A Mechanism for the Inhibition
of Neural Progenitor Cell Proliferation
Chun-Ting Lee1*, Jia Chen1, Teruo Hayashi1, Shang-Yi Tsai1, Joseph F. Sanchez1, Stacie L. Errico1, Rose Amable1,
Tsung-Ping Su1, Ross H. Lowe2, Marilyn A. Huestis2, James Shen3, Kevin G. Becker4, Herbert M. Geller5,
William J. Freed1
1 Cellular Neurobiology Research Branch, Intramural Research Program (IRP), National Institute on Drug Abuse (NIDA), National Institutes of Health (NIH), Department of
Health and Human Services (DHHS), Baltimore, Maryland, United States of America, 2 Chemistry and Drug Metabolism, IRP, NIDA, NIH, DHHS, Baltimore, Maryland, United
States of America, 3 ScienCell Research Laboratories, San Diego, California, United States of America, 4 Gene Expression and Genomics Unit, Research Resources Branch,
National Institute on Aging (NIA), NIH, DHHS, Baltimore, Maryland, United States of America, 5 Developmental Neurobiology Section, Cell Biology and Physiology Center,
Division of Intramural Research, National Heart, Lung and Blood Institute (NHLBI), NIH, DHHS, Bethesda, Maryland, United States of America
Funding: Research supported by the
IRPs of NIDA, NIA, and NHLBI, NIH,
DHHS. The funding organizations
had no role in the study design,
collection, analysis, interpretation of
data, writing of the paper, or
decision to submit it for publication.
Competing Interests: US
Provisional Application Number 60/
893,218 filed March 6, 2007:
‘‘Cytochrome P450 Inhibitors for
Treatment of Cocaine-Induced Fetal
Brain Injury,’’ C-TL and WJF.
Academic Editor: Manuel Graeber,
Imperial College London, United
Citation: Lee C-T, Chen J, Hayashi T,
Tsai S-Y, Sanchez JF, et al. (2008) A
mechanism responsible for the
inhibition of neural progenitor cell
proliferation by cocaine. PLoS Med
5(6): e117. doi:10.1371/journal.
Received: July 6, 2007
Accepted: April 16, 2008
Published: June 10, 2008
Copyright: ? 2008 Lee et al. This is
an open-access article distributed
under the terms of the Creative
Commons Public Domain
declaration which stipulates that,
once placed in the public domain,
this work may be freely reproduced,
distributed, transmitted, modified,
built upon, or otherwise used by
anyone for any lawful purpose.
Abbreviations: BrdU, 5-bromo-29-
deoxyuridine; CNS, central nervous
system; ER, endoplasmic reticulum;
IP, intraperitoneally; LDH, lactate
dehydrogenase; ROS, reactive
oxygen species; RT-PCR, reverse
transcription PCR; SEM, standard
error of the mean; SVZ,
subventricular zone; VZ, ventricular
* To whom correspondence should
be addressed. E-mail: firstname.lastname@example.org.
A B S T R A C T
Prenatal exposure of the developing brain to cocaine causes morphological and behavioral
abnormalities. Recent studies indicate that cocaine-induced proliferation inhibition and/or
apoptosis in neural progenitor cells may play a pivotal role in causing these abnormalities. To
understand the molecular mechanism through which cocaine inhibits cell proliferation in
neural progenitors, we sought to identify the molecules that are responsible for mediating the
effect of cocaine on cell cycle regulation.
Methods and Findings
Microarray analysis followed by quantitative real-time reverse transcription PCR was used to
screen cocaine-responsive and cell cycle-related genes in a neural progenitor cell line where
cocaine exposure caused a robust anti-proliferative effect by interfering with the G1-to-S
transition. Cyclin A2, among genes related to the G1-to-S cell cycle transition, was most strongly
down-regulated by cocaine. Down-regulation of cyclin A was also found in cocaine-treated
human primary neural and A2B5þ progenitor cells, as well as in rat fetal brains exposed to
cocaine in utero. Reversing cyclin A down-regulation by gene transfer counteracted the
proliferation inhibition caused by cocaine. Further, we found that cocaine-induced
accumulation of reactive oxygen species, which involves N-oxidation of cocaine via cytochrome
P450, promotes cyclin A down-regulation by causing an endoplasmic reticulum (ER) stress
response, as indicated by increased phosphorylation of eIF2a and expression of ATF4. In the
developing rat brain, the P450 inhibitor cimetidine counteracted cocaine-induced inhibition of
neural progenitor cell proliferation as well as down-regulation of cyclin A.
Our results demonstrate that down-regulation of cyclin A underlies cocaine-induced
proliferation inhibition in neural progenitors. The down-regulation of cyclin A is initiated by N-
oxidative metabolism of cocaine and consequent ER stress. Inhibition of cocaine N-oxidative
metabolism by P450 inhibitors may provide a preventive strategy for counteracting the adverse
effects of cocaine on fetal brain development.
The Editors’ Summary of this article follows the references.
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P PL Lo oS S MEDICINE
Abuse of cocaine during pregnancy exposes several
hundred thousand infants per year to cocaine in the United
States alone . A variety of disorders of central nervous
system (CNS) development, e.g., intrauterine growth retarda-
tion , interference with neuronal migration and differ-
entiation , and neurobehavioral deficits [4,5], have been
associated with prenatal exposure to cocaine. Adverse effects
of cocaine on brain development have also been demon-
strated in nonhuman primates. Prenatal cocaine exposure
results in neurobehavioral deficits in subhuman primate
infants or adolescents, including deficits in attention and
motor maturity . At the cellular level, cocaine exposure
induces neocortical cytoarchitectural abnormalities includ-
ing a decrease in the number of cortical neurons and
abnormal positioning of cortical neurons in the primate
embryonic cerebral wall [7,8]. Notably, these abnormalities
are found only when cocaine is administered during the
second trimester (E40–E102), the period when proliferation
of neural progenitors is most active . The specific actions of
cocaine in the second trimester and the decrease of neuron
numbers in the cortex suggest that cocaine may affect
important cellular functions of neural progenitor cells.
In vitro, cocaine has been shown to influence several cell
biological functions such as cell survival and mitogenesis
independent of its action on monoaminergic systems. One in
vitro study showed that a single 30-min exposure to 1 lM
cocaine results in late-onset (.72 h) cell death in differ-
entiated human neuronal progenitor cells . On the other
hand, accumulating evidence highlights an inhibitory effect
of cocaine on neural progenitor cell proliferation. Cocaine
(1–100 lM, 7 d) was shown in an in vitro study to inhibit the
proliferation of human neural precursor cells without
producing a cytotoxic effect . Cocaine has also been
shown to cause genetic toxicity and disturbances in chromo-
some segregation during meiosis [12,13]. These findings
suggest that cocaine may influence cell cycle control. Because
the proliferation of neural progenitors is an important factor
that eventually contributes to determining numbers of
neurons and brain cytoarchitecture, clarifying the action of
cocaine on cell cycle control might provide an avenue for
understanding the mechanisms underlying cocaine-induced
retardation of brain development.
