Involvement of Microtubules in the Regulation of Bcl2 Phosphorylation and Apoptosis through Cyclic AMP-Dependent Protein Kinase

Abstract

The Bcl2 family of proteins plays a significant role in regulation of apoptosis. In this study, the microtubuledamaging drugs paclitaxel, vincristine, and vinblastine induced Bcl2 hyperphosphorylation and apoptosis in MCF-7 and MDA-MB-231 cells and reduced Bcl2-Bax dimerization. Paclitaxel or vincristine induced increased expression of Bax, while overexpression of Bcl2 in these cell lines counteracted the effects of low doses of these drugs. In addition, paclitaxel-and vincristine-induced activation of cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) induced Bcl2 hyperphosphorylation and apoptosis, which were blocked by the PKA inhibitor Rp diastereomers of cAMP (Rp-cAMP). This finding suggests that activation of PKA due to microtubule damage is an important event in Bcl2 hyperphosphorylation and induction of apoptosis. These microtubule-damaging drugs caused growth arrest in G 2 -M phase of the cell cycle and had no effect on p53 induction, suggesting that hyperphosphorylation mediated inactivation of Bcl2 and apoptosis without the involvement of p53. By comparison, the DNA-damaging drugs methotrexate and doxorubicin had no effect on Bcl2 hyperphosphorylation but induced p53 expression. Interestingly, paclitaxel or vincristine induced activation of caspase 3 and cleavage of poly(ADP-ribose) polymerase downstream of Bcl2 hyperphosphorylation. These data suggest that there may be a signaling cascade induced by agents that disrupt or damage the cytoskeleton that is distinct from (i.e., p53 independent), but perhaps related to (i.e., involves kinase activation and leads to apoptosis), the cellular response to DNA damage.

Dual Anticancer Activity of 8-Cl-cAMP: Inhibition of Cell Proliferation and Induction of Apoptotic Cell Death

“…cAMP decreases Bcl-2 in SH-SY5Y cells and enhances apoptotic cell death induced by serum deprivation (26). Contrary to this report, cAMP increases bcl-2 promoter activity in other system through cAMPresponse element (27). Bcl-2 protein is also phosphorylated and inactivated by PKA during the apoptosis induced by microtubule-damaging agents (28).…”

Aluminum Maltolate-Induced Toxicity in NT2 Cells Occurs Through Apoptosis and Includes Cytochrome c Release

“…Apoptosis has been suggested to play a role in AD pathology (Cotman and Anderson, 1995;Su et al, 1997) however, either the full cascade is not activated or appears to be aborted (Perry et al, 1998a(Perry et al, , 1998bRaina et al, 2001) An intriguing possibility is that apoptosis-related changes, oxidative stress, and axonal transport disruption are linked in a way that promotes neurodegeneration and involves cytoskeletal disruption (Srivastava et al, 1998). Since Al contributes to all of these factors (Ghribi et al, 2001b;Kashiwagi et al, 1998;Pratico et al, 2002), the model presented here may prove useful in understanding the mechanisms leading to neuronal death in neurodegenerative diseases.…”

Kinetic stabilization of microtubule dynamic instability by benomyl increases the nuclear transport of p53

“…Both the floating and attached cells were harvested with the help of a cell scrapper and collected by centrifugation. The total cell lysates were collected as described earlier [15,30,31]. The cell lysates containing polymeric and soluble tubulin (monomeric tubulin) were prepared as described earlier [32,33].…”