Cocaine Inhibits Cell Proliferation without Affecting Cell Survival
Log-phase cultures of AF5 cells were treated with varying concentrations of cocaine (1–100 μM) for 24 h. The number of cells in each condition was then measured and compared to the number at the initiation of the treatment. As shown in A, the number of AF5 cells in untreated cultures doubled in 24 h, while numbers of cells in cocaine-treated cultures were significantly reduced in a dose-dependent manner as compared to controls. When exposure of AF5 cells to 100 μM cocaine was extended to 6 d, the inhibitory effect was even more pronounced (Figure S2
). Because cocaine substantially inhibited proliferation of AF5 cells after only 24 h exposure, we employed this exposure duration for subsequent experiments.
The effect of cocaine was not due to an increase in cell death, as we did not observe a change in either extracellular LDH activity (B) or numbers of apoptotic nuclei immunoreactive for single-strand DNA (C). We also did not observe any morphological changes indicative of necrosis or apoptosis in cocaine-treated AF5 cells. Therefore, as has been reported for several other cell types [11
], cocaine inhibits proliferation of AF5 neural progenitor cells.
Cocaine Interferes with the G1/S Transition
We next examined the cell cycle distribution of cocaine-treated cells by FACS (D). Cocaine resulted in a dose-dependent increase in cells in G1 phase and a reduction in the number of cells in S phase, indicating that cocaine suppresses the G1-to-S phase transition (D). To further clarify the transition suppression, cell populations going through S phase were monitored by BrdU incorporation over 24 h. Both 10 μM and 100 μM cocaine significantly decreased the percentage of BrdU-positive cells that had entered S phase, supporting the notion that cocaine interferes with the G1/S cell cycle transition (E). The percentage of mitotic cells was low (<3%) as measured by phospho-histone H3 immunocytochemistry, and was not affected by cocaine (E).
Microarray Screening Identifies Cyclin A2 as a G1/S Phase Transition Controller Affected by Cocaine
To identify molecules that could mediate the cocaine-induced G1/S transition impairment, we used a microarray that contains 93 cell cycle-related genes including 16 G1/S phase transition controllers (cyclin A2, C, D1, D2, D3, E1, E2, G1, CDK2, p12CDK2-AP1, CDK4, p27Kip1, p57Kip2, p18INK4c, PITSLRE/CDK11p58, and p53). Of these, cocaine (10 and 100 μM, 24 h) significantly down-regulated only cyclin A2 (A). The decrease in cyclin A2 expression by 10 or 100 μM cocaine was confirmed by quantitative real-time RT-PCR (B).
Microarray and Quantitative Real-Time RT-PCR Analyses of Cocaine-Treated AF5 Cells
To characterize the time course of cocaine-induced down-regulation of cyclin A, we treated AF5 cells with 100 μM cocaine for 6 d and found that the cyclin A protein level, as measured by Western blotting, had started to decrease by day 1, continued to decline at day 3, and finally resulted in an undetectable amount of cyclin A protein by day 6 (Figure S3
). These data show that cocaine-induced inhibition of AF5 cell proliferation is correlated with down-regulation of cyclin A.
Cocaine Down-Regulates Cyclin A2 Expression in Primary Human Fetal CNS Progenitor Cells in Culture
To determine whether our findings in AF5 cells are relevant to primary cells, we measured cyclin A2 mRNA in several types of primary human fetal CNS cells obtained from ~20-wk (second trimester) human fetal cerebral cortexes. As measured by quantitative real-time RT-PCR, in vitro cocaine exposure (100 μM, 24 h) significantly decreased cyclin A2 mRNA level in both human neural progenitor cells (>95% of cells are Nestin-positive and A2B5-negative, unpublished data) and A2B5+ progenitor cells (>90% of cells are A2B5-positive and Nestin-positive, unpublished data), whereas cyclin A2 mRNA was not altered in neurons or microglia (A). In contrast, cocaine increased the cyclin A2 transcript in human astrocytes (A).