The aim of the present study is to clarify the effect of
cocaine on proliferation of neural progenitors and elucidate
the underlying molecular mechanisms. Both human and
animal studies have demonstrated that cocaine can cross the
placental barrier and enter the fetal brain rapidly after
maternal cocaine use [14,15]. Plasma cocaine concentrations
after intranasal application of 1.5 mg/kg cocaine in human
volunteers were between 0.4 and 1.6 lM , while plasma
cocaine concentrations are often considerably higher in
tolerant abusers, reaching ;13 lM . A previous study
found that concentrations of cocaine in maternal rat brain
are higher (3- to 4-fold) than in plasma , and cocaine
concentrations in fetal brain are 50%–90% of those found in
the maternal brain , indicating that the high range of
cocaine concentrations in the fetal brain may reach ;20–47
lM. Moreover, cocaine concentrations up to 100 lM and
higher have been reported in postmortem brains of chronic
human cocaine users after acute intoxication . Based on
these reports, we considered the cocaine dose range from 1 to
100 lM to be comparable to the range of exposure of the fetal
brain to cocaine. Therefore, we employed cocaine in this
concentration range to investigate its effects on neural
progenitor cell proliferation.
Materials and Methods
Cocaine hydrochloride was provided by the National
Institute on Drug Abuse. SKF-525A, cimetidine, a-tocopherol,
3(2)-tert-butyl-4-hydroxyanisole (BHA), and deferoxamine
(DFO) were obtained from Sigma-Aldrich.
The AF5 neural progenitor cell line was maintained as
previously described . The AF5 cell line is homogenous
and over long-term culture maintains growth stability ,
differentiation capacity [19,20], and an intact p53 function
[19,21]. Notably, the total length of the cell cycle of AF5 cells
(24 h) is close to that in the rodent ventricular zone (VZ) (18 h
at E15 or later) . 5 3 103cells/well were plated in 96-well
plates for cell proliferation and cytotoxicity assays, and 43104
cells/well in 12-well plates for immunostaining and reactive
oxygen species (ROS) measurement 24 h prior to use. Primary
human fetal CNS cells (ScienCell Research Laboratories) were
from ;20-wk human fetal cerebral cortexes, obtained in
accordance with principles embodied in the Declaration of
Helsinki (Code of Ethics of the World Medical Association)
and were cultured at 37 8C, 5% CO2using the recommended
human cell media obtained from ScienCell Research Labo-
ratories, except that human neural progenitor cells were
maintained in DMEM/F12 (1:1, Invitrogen) supplemented with
N2 supplement (R & D Systems), 20 ng/ml EGF (R & D
Systems), 20 ng/ml bFGF (R & D Systems), 5 lg/ml heparin
(Sigma-Aldrich), 100 U/ml penicillin G, 100 lg/ml streptomy-
cin, and 50 lg/ml gentamicin (Sigma-Aldrich). Purity of
respective types of CNS cells was evaluated by immunocyto-
chemistry using antibodies against specific cell markers. For
evaluation of neural progenitors, neurospheres were first
dissociated into single cells using papain (Worthington
Biochemical Corporation) for 10 min at 37 8C, and plated
onto laminin/poly-L-ornithine-coated slides. Once attached,
cells were fixed with 4% paraformaldehyde and evaluated by
immunocytochemistry. A2B5 progenitor cells differ from
neural progenitor cells in that they are committed to a glial
cell lineage, eventually differentiating to type-2 astrocytes or
oligodendrocytes, whereas neural progenitor cells can become
both neurons and glial cells. The human neurons used in this
study were MAP-2 positive, and immunoreactive for glutamate
(44% 6 3%) and GABA (69% 6 5%), but not for tyrosine
hydroxylase (TH), choline acetyltransferase (ChAT), or 5-
hydroxytryptamine (5-HT) (unpublished data).
Cell Proliferation and Cytotoxicity Assays
AF5 cells were treated with cocaine at various concen-
trations, and cell proliferation was measured using CyQUANT
cell proliferation assay (Invitrogen). Cocaine-induced cyto-
toxicity was evaluated by lactate dehydrogenase (LDH) release
from the cytosol into the medium after exposure of AF5 cells
to various concentrations of cocaine for 24 h, according to the
manufacturer’s protocol (Roche Applied Science). For single-
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Cocaine and Neural Progenitor Proliferation
stranded DNA immunostaining, cells were fixed with meth-
anol/PBS (6:1) for 24 h at ?20 8C followed by incubation in
formamide at 70 8C for 5 min. Fixed cells were immunos-
tained with mouse anti-single-stranded DNA monoclonal
antibodies (1:10, Chemicon) and fluorescein-conjugated anti-
mouse IgM (1:200, Jackson Immunoresearch). Data were
represented as: (number of single-stranded DNA-positive
nuclei/number of DAPI-positive total nuclei) 3 100%.
Cultures were synchronized by maintaining in serum-free
medium for 24 h, followed by exposure to 0, 10, or 100 lM
cocaine in serum-containing medium for 24 h. AF5 cells were
analyzed by flow cytometry on a FACS Calibur flow cytometer
(Becton Dickinson). Proportions of cells in the G1, S, and G2/
M phases of the cell cycle were determined by using ModFit
LT software (Verity Software House).
5-Bromo-29-Deoxyuridine Incorporation and Mitosis Assay
AF5 cultures were treated with 20 lM 5-bromo-29-
deoxyuridine (BrdU) (BD Biosciences) in the presence/
absence of cocaine for 24 h, fixed with 95% ethanol, and
permeabilized with 2 N HCl. Nonspecific staining was blocked
with 5% normal goat serum and 0.1% Nonidet P-40 in PBS
for 20 min at room temperature. Cells were double-labeled
with monoclonal mouse anti-BrdU (1:100, BD Biosciences)
and polyclonal rabbit anti-phospho-histone H3 (1:200,
Upstate Biotechnology) overnight at 4 8C. After washing,
secondary antibodies Alexa Flour 594 goat anti-mouse and
Alexa Flour 488 goat anti-rabbit (1:500, Invitrogen) were
applied, and nuclei were labeled with DAPI. Data were
obtained by dividing numbers of nuclei positive for BrdU or
phospho-histone H3 by total numbers of nuclei.
Total RNA was extracted from AF5 cultures using RNA
STAT-60 (TEL-TEST). cDNA microarray analysis was per-
formed using a mouse developmental cDNA microarray
containing 15k clones derived from early Kargul libraries
using procedures for processing as previously described .
z-Score transformation was employed to compare array data
between different treatments . z-Score transformation
allows analysis of array data independent of the original pixel
intensities and can be used in calculation of p-values for
significance estimates. To calculate gene expression changes
after cocaine treatment, z scores were converted to z ratios,
which represent fold-like changes for each gene.
