from three experiments (mean ± SEM) with and without flavopiridol cotreatment is shown. D, So that the specific nature of the EMSA band could be ensured, cell extracts from wild-type or p53-deficient neurons were untreated or treated with camptothecin (10 μ
m
) for 8 hr and analyzed for p53 binding to a labeled p53 oligonucleotide probe by EMSA.cold comp, The addition of unlabeled probe as a competitor for radiolabeled probe. The band labeled p53 is the only band present in the camptothecin-treated wild-type or heterozygous extracts and is missing in the untreated and p53-deficient extracts.
Fig. 3.
Nuclear localization of p53 is not inhibited by the inhibition of CDK. A, Immunofluorescence image of cultured cortical neurons stained with nuclear dye or anti-p53 antibody. Where indicated, the cultures were treated with camptothecin (campto; 10 μ
m
) for 5 hr with and without flavopiridol (1 μm) treatment. B, Quantitation of neurons with positive nuclear staining for p53. Data are the mean ± SEM from three cultures.
Fig. 4.
Phosphorylation of pRb is not inhibited by p53 deficiency during DNA damage-induced neuronal apoptosis.A, Western immunoblots (probed with anti-phospho-Rb Ser795 antibody) of whole-cell lysates of cortical neurons from p53-heterozygous or p53-nullizygous embryos after treatment with camptothecin (campto; 10 μ
m
) for the indicated times. B, Densitometric analyses of the Western immunoblots. All data points were normalized to actin expression levels. Data are the mean ± SEM from three experiments and are expressed relative to the initial amount of phosphorylated pRb at time 0.
Fig. 5.
DNA damage-induced Bax translocation is inhibited by either p53 deficiency or CDK inhibition. A, Bax protein levels do not change during the death of cortical neurons evoked by DNA damage. Shown are Western immunoblots of whole-cell lysates of cortical neurons after various periods of treatment with camptothecin (campto; 10 μ
m
). Antibody specificity is demonstrated for Western analysis by using lysates from Bax-deficient neurons. Control for protein loading is indicated by actin expression. B, Immunofluorescence micrograph of Bax translocation in cortical neurons of Bax (−/−), p53 (−/+), or p53 (−/−) backgrounds. Where indicated, the cultures were treated with camptothecin (CA; 10 μ
m
; 10 hr) alone or cotreated with the caspase inhibitor zVAD (100 μ
m
) or the CDK inhibitor flavopiridol (FL; 1 μ
m
). Punctate staining is indicative of Bax translocation from the cytosol and is quantitated in C. Data are the mean ± SEM from three cultures.
Fig. 6.
Cytochrome c (cytC) release is blocked by p53 deficiency during DNA damage-induced neuronal apoptosis. Cortical neurons from p53-heterozygous or p53-nullizygous embryos were cultured and treated with camptothecin (campto; 10 μ
m
) for 12 hr. Quantitation of cytochrome_c_ release (bottom; as indicated by punctate-to-diffuse expression in top) is shown. Data are the mean ± SEM from three cultures. * indicates significance (p < 0.05, as derived from Student's _t_test).
Fig. 7.
Bax deficiency does not inhibit p53 induction or DNA binding in neurons exposed to DNA damage. Bax-heterozygous or Bax-nullizygous neurons were treated with camptothecin (campto; 10 μ
m
) for the indicated times and analyzed for p53 protein levels by Western blot analyses (A) and for DNA binding by EMSA (B). For B, representative radiographs and densitometric analysis from three experiments (mean ± SEM) are shown.
Fig. 8.
The general caspase inhibitor BAF does not inhibit p53 induction or DNA binding in neurons exposed to DNA damage. Cortical neurons were treated with camptothecin (campto; 10 μ
m
) alone or cotreated with BAF (100 μ
m
) for the indicated times and analyzed for p53 protein levels by Western blot analyses (A) and DNA binding by EMSA (B). For B, representative radiographs and densitometric analysis from three experiments (mean ± SEM) are shown.
Fig. 9.
Delayed death in flavopiridol-treated, p53-deficient, or Bax-deficient mice is not accompanied by caspase-3-like activity. DNA damage-induced caspase activation in neurons is CDK-, p53-, and Bax-dependent. Cortical neurons were treated with camptothecin (campto; 10 μ
m
) and assessed for survival (left column) or DEVD-AFC cleavage activity (right column). For survival experiments, each_point_ is the mean ± SEM of data from three cultures. Cleavage activity in camptothecin-treated wild-type or p53/Bax littermate cultures was assessed only at 0 and 24 hr because of low levels of total protein observed at later time points.A, Wild-type cortical neurons were cotreated with flavopiridol (1 μ
m
). B, p53-Deficient neurons or littermate controls. C, Bax-deficient neurons or littermate controls.
Fig. 10.
Proposed model for role of CDK, pRb, p53, Bax, and caspases in the death of cortical neurons evoked by DNA damage (see Discussion for summary).
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