Expression of Cyclin A in Primary Human Fetal CNS Cells Treated with Cocaine
The cyclin A protein level was also significantly decreased by cocaine (10 and 100 μM, 24 h) in both human neural and A2B5+ progenitor cells (B and C). Cocaine caused a maximum down-regulation of cyclin A protein level at 10 μM in human neural progenitor cells (B), but decreased expression of cyclin A in a dose-dependent manner in A2B5+ progenitor cells (C). These data verify that cocaine down-regulates cyclin A in both types of progenitor cells derived from human fetal cerebral cortexes.
Cocaine Down-Regulates Cyclin A2 Expression and Inhibits Cell Cycle Progression in Fetal Rat Brains
We next examined cyclin A expression in fetal rat brains exposed to cocaine in utero. Neocortical neurogenesis occurs within two proliferative strata of the embryonic cerebral wall, which is adjacent to the ventricle. Neocortical neurogenesis starts at E12 and ends at E19 in the rat [29
]. During this process, the VZ appears first and is followed by the SVZ. To investigate the time-frame within which neocortical development is susceptible to prenatal cocaine exposure, we examined three different time periods: the early neurogenesis period from E13 to E15, the middle period of neurogenesis from E15 to E17, and the late neurogenesis period from E17 to E19. Pregnant animals received cocaine according to the injection schedule shown in A, and the frontal cortex of developing fetuses was dissected as shown in B.
Effects of Cocaine on Cyclin A Expression in the Developing Rat Neocortex
To examine cocaine concentrations in the fetal neocortex under our injection schedule and for comparison to our in vitro studies, tissue concentrations of cocaine were analyzed at the early period of neurogenesis after the second cocaine administration. Cocaine concentrations in fetal neocortex reached at least 30 μM (9,812 ng/g) 0.5 h after injection, dropped to ~2 μM (median value of 594 ng/g) at 1 h, and gradually declined to ~0.2 μM (median value of 63 ng/g) at 6 h (C). Thus, the injection schedule used produced exposure of fetal brains to maximum concentrations of cocaine within the range of doses that we found to inhibit proliferation of progenitor cells in vitro.
Down-regulation of cyclin A2
mRNA was seen in prefrontal cortex of the developing fetuses when cocaine was injected at either early or middle periods of neurogenesis (D and S4
A). Cyclin A2
mRNA was not, however, changed by injections during the late period of neurogenesis (D and S4
A). Similarly, cyclin A protein was also significantly decreased by cocaine in both early and middle neurogenesis periods, but not during the late period of neurogenesis (E and S4
B). This result suggests that cocaine down-regulates cyclin A expression only during earlier periods of neocortical neurogenesis that involve active proliferation of neural progenitor cells in the VZ.
Since cocaine causes down-regulation of cyclin A in fetal brains, we also examined cell cycle progression of neural progenitors in the VZ and SVZ in fetal brains in utero exposed to cocaine. Pulse labeling with BrdU was used to quantify cortical progenitor cells that had entered S phase during a period of 2 h, whereas Ki67 immunocytochemistry was used to monitor the total fraction of progenitors that are in any phase of the cell cycle except for G0
]. Cocaine decreased the number of BrdU-labeled progenitor cells in the VZ during both the early and middle neurogenesis periods (F, G, S4
C, and S5
), suggesting that cocaine inhibits the proliferation of progenitor cells in the VZ.
In contrast to the findings in the VZ, BrdU positive progenitor cells in the SVZ were not changed by cocaine (F, H, S4
D, and S5
). Also, since cortical germinal zones in the late period of neurogenesis comprise SVZ only, cocaine did not change BrdU labeling during this period (H, S4
D, and S5
). Taken together, these data suggest that cocaine promotes cyclin A down-regulation and cell proliferation inhibition both in vitro and in vivo.