Primary human fetal CNS cells were fixed in 4%
paraformaldehyde in PBS for 10 min and processed for
immunostaining . The following primary antibodies were
used: rabbit anti-nestin (1:200, Chemicon); mouse anti-A2B5
(supernatant, clone 105, 1:3, ATCC); mouse anti-MAP2 (1:500,
BD Biosciences); mouse anti-OX42 (1:200, BioLegend); and
mouse anti-GFAP (1:200, Sigma-Aldrich). Cells were devel-
oped using Alexa Fluor 488 goat anti-rabbit or mouse
secondary antibody (1:500, Invitrogen), and nuclei were
labeled with DAPI.
Quantitative Real-Time Reverse Transcription-PCR
Reverse transcription was performed as described previ-
ously . To quantify the cyclin A2 transcript, quantitative
real-time reverse transcription (RT)-PCR using the DNA
Engine Opticon Fluorescence Detection System (MJ Re-
search) was performed using SYBR green according to the
manufacturer’s protocol. The primer sequences and sizes of
the PCR products for rat cyclin A2 were ATATGAAGAGG-
CAGCCAGACA (sense), AGGCAGCTCCAGCAATAAGCG
(antisense), 483 bp; and for human cyclin A2 were GCAAA-
CAGTAAACAGCCTGCG (sense), TCAACTAACCAGTCCAC-
GAGG (antisense), 386 bp. The results were analyzed using
Opticon software. Relative expression was determined by
normalizing to 18S ribosomal RNA (Ambion) using 1.0 for the
Animals and Experimental Design
Pregnant Sprague-Dawley rats (Charles River Laboratories)
received cocaine at early (E13 and E14), middle (E15 and E16),
or late periods (E17 and E18) of neocortical neurogenesis.
Rats received 20 mg/kg cocaine (intraperitoneally [IP]) twice
at an interval of 12 h followed by 50 mg/kg BrdU (Sigma-
Aldrich), IP, 24 h after the last injection of cocaine. Rats were
euthanized by CO2inhalation 2 h after BrdU. Control animals
received physiological saline. All animal procedures were
performed according to the ‘‘Guide for the Care and Use of
Laboratory Animals,’’ according to an animal protocol
approved by the Institutional Animal Care and Use Commit-
tee of the NIDA Intramural Research Program.
Quantitation of Cocaine in the Fetal Rat Brain
For measurements of cocaine concentrations, prefrontal
cortex and peri-ventricular region were dissected from fetal
rat brains at the early period of neurogenesis (E15). Tissues
from all fetuses of each pregnant dam were pooled to become
one individual sample. Detection and quantification of
cocaine in fetal rat brain was accomplished utilizing a
modification of a previously published method . The
method employed ultrasonic homogenization of brain tissue
in pH 4.0 sodium acetate buffer followed by solid phase
extraction. Extracts were derivatized with N,O-bis-[trimethyl-
silyl]trifluoroacetamide (BSTFA). Cocaine was separated by
capillary gas chromatography and simultaneously quantified
by electron impact mass spectrometry in selected ion mode.
The calibration curve for cocaine was linear to 15,000 ng/g of
brain, and the limit of quantification was 50 ng/g. This
method provided sufficient analytical sensitivity to allow
quantification of cocaine in small amounts of tissue.
Cyclin A Expression and BrdU Labeling In Vivo
For cyclin A expression studies, tissues (prefrontal cortex
and peri-ventricular region) from fetal rat brain were
dissected. Tissues from three fetal rats were pooled for each
individual assay to obtain sufficient material. RNA and
proteins were extracted with RNA STAT-60 (TEL-TEST)
and lysis buffer, respectively. For BrdU labeling, coronal
brain sections were labeled with monoclonal mouse anti-
BrdU (1:200, BD Biosciences) and polyclonal rabbit anti-Ki67
(1:500, Novocastra Laboratories) overnight at 4 8C and
visualized using Alexa Flour 594 goat anti-mouse and Alexa
Flour 488 goat anti-rabbit antibodies (Invitrogen). BrdU
labeling index [(number of BrdU-positive nuclei/number of
Ki67-positive nuclei) 3 100%] was calculated in the regions
with 160 lm width (red rectangles in Figure S1) that locate on
one-third and two-thirds of the cerebral cortex defined by
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Cocaine and Neural Progenitor Proliferation
arrowheads at the superior sagittal sinus (SSS) and the causal
pole of the internal capsule in Figure S1. Six brain sections
from both brain hemispheres were examined in each animal.
The outer half of the VZ (the half farthest from the ventricle)
contains a layer of closely packed cells positive for Ki67 and
BrdU S-phase incorporation. The outer edge of this
epithelium-like layer was used to define the boundary
between VZ and subventricular zone (SVZ).
Western Blot Analysis
Western blotting was performed as previously described
 using antibodies to CDK2 and pRb (BD Biosciences), a-
tubulin (Sigma-Aldrich), phospho-CDK2 (Thr-160), phospho-
eIF2a (Ser-51), and eIF2a (Cell Signaling), cyclin A, ATF1,
ATF2, ATF3, ATF4, phospho-CREB (Ser-133), CREB, JunB,
JunD, c-Jun, c-Fos, p21, and p27 (Santa Cruz Biotechnology).
Immunoreactive bands were densitometrically quantitated
using Kodak Image Station 440 CF.
AF5 cells were transfected with a plasmid encoding cyclin A
(pRc/CMV-CycA; gift of P. Hinds, Harvard Medical School,
Boston, Massachusetts, United States of America ) or a
control plasmid (pcDNA3.1; Invitrogen). Briefly, one million
AF5 cells in suspension (100 ll) were mixed with 2 lg of either
plasmid, and electroporation was performed using nucleo-
fection protocol T-20 (Amaxa Biosystems). Immediately after
nucleofection, cells were plated in six-well plates for the
Western blot analysis and 96-well plates for the CyQUANT
cell proliferation assay.
Analysis of Endogenous ROS Formation
Endogenous ROS were measured by incubating AF5 cells
with 100 lM 29, 79-dichlorofluorescein diacetate (DCFH-DA)
(Sigma-Aldrich) during the last 20 min of indicated treat-
ments. The treated AF5 cells were washed, dissolved with 1%
Triton X-100 in PBS, and fluorescence was measured at an
excitation wavelength of 485 nm, and an emission wavelength
of 530 nm using a fluorescence microplate reader.
All values were expressed as means 6 standard error of the
mean (SEM). Mean values were compared using the Student’s
t-test for two-sample comparisons, or analysis of variance
(ANOVA) followed by Newman-Keuls tests for multiple
comparisons as indicated in the legends for Figures 1–7, S2,
and S6. The criterion for statistical significance was p , 0.05.
