A Mechanism for the Inhibition of Neural Progenitor Cell Proliferation by Cocaine
Figure 5
Role of Cyclin A Expression in Cocaine-Induced Proliferation Inhibition
(A) Western blot analysis of cyclin A and its downstream proteins in cocaine-treated AF5 cells. AF5 cells were treated with vehicle or 100 μM cocaine for 24 h. Signals of cyclin A were normalized to α-tubulin. Phosphorylation status of CDK2 and pRb were determined by normalizing phosphorylated forms to total CDK2 proteins and unphosphorylated forms of pRb, respectively. Intensities of bands were densitometrically analyzed. Data represent means of three to five independent experiments. Cyclin A, *, p = 0.031 compared to control; p-CDK2, **, p = 0.002 compared to control; p-pRB, **, p = 0.002 compared to control.
(B) Time course of cyclin A2 mRNA levels in AF5 cells treated with 10 and 100 μM cocaine, as measured by quantitative real-time RT-PCR. The expression of cyclin A2 was expressed as fold changes in relationship to the control values. For 10 μM cocaine, 6 h, ***, p < 0.001 compared to control; 12 h, *, p = 0.012 compared to control; 24 h, **, p = 0.004 compared to control. For 100 μM cocaine, ***, p < 0.001 compared to each control. n = 3–7.
(C) Time course of cyclin A protein levels in AF5 cells treated with 10 and 100 μM cocaine. The expression of cyclin A was normalized to α-tubulin and expressed as ratios to the control values. For 10 μM cocaine, 12 h, **, p = 0.007 compared to control; 24 h, *, p = 0.012 compared to control. For 100 μM cocaine, 12 h, **, p = 0.003 compared to control; 24 h, **, p = 0.002 compared to control. n = 3–6.
(D) Effect of cyclin A overexpression on cocaine-induced proliferation inhibition. Cyclin A protein levels and cell proliferation were measured 24 h after electroporation of the CMV-Cyc A vector and 100 μM cocaine treatment. Cyclin A: cells transfected with pRc/CMV-CycA. Control: cells transfected with the empty vector. Data are presented as percentage of control cell numbers at 0 h. ***, p < 0.001 compared to control; +++, p < 0.001 compared to treatment with cocaine only. n = 8.