Cocaine Inhibits Cyclin A2 Downstream Cell Cycle Effectors: Decreased Phosphorylated CDK2 and pRb
Western blotting confirmed that cocaine (100 μM, 24 h) decreases cyclin A2 protein in AF5 cells (A). Cyclin A induces a conformational change in CDK2, and thereby permits activation of CDK2 through phosphorylation of Thr-160 catalyzed by CDK-activating kinase (CAK) [31
]. Activated CDK2 phosphorylates the downstream molecule pRb, which in turn promotes the expression of genes required for progression from G1 to S. Western blotting showed that cocaine significantly decreased the phosphorylated forms of CDK2 and pRb (A), indicating that cocaine-induced down-regulation of cyclin A results in a hypoactive state of the downstream cell cycle signaling pathway.
Role of Cyclin A Expression in Cocaine-Induced Proliferation Inhibition
Reversal of Cocaine-Induced Proliferation Inhibition by Cyclin A Transfection
To demonstrate a causal relationship between cyclin A down-regulation and cocaine-induced inhibition of AF5 cell proliferation, we attempted to compensate for cyclin A down-regulation by gene transfer using an expression vector encoding cyclin A (pRc/CMV-CycA).
To examine the appropriate timing of transfection, we first examined the time course of cyclin A expression after either cocaine treatment or vector transfection. Quantitative real-time RT-PCR showed that cyclin A2 mRNA was decreased as early as 6 h after 10 or 100 μM cocaine, and the down-regulation lasted up to 24 h (B). Cyclin A protein was significantly decreased 12 h after 10 or 100 μM cocaine with nearly maximum effects (64%) at 12 h for 10 μM cocaine, and a 48% decrease at 24 h for 100 μM cocaine (C). Since pRc/CMV-CycA transfection (with ~80% transfection efficiency) also showed the maximum level of overexpression at 24 h post-transfection (unpublished data), we exposed cells to 100 μM cocaine immediately after electroporation of the CMV-Cyc A vector, and measured cyclin A protein and cell proliferation 24 h later. As shown in D, cyclin A transfection counteracted the cocaine-induced down-regulation of cyclin A, as well as the inhibition of proliferation caused by cocaine. These data suggest that cocaine-induced down-regulation of cyclin A contributes to the proliferation inhibition seen in cocaine-exposed AF5 cells.
Identification of the eIF2α-ATF4 Pathway in Transcriptional Down-Regulation of Cyclin A by Cocaine
We next examined the levels of several transcription factors involved in regulation of the cyclin A2
promoter activity. Western blot analysis showed that cocaine (100 μM, 3 h) significantly up-regulated only ATF4 among a total of nine candidate transcription factors tested, including ATF1–4, CREB, JunB, JunD, c-Jun, and c-Fos (A). The phosphorylation status of CREB, which is involved in activation of cyclin A
transcription at G1/S [32
], was not affected by 100 μM cocaine at 3 h (A). In addition, cocaine did not change levels of p21 and p27, CDK inhibitors that decrease transcription of cyclin A2
Cocaine-Induced Cyclin A Down-Regulation and Proliferation Inhibition Involves Oxidative ER Stress Signaling
An increase in ATF4 protein occurred as early as 1 h after 100 μM cocaine exposure, and reached the maximal level at 3 h (B). Cocaine at concentrations higher than 1 μM resulted in dose-dependent induction of ATF4 (B). The phosphorylated form of the alpha subunit of translation initiation factor 2 (eIF2α) is known to promote ATF4 induction [35
]. Indeed, we found that 10 μM cocaine significantly increased the phosphorylated form of eIF2α 0.5 h after exposure to cocaine, preceding induction of ATF4 (C).