It should be noted that this study includes a large number of
statistical tests; thus, it is possible that some differences, even
though statistically significant, are due to chance. For all
differences found to be statistically significant by Student’s t-
tests, the differences were additionally assessed by non-
parametric Mann-Whitney U tests, the results of which are
listed in Table S1. p-Values obtained by both tests were
similar. Also, as compared to the experiments performed in
vitro, the variability between animals was relatively large;
Figure 1. Cocaine-Induced Proliferation Inhibition in AF5 Cells
(A) AF5 cells were treated with cocaine at concentrations from 1–100 lM for 24 h. Inset: dose-dependent inhibition of cell proliferation by cocaine (24
h). Data are presented as means 6 SEM of four replicates from ten separate experiments. *, p ¼ 0.010; ***, p , 0.001 compared to control.
(B) LDH activity in the medium from cocaine-treated AF5 cells (24 h). Cytotoxicity was expressed as a percentage of the maximum LDH activity (LDH
released by 2% Triton X-100). Data represent six separate experiments (means 6 SEM of triplicate observations).
(C) Effect of cocaine (24 h) on single-stranded DNA (ssDNA) immunostaining (red). DAPI in blue. Data are presented as percentage of ssDNA positive
cells from 15 fields of three wells in each group (.450 cells/group). Scale bar ¼ 10 lm.
(D) Cell cycle distribution measured by FACS in AF5 cells treated with cocaine (24 h). Note changes in the scales in y-axis. A graph shows means 6 SEM
from six independent experiments. **, p ¼ 0.004; ***, p , 0.001 as compared to each control.
(E) BrdU-positive (red) and mitotic (green staining, phospho-histone H3-positive) cells in cocaine-treated AF5 cultures (24 h with BrdU). Cells that
coexpress BrdU and phospho-histone H3 appear yellow, DAPI in blue. Scale bar¼10 lm. Sixteen fields from four wells were examined for quantification
(.1,000 cells/group). *, p ¼ 0.013; **, p ¼ 0.004 compared to control.
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Cocaine and Neural Progenitor Proliferation
therefore, scattergram plots showing the data for individual
animals are included as Figures S4 and S7.
Cocaine Inhibits Cell Proliferation without Affecting Cell
Log-phase cultures of AF5 cells were treated with varying
concentrations of cocaine (1–100 lM) 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
Figure 1A, 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 lM 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 (Figure 1B) or numbers of apoptotic nuclei
immunoreactive for single-strand DNA (Figure 1C). 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 [11,27,28],
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 (Figure 1D). 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 (Figure 1D). To
further clarify the transition suppression, cell populations
going through S phase were monitored by BrdU incorpo-
ration over 24 h. Both 10 lM and 100 lM 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 (Figure 1E).
The percentage of mitotic cells was low (,3%) as measured
by phospho-histone H3 immunocytochemistry, and was not
affected by cocaine (Figure 1E).
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
lM, 24 h) significantly down-regulated only cyclin A2 (Figure
2A). The decrease in cyclin A2 expression by 10 or 100 lM
cocaine was confirmed by quantitative real-time RT-PCR
To characterize the time course of cocaine-induced down-
regulation of cyclin A, we treated AF5 cells with 100 lM
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 lM, 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 A2B5þ progenitor cells (.90% of cells are A2B5-
positive and Nestin-positive, unpublished data), whereas cyclin
A2 mRNA was not altered in neurons or microglia (Figure
3A). In contrast, cocaine increased the cyclin A2 transcript in
human astrocytes (Figure 3A).
The cyclin A protein level was also significantly decreased
by cocaine (10 and 100 lM, 24 h) in both human neural and
A2B5þprogenitor cells (Figure 3B and 3C). Cocaine caused a
Figure 2. Microarray and Quantitative Real-Time RT-PCR Analyses of Cocaine-Treated AF5 Cells
(A) Microarry analysis of G1/S phase transition regulators after cocaine treatment (10 and 100 lM, 24 h). ***, p , 0.001 compared to control, n ¼ 4.
(B) Confirmation of changes in cyclin A2 expression (10 and 100 lM cocaine, 24 h) by quantitative real-time RT-PCR. Data are expressed as fold changes
in relationship to the control condition. **, p ¼ 0.004; ***, p , 0.001 compared to control, n ¼ 3–7.
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Cocaine and Neural Progenitor Proliferation
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Cocaine and Neural Progenitor Proliferation
maximum down-regulation of cyclin A protein level at 10 lM
in human neural progenitor cells (Figure 3B), but decreased
expression of cyclin A in a dose-dependent manner in A2B5þ
progenitor cells (Figure 3C). 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
Figure 3. Expression of Cyclin A in Primary Human Fetal CNS Cells Treated with Cocaine
(A) Five different types of human primary cells from cerebral cortex (8–21 d in vitro), characterized by the CNS cell type-specific markers including nestin
(green) for neural progenitor cells, A2B5 (green) for A2B5þprogenitor cells, MAP2 (green) for neurons, OX42 (green) for microglia, and GFAP (green) for
astrocytes, were treated with 100 lM cocaine for 24 h, and cyclin A2 was measured by quantitative real-time RT-PCR analysis. DAPI nuclear staining is
shown in blue. Human neural progenitor cells, *, p ¼ 0.039 compared to control; human A2B5þ progenitor cells, *, p ¼ 0.040 compared to control;
human astrocytes, **, p¼0.001 compared to control. n¼3–5, from three to five individual brains. All scale bars are 25 lm, except for morphology and
marker expression of A2B5þ progenitor cells scale bars are 15 and 35 lm, respectively.
(B–C) Western blot analysis of cyclin A in cocaine (24 h)-treated human neural and A2B5þprogenitor cells. The expression of cyclin A was normalized to
a-tubulin and expressed as ratios to the control values. For human neural progenitor cells, 10 lM cocaine, *, p ¼ 0.013 compared to control; 100 lM
cocaine, *, p¼0.027 compared to control. For human A2B5þprogenitor cells, 10 lM cocaine, **, p¼0.006 compared to control; 100 lM cocaine, **, p¼
0.001 compared to control. n ¼ 3 from three individual brains.
Figure 4. Effects of Cocaine on Cyclin A Expression in the Developing Rat Neocortex
(A) Experimental paradigm for cocaine and BrdU injections. Twenty mg/kg of cocaine was injected twice (IP) to pregnant rats at an interval of 12 h. BrdU
was injected (IP) 24 h after cocaine.
(B) Diagrams of E15 (sagittal), E17 (coronal), and E19 (coronal) fetal brains showing the areas used for fetal brain cocaine concentration measurements
and cyclin A assays (inside red dotted lines). Further anatomical detail can be obtained from Paxinos et al. .
(C) Time course of cocaine concentrations in developing rat neocortex at early period of neurogenesis determined by gas chromatography-mass
spectrometry (GC-MS). The maximum concentration shown is 15,000 ng/g, indicating that higher concentrations are over the limit (15,000 ng/g) of
quantification. n ¼ 3 from three individual pregnant dams.
(D) Cyclin A2 mRNA expression in cocaine-treated (closed bars) fetal cortex measured by quantitative real-time RT-PCR. Data are expressed as fold
changes in relationship to the control condition (open bars). E15, **, p¼0.00995 compared to control; E17, ***, p , 0.001 compared to control. n¼4–7
from three individual pregnant dams.
(E) Western blot analysis of cyclin A in developing neocortex. The expression of cyclin A was normalized to a-tubulin and expressed as ratios to the
control values. E15, **, p ¼ 0.001 compared to control; E17, *, p ¼ 0.010 compared to control. n ¼ 4–7 from three individual pregnant dams.
(F) BrdU incorporation in peri-ventricular region of cocaine-exposed fetuses during the early period of neurogenesis (E15). Images showing
immunoreactivity of BrdU (red) and Ki67 (green). Scale bar is 20 lm.
(G) Percentage of cortical progenitor cells (Ki67-positive cells) in VZ that entered S phase (BrdU-positive). E15, *, p¼0.014 compared to control; E17, *, p
¼ 0.025 compared to control. n ¼ 6 from three to four individual pregnant dams.
(H) Percentage of cortical progenitor cells (Ki67-positive cells) in SVZ that entered S phase (BrdU-positive). n¼6 from three to four individual pregnant
dams. These differences were not statistically significant.
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Cocaine and Neural Progenitor Proliferation
starts at E12 and ends at E19 in the rat . During this
process, the VZ appears first and is followed by the SVZ. To
investigate the time-frame within which neocortical develop-
ment 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 Figure 4A, and the frontal cortex
of developing fetuses was dissected as shown in Figure 4B.
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 lM (9,812 ng/g) 0.5 h after injection,
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 lM cocaine for
24 h. Signals of cyclin A were normalized to a-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 ¼ 0.031 compared to control; p-CDK2, **, p ¼ 0.002 compared to control; p-pRB, **, p ¼ 0.002
compared to control.
(B) Time course of cyclin A2 mRNA levels in AF5 cells treated with 10 and 100 lM 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 lM cocaine, 6 h, ***, p , 0.001 compared to control; 12 h, *, p¼
0.012 compared to control; 24 h, **, p ¼ 0.004 compared to control. For 100 lM cocaine, ***, p , 0.001 compared to each control. n ¼ 3–7.
(C) Time course of cyclin A protein levels in AF5 cells treated with 10 and 100 lM cocaine. The expression of cyclin A was normalized to a-tubulin and
expressed as ratios to the control values. For 10 lM cocaine, 12 h, **, p¼0.007 compared to control; 24 h, *, p¼0.012 compared to control. For 100 lM
cocaine, 12 h, **, p ¼ 0.003 compared to control; 24 h, **, p ¼ 0.002 compared to control. n ¼ 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 lM 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;þþþ, p , 0.001 compared to
treatment with cocaine only. n ¼ 8.
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Cocaine and Neural Progenitor Proliferation
dropped to ;2 lM (median value of 594 ng/g) at 1 h, and
gradually declined to ;0.2 lM (median value of 63 ng/g) at 6 h
(Figure 4C). 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 (Figures 4D
and S4A). Cyclin A2 mRNA was not, however, changed by
injections during the late period of neurogenesis (Figures 4D
and S4A). 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
(Figures 4E and S4B). 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
Figure 6. Cocaine-Induced Cyclin A Down-Regulation and Proliferation Inhibition Involves Oxidative ER Stress Signaling
(A) Expression of transcription factors, CDK inhibitors, and the activity of CREB following cocaine treatment. AF5 cells were exposed to cocaine for 3 h,
and transcription factors and CDK inhibitors that regulate the transcription of cyclin A2 were measured by immunoblotting. Signals were normalized to
a-tubulin and expressed as percent of control. Phosphorylated CREB was normalized to total CREB proteins. **, p¼0.001 compared to control. n¼4–6.
(B) Time course of ATF4 up-regulation in AF5 cells treated with cocaine. For dose-response experiments, AF5 cells were exposed to cocaine for 3 h. The
expression of ATF4 was normalized to a-tubulin and expressed as percentage of the control values. For time course experiments, 1h and 3 h, ***, p ,
0.001 compared to the respective controls; 6 h, *, p ¼ 0.039 compared to control; 12 h, **, p ¼ 0.008 compared to control. For dose-response
experiments, 10 lM cocaine, *, p ¼ 0.031 compared to control; 100 lM cocaine, ***, p , 0.001 compared to control. n ¼ 3.
(C) Time course of phosphorylation of eIF2a in AF5 cells treated with 10 lM cocaine. The phosphorylation status of eIF2 a was determined by
normalizing phosphorylated forms to total eIF2 a proteins and expressed as percentage of the control values. 0.5 h, **, p¼0.005 compared to control; 1
h, *, p ¼ 0.031 compared to control; 3 h, *, p ¼ 0.045 compared to control. n ¼ 3.
(D) ROS formation in AF5 cells treated with cocaine determined by DCFH-DA. Data are presented as percentage of control. **, p¼0.003; ***, p , 0.001
compared to each control. n ¼ 4–9.
(E–H) Effects of P450 inhibitors on ROS formation (E), expression of ATF4 (F) and cyclin A (G), and proliferation of AF5 cells (H). SKF-525A (100 lM) or
cimetidine (100 lM) were applied 30 min before 100 lM cocaine. ROS and ATF4 protein levels were measured 30 min and 3 h after cocaine,
respectively, whereas cyclin A protein levels and cell proliferation were measured 24 h after cocaine. For Western blot analysis, the expression of ATF4
and cyclin A was normalized to a-tubulin. For the cell proliferation assay, data are shown as percentages of control cell numbers at 0 h. ***, p , 0.001
compared to each control;þþþ,p , 0.001 compared to each cocaine treatment. n ¼ 6–8 for ROS measurement; n ¼ 3–6 for ATF4 and cyclin A
measurements; and n ¼ 6 for the cell proliferation assay.
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Cocaine and Neural Progenitor Proliferation
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
(Figures 4F, 4G, S4C, 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
(Figures 4F, 4H, S4D, 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
(Figures 4H, S4D, 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 lM, 24 h)
decreases cyclin A2 protein in AF5 cells (Figure 5A). 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) . 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 (Figure 5A), indicating that cocaine-induced
down-regulation of cyclin A results in a hypoactive state of
the downstream cell cycle signaling pathway.
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-
Figure 7. Recovery of Cocaine-Induced Changes in the Developing Neocortex by the P450 Inhibitor Cimetidine
Pregnant rats at early period of neurogenesis (E13–E15) were pretreated with 100 mg/kg of cimetidine (IP) 1 h before receiving 20 mg/kg of cocaine
using the regimen described in Figure 4A.
(A and B) Effects of cimetidine on the BrdU incorporation in the developing rat neocortical VZ and SVZ of cocaine-exposed fetuses. (A) Percentage of
cortical progenitor cells (Ki67-positive cells) in VZ that entered S phase (BrdU-positive). **, p ¼ 0.009 compared to control;þ, p ¼ 0.018 compared to
cocaine treatment. n ¼ six to nine samples from three pregnant dams.
(B) Percentage of cortical progenitor cells (Ki67-positive cells) in SVZ that entered S phase (BrdU-positive). n¼6–9 from three individual pregnant dams.