Role of N-Oxidative Metabolism of Cocaine in Generation of ROS, Endoplasmic Reticulum Stress, Induction of ATF4, Inhibition of Cyclin A, and Cell Proliferation
The eIF2α-ATF4 pathway is activated by PERK, an endoplasmic reticulum (ER) stress sensor protein [35
]. The ER stress pathway has been shown to be activated by a variety of chemicals or pathological stress including oxidative ER stress [35
]. Notably it has been demonstrated that the cytochrome P450 dependent N-oxidative pathway is responsible for generation of ROS and glutathione (GSH) depletion during cocaine biotransformation in the liver [37
]. We therefore hypothesized that ROS generation caused by the N-oxidative metabolism of cocaine may trigger activation of the eIF2α-ATF4 pathway.
Accordingly, we first examined whether cocaine induces ROS production in progenitor cells. As shown in D, 10 μM cocaine caused a significant increase in ROS 30 min after treatment. Cocaine at 100 μM caused ROS generation even earlier, with a significant increase 15 min after the exposure.
Because the rise of cocaine concentrations over 10 μM lasts for less than 1 h in fetal brains after cocaine injection (C), AF5 cells were next treated with 30 μM cocaine for 30 min followed by incubation in cocaine-free medium for 2.5 h to examine ATF4 protein levels. We found that ATF4 was significantly up-regulated by this transient 30 min exposure to cocaine (Figure S6
). Thus, exposure to cocaine for a period of only 30 min can generate ROS sufficient to initiate ER stress, and result in up-regulation of ATF4 2.5 h later.
Pretreatment with the cytochrome P450 inhibitors SKF-525A or cimetidine, drugs that have been shown to potently block N-oxidative metabolism of cocaine, completely blocked cocaine-induced ROS formation (E), confirming that N-oxidative metabolism of cocaine is involved in ROS formation in AF5 cells. Further, ROS generation appears to be the source of cocaine-induced ER stress, as both SKF-525A and cimetidine also completely inhibited cocaine-induced ATF4 up-regulation and cyclin A down-regulation (F and G).
Finally, we tested effects of SKF-525A and cimetidine on the inhibition of cell proliferation by cocaine. Both drugs significantly diminished cocaine-induced proliferation inhibition (100 μM for 24 h) (H). On the other hand, the lipophilic free radical scavengers α-tocopherol and BHA and the iron chelator DFO (each 50 μM) did not prevent cocaine-induced proliferation inhibition (unpublished data). These data indicate that cytochrome P450-dependent ROS formation occurring during cocaine metabolism is responsible for both cocaine-induced proliferation inhibition and cyclin A down-regulation.
Cimetidine Reverses Effects of Cocaine In Utero in a Rat Model
To determine whether P450 inhibitors can block cocaine-induced proliferation inhibition in neural progenitor cells in the developing neocortex, pregnant rats at the early period of neurogenesis (E13–E15) were pretreated with 100 mg/kg cimetidine IP 1 h before each cocaine administration. Cimetidine is known to cross the placenta [38
]. Cimetidine itself did not affect neural progenitor cell proliferation, survival, density, or fetal mortality (A, B, S7
B, and S8
E). As shown in A and S7A, pretreatment of pregnant rats with cimetidine resulted in recovery of the cocaine-induced decease in BrdU-positive progenitor cells in the VZ. As expected, no differences in BrdU-positive progenitor cells were observed in the SVZ (B and S7
To determine whether the protection afforded by cimetidine was due to the recovery of cocaine-induced down-regulation of cyclin A and mediation by ER stress, the effects of cimetidine on expression of ATF4 and cyclin A were also measured in prefrontal cortex of cocaine-treated fetuses. Pretreatment of pregnant rats with cimetidine significantly inhibited the cocaine-induced up-regulation of ATF4 and the down-regulation of cyclin A (C, D, S7
D). These results suggest that blockade of cocaine N-oxidative metabolism by the P450 inhibitor cimetidine reverses cocaine-induced proliferation inhibition of neural progenitor cells in the VZ through normalizing cocaine-induced oxidative ER stress and consequent cyclin A down-regulation.