These differences were not statistically significant.
(C and D) Effects of cimetidine on expression of ATF4 and cyclin A in prefrontal cortex of cocaine-exposed fetuses. ATF4 protein levels were measured 3
h after the last injection of cocaine. Cyclin A protein was measured 24 h after the last injection of cocaine. For Western blot analysis, the expression of
ATF4 and cyclin A was normalized to a-tubulin. For ATF4, **, p¼0.001 compared to control;þþ, p¼0.002 compared to cocaine treatment. For cyclin A,
**, p ¼ 0.007 compared to control;þþ, p ¼ 0.008 compared to cocaine treatment. n ¼ five to six samples from three pregnant dams for ATF4
measurement, and n ¼ 6 samples from three pregnant dams for cyclin A measurement.
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Cocaine and Neural Progenitor Proliferation
time RT-PCR showed that cyclin A2 mRNA was decreased as
early as 6 h after 10 or 100 lM cocaine, and the down-
regulation lasted up to 24 h (Figure 5B). Cyclin A protein was
significantly decreased 12 h after 10 or 100 lM cocaine with
nearly maximum effects (64%) at 12 h for 10 lM cocaine, and
a 48% decrease at 24 h for 100 lM cocaine (Figure 5C). Since
pRc/CMV-CycA transfection (with ;80% transfection effi-
ciency) also showed the maximum level of overexpression at
24 h post-transfection (unpublished data), we exposed cells to
100 lM cocaine immediately after electroporation of the
CMV-Cyc A vector, and measured cyclin A protein and cell
proliferation 24 h later. As shown in Figure 5D, cyclin A
transfection counteracted the cocaine-induced down-regu-
lation 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 eIF2a-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 lM, 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 (Figure 6A). The
phosphorylation status of CREB, which is involved in
activation of cyclin A transcription at G1/S , was not
affected by 100 lM cocaine at 3 h (Figure 6A). In addition,
cocaine did not change levels of p21 and p27, CDK inhibitors
that decrease transcription of cyclin A2 (Figure 6A) [33,34].
An increase in ATF4 protein occurred as early as 1 h after
100 lM cocaine exposure, and reached the maximal level at 3
h (Figure 6B). Cocaine at concentrations higher than 1 lM
resulted in dose-dependent induction of ATF4 (Figure 6B).
The phosphorylated form of the alpha subunit of translation
initiation factor 2 (eIF2a) is known to promote ATF4
induction . Indeed, we found that 10 lM cocaine
significantly increased the phosphorylated form of eIF2a 0.5
h after exposure to cocaine, preceding induction of ATF4
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 eIF2a-ATF4 pathway is activated by PERK, an
endoplasmic reticulum (ER) stress sensor protein [35,36].
The ER stress pathway has been shown to be activated by a
variety of chemicals or pathological stress including oxidative
ER stress . Notably it has been demonstrated that the
cytochrome P450 dependent N-oxidative pathway is respon-
sible for generation of ROS and glutathione (GSH) depletion
during cocaine biotransformation in the liver . We
therefore hypothesized that ROS generation caused by the
N-oxidative metabolism of cocaine may trigger activation of
the eIF2a-ATF4 pathway.
Accordingly, we first examined whether cocaine induces
ROS production in progenitor cells. As shown in Figure 6D,
10 lM cocaine caused a significant increase in ROS 30 min
after treatment. Cocaine at 100 lM caused ROS generation
even earlier, with a significant increase 15 min after the
Because the rise of cocaine concentrations over 10 lM lasts
for less than 1 h in fetal brains after cocaine injection (Figure
4C), AF5 cells were next treated with 30 lM 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.
or cimetidine, drugs that have been shown to potently block N-
oxidative metabolism of cocaine, completely blocked cocaine-
induced ROS formation (Figure 6E), 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 (Figure 6F and 6G).
Finally, we tested effects of SKF-525A and cimetidine on
the inhibition of cell proliferation by cocaine. Both drugs
significantly diminished cocaine-induced proliferation inhib-
ition (100 lM for 24 h) (Figure 6H). On the other hand, the
lipophilic free radical scavengers a-tocopherol and BHA and
the iron chelator DFO (each 50 lM) did not prevent cocaine-
induced proliferation inhibition (unpublished data). These
data indicate that cytochrome P450-dependent ROS forma-
tion occurring during cocaine metabolism is responsible for
both cocaine-induced proliferation inhibition and cyclin A
Cimetidine Reverses Effects of Cocaine In Utero in a Rat
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 [38–40]. Cimetidine
itself did not affect neural progenitor cell proliferation,
survival, density, or fetal mortality (Figures 7A, 7B, S7A, S7B,
and S8A–S8E). As shown in Figures 7A 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 progen-
itor cells were observed in the SVZ (Figures 7B and S7B).
To determine whether the protection afforded by cimeti-
dine 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 (Figures 7C, 7D, S7C, S7D). These
results suggest that blockade of cocaine N-oxidative metab-
olism 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.
In the present study, we found that (1) cocaine causes
proliferation inhibition and cyclin A down-regulation in
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Cocaine and Neural Progenitor Proliferation
neural progenitor cells both in vitro and in vivo; (2) restoring
cyclin A reverses proliferation inhibition induced by cocaine;
and (3) ROS-induced ER stress, activating the eIF2a-ATF4
pathway, is involved in cyclin A down-regulation induced by
cocaine. Thus, this study identifies ES stress-induced cyclin A
down-regulation as an important molecular event involved in
cocaine-induced proliferation inhibition in neural progenitor
cells. A diagram, illustrating this pathway is shown in Figure 8.
The Role of Cyclin A in Neural Progenitor Cells Exposed to
Using the AF5 neural progenitor cell line, we determined
that cocaine treatment for 24 h causes proliferation
inhibition at concentrations higher than 1 lM. Similar results
were reported by Hu et al.  showing that cocaine ranging
1–100 lM (7 d) suppresses growth of human neural precursor
cells, as measured by thymidine incorporation. Nevertheless,
Figure 8. Schematic Illustrating the Mechanism of Cocaine’s Effect on Brain Development Described in this Study
During cortical neurogenesis, ventricular progenitors (blue) withdraw from the cell cycle (pink) and start to differentiate and migrate to their specific
destinations to form cortical layers. Exposure to cocaine during neocortical development causes rapid ROS accumulation in ER via cytochrome P450
dependent N-oxidative metabolism. ROS-induced oxidative ER stress leads to activation of the PERK/eIF2a/ATF4 pathway. ATF4 represses the
transcription of cyclin A2 in neural progenitor cells. Blocking cell cycle progression by cyclin A down-regulation may result in fewer immature neurons
migrating into the cortex to form the cortical layers. I, III, IV, V, and VI, cortical layers I, III, IV, V, and VI; IZ, intermediate zone; SP, superficial portion of the
intermediate zone; SVZ, subventricular zones; VZ, ventricular zone. Up-regulation or activation of transcripts or proteins is denoted by "; down-
regulation or inactivation of transcripts or proteins is denoted by #. Changes indicated by red arrows were confirmed by quantitative real-time RT-PCR
or Western blot analysis in this study.
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Cocaine and Neural Progenitor Proliferation
this group also did not detect proliferation inhibition by 1
lM cocaine for 24 h, although a significant effect was
produced by treatment with 1 lM cocaine for 5 d . The
findings of Hu et al.  are therefore consistent with our
Poon et al.  found that 1 lM cocaine treatment for 30
min induced late-onset cell death (72 h later) in differentiated
human neuronal progenitor cells. We did not observe cell
death in cocaine (1–100 lM)-treated AF5 cells (Figure 1B and
1C) or in primary human fetal neural and A2B5þ progenitor
cells (unpublished data) 24 h after treatment, although we did
not examine cell death at later time points such as 72 h. In
agreement with our findings, cocaine treatment (1 pM–100
lM) for 7 d did not induce cell death in undifferentiated
human neural precursor cells . Under certain conditions,
cocaine can selectively induce cell death in neurons without
affecting viability of other types of CNS cells . The
differentiated neuronal progenitor cells employed by Poon et
al.  may therefore be relatively more sensitive to an effect
of cocaine in promoting cell death as compared to
undifferentiated neural progenitor cells.
Although cocaine has been shown to inhibit DNA synthesis
as measured by thymidine incorporation in human neural
precursor cells , the mode of action of cocaine on cell
cycle progression has not previously been determined. We
found that cocaine causes cell proliferation inhibition by
interfering with the G1-to-S transition. Although one report
suggested a disturbance in chromosome segregation induced
by 2,500 lM cocaine treatment (14 or 24 h) of mouse oocytes
, our flow cytometric and immunocytochemical analyses
show that cocaine (10–100 lM) does not have any effect on
the mitotic phase at least in our system (Figure 1D and 1E). A
cDNA microarray identified cyclin A2 as a candidate molecule
related to cocaine-mediated G1/S phase arrest. Cocaine
causes specific down-regulation of cyclin A2 in AF5 cells,
primary neural and A2B5þ progenitor cells, and fetal brains
exposed to cocaine in utero. Further, compensating for
cocaine-induced cyclin A down-regulation by gene transfer
counteracted the growth-suppressing action of cocaine in
AF5 cells (Figure 5D).
Reversal of cocaine-induced proliferation inhibition by
cyclin A transfection is not, in itself, proof that the effect of
cocaine on neural progenitor cell proliferation is caused by
down-regulation of cyclin A. Nevertheless, among known
modulators of the G1-to-S transition, microarray analysis
identified only cyclin A2 as being down-regulated by cocaine.
The cyclin A transfection results are consistent with the
hypothesis that cocaine decreases proliferation via down-
regulation of cyclin A. Moreover, the fact that the ROS-
induced ER stress consequently promoted transcriptional
down-regulation of cyclin A is further evidence that
reductions in cyclin A signaling are responsible for decreased
proliferation. These findings, taken together, suggest that
cyclin A down-regulation constitutes at least one molecular
mechanism by which cocaine causes dysfunction of neural
Cocaine-induced cyclin A2 down-regulation was observed in
human neural progenitor and A2B5þ progenitor cells, but
not in human neurons and microglia. In contrast to
progenitor cells, the cyclin A2 transcript was increased by
cocaine in human astrocytes. Although we do not know the
mechanism underlying the proliferative effect of cocaine on
astrocytes, it is worth noting that cellular stress inhibits
proliferation in CNS progenitors, whereas it activates
proliferation in astrocytes, leading to gliosis in the brain
. Cocaine thus has at least two pharmacological actions on
cellular proliferation in a cell type-specific manner. More-
over, in utero cocaine exposure down-regulated cyclin A
expression in progenitor cells in the VZ, but not in the
progenitor cells in the SVZ. Although the reason for this
specific mode of action is unknown, variations in expression
and metabolic capacities of cytochrome P450s, plasma
membrane permeability of cocaine, and variations in cellular
stress response mechanisms could be involved. Future studies
exploring the activity of cytochrome p450s and oxidative
stress in different types of CNS cells will address this issue.
Oxidative ER Stress in Cocaine-Induced Cyclin A Down-
Screening of molecules that can regulate the promoter
activity of cyclin A2 demonstrated that ATF4 is specifically up-
regulated by cocaine exposure; this effect of cocaine occurs 3
h prior to cyclin A2 down-regulation. Upon ER stress such as
ER oxidation, the ER sensor protein PERK phosphorylates
eIF2a, subsequently promoting translational activation of
ATF4 [35,36]. ATF4 promotes not only up-regulation of genes
involved in redox control (e.g., glutathione biosynthesis) ,
but also promotes down-regulation of cyclin A during G1-to-
S progression, which can lead to cell cycle arrest . Thus,
ER oxidation is capable of causing cell cycle arrest via the
In addition to promoting dopamine auto-oxidation,
cocaine can itself get through the cell membrane in its
nonprotonated form, where it produces ROS via N-oxidative
metabolism catalyzed by cytochrome P450 at the ER .
Cocaine is first N-demethylated to norcocaine [44,45],
followed by oxidation to N-hydroxynorcocaine [44–47] by
cytochrome P450 and flavin-containing monooxygenases. N-
hydroxynorcocaine is further converted to norcocaine nitro-
xide by one-electron oxidation, also through cytochrome
P450 activity [44,48]. Norcocaine nitroxide can be rapidly
reduced to N-hydroxynorcocaine again by flavoproteins such
as cytochrome P450 reductase and FAD-monooxygenase
[44,48]. Redox cycling between norcocaine nitroxide and N-
hydroxynorcocaine is responsible for generation of ROS such
as superoxide anion and hydrogen peroxide [37,44]. In
addition, depletion of NADPH, an essential cofactor for
maintenance of reduced glutathione, during the futile redox
cycling of N-hydroxynorcocaine/norcocaine nitroxide is able
to disrupt the homeostatic effect of cellular glutathione ,
possibly also contributing to cellular oxidative stress.
Indeed, we found that cocaine induces endogenous ROS
accumulation as early as 15 min after treatment of AF5 cells
(Figure 6D). Further, the P450 inhibitors SKF-525A and
cimetidine, which have been shown to inhibit N-oxidative
metabolism of cocaine, blocked cocaine-induced endogenous
ROS generation, translational activation of ATF4, down-
regulation of cyclin A, and proliferation inhibition in AF5
cells (Figure 6E–6H). In contrast, lipophilic free radical
scavengers and the iron chelator DFO did not block
cocaine-induced inhibition of proliferation, suggesting that
lipid peroxidation and iron-mediated ROS production
(Fenton reaction) in mitochondria are not primarily involved
in cocaine-induced ROS production in AF5 cells. We there-
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Cocaine and Neural Progenitor Proliferation
fore conclude that cocaine biotransformation by microsomal
cytochrome P450 is the source of cocaine-induced ROS
generation, which promotes cell cycle arrest via ER stress-
induced cyclin A down-regulation (Figure 6E–6H).
In cultured human neural precursor cells, cocaine-induced
(1–100 lM for 7 d) cell proliferation inhibition has been
suggested to be related to increased expression of p21, a
major transcriptional target of p53, as well as down-
regulation of nuclear antigen (PCNA), an essential DNA
replication factor . Activation of the p53-p21 pathway has
been implicated in hyperoxia-induced inhibition of G1/S
progression, through suppression of CDK activity and
subsequent repression of cyclin A2 transcription and inacti-
vation of PCNA [34,49]. Increased expression of p21 was not
observed 24 h after exposure to cocaine in our study;
however, we did detect an increase of p21 48 h after exposure
to cocaine (unpublished data). These data suggest that the
p53-p21 pathway may be involved in longer-term effects of
cocaine, but not in the short-term action of cocaine that
promotes down-regulation of cyclin A2 within 24 h.
Cocaine has been shown to inhibit protein synthesis in rat
fibroblasts, associated with cocaine-induced inhibition of cell
proliferation . One of the cellular adaptive responses to
oxidative ER stress is inhibition of translation, which may
explain the inhibition of protein synthesis observed in
cocaine-treated fibroblasts. Also, cocaine acts as a local
anesthetic by inhibiting sodium influx into cells. An in vitro
study, however, has shown that cocaine-induced proliferation
inhibition in lymphocytes was not related to sodium channel
Effects of Cocaine in Developing Cerebral Cortex
Neural progenitor cells in ventricular proliferating zones
are multipotent, having the potential to differentiate to
neurons destined for different layers of the cortex. Alter-
ations in cell cycle dynamics of neural progenitor cells have
been linked to the production of various sizes of cortical
areas and to the thickness of cortical layers [51,52]. In
addition, disruption of neural progenitor proliferation can
result in microcephaly , which has been reported in
infants born to cocaine-abusing mothers . We demonstra-
ted here that oxidative ER stress-mediated down-regulation
of cyclin A contributes to cocaine-induced inhibition of
neural progenitor cell proliferation in the developing neo-
cortex (Figure 8). Because cell cycle duration is regulated
mainly via G1 phase and/or the G1-to-S transition during
embryonic neurogenesis [54,55], cocaine-induced G1/S cell
cycle arrest mediated by down-regulation of cyclin A may
have a substantial impact on proliferation of progenitor cells,
as well as on ensuing migration and differentiation (Figure 8).
Cocaine inhibited proliferation of AF5 cells in concen-
trations as low as 10 lM for 24 h of exposure. As shown in
Figure 4C, the concentrations of cocaine in the developing
neocortex following the second IP administrations of 20 mg/
kg cocaine peaked higher than 30 lM within 30 min. A short
duration of exposure of the fetal brain to cocaine may in fact
be deleterious since, as shown in Figure S6, exposure of AF5
cells to 30 lM cocaine for 30 min significantly up-regulated
the expression of ATF4 at 3 h. Thus, exposure to 30 lM
cocaine for 30 min is sufficient to increase endogenous ROS
to a degree that activates the ATF4 signaling pathway.
Therefore, it is conceivable that exposure to cocaine for
fairly short durations could initiate the molecular mecha-
nisms underlying cocaine-induced inhibition of neural
progenitor cell proliferation.
Maternal cocaine exposure has been associated with
uterine vasoconstriction , which could cause low oxygen-
ation of the developing fetal brain. A number of studies
indicate, however, that cocaine-induced vasoconstriction is
unlikely to mediate impairments in brain development [57–
59] as measured by brain structure and DNA synthesis. Also,
Lidow and Rakic  have shown that neurotransmitter
receptors expressed in neural progenitor cells, including
dopaminergic, serotonergic, and adrenergic receptors, are
related to cell proliferation stimulation or inhibition in the
developing primate occipital lobe. Therefore, it is possible
that cocaine-induced accumulation of monoamine neuro-
transmitters and activation of corresponding receptors also
contribute to the inhibition of cell cycle progression by
cocaine, especially in vivo. Moreover, in addition to ROS
induced by N-oxidation of cocaine, auto-oxidation of mono-
amines could contribute to ATF4-mediated cyclin A down-
regulation in fetal brains. Nevertheless, our data demonstra-
ted that cytochrome P450-dependent N-oxidation of cocaine
promotes cyclin A2 down-regulation by causing ER stress,
which constitutes at least one mechanism underlying cocaine-
induced inhibition of ventricular progenitor cell prolifer-
ation in the developing neocortex.
Possible Clinical Use of Cimetidine
We have shown that pretreatment with the P450 inhibitor
cimetidine effectively abolished both the cocaine-induced
inhibition of neural progenitor cell proliferation in the VZ of
the developing rat brain, and cyclin A down-regulation
(Figures 7A, 7D, S7A, and S7D). This effect of cimetidine
appeared to be mediated by prevention of N-oxidative
metabolism of cocaine and consequent ER stress, as cimeti-
dine also prevented the cocaine-induced increase in ATF4
(Figures 7C and S7C). These effects of cimetidine in the
developing rat brain were consistent with the efficacy of
cimetidine in blocking effects of cocaine on the AF5 cell line
Cimetidine belongs to a class of drugs called histamine H2
receptor antagonists, and is often prescribed for the treat-
ment of gastroesophageal reflux diseases and peptic ulcer
diseases. Cimetidine crosses the placenta by passive diffusion
 and has been allocated to pregnancy category B by the
FDA, which means that cimetidine is not expected to be
harmful to the fetus. There are conflicting reports on anti-
androgenic effects of cimetidine in animals exposed in utero
[61–64]; however, no anti-androgenic effects of cimetidine
have so far been reported in human pregnancy.
We demonstrated that giving cimetidine (100 mg/kg IP
twice at an interval of 12 h) to pregnant rats at an early
period of neurogenesis did not affect proliferation (Figures
7A, 7B, S7A, and S7B), survival (Figure S8A and S8B), or
density (Figure S8C and S8D) of neural progenitor cells in the
VZ or SVZ. We also did not observe an increase in perinatal
mortality in cimetidine-treated animals (Figure S8E). The
expression of histamine H2receptors in the cerebral cortex is
very low [65,66]; therefore, H2receptor activity is not likely to
be involved in the ability of cimetidine to decrease cocaine-
induced changes in the fetal brain.
The question naturally arises as to whether cimetidine or a
PLoS Medicine | www.plosmedicine.org June 2008 | Volume 5 | Issue 6 | e1171000
Cocaine and Neural Progenitor Proliferation
similar drug could be employed to prevent the adverse effects
of cocaine on brain development. Presumably, the manner in
which this would be accomplished would be that women of
child-bearing age with a history of cocaine abuse, and who are
at risk for subsequent cocaine abuse, would be asked to take
cimetidine as a prophylactic measure. Several issues would
have to be addressed before this could be done. Most
importantly, the possibility that cimetidine increases the
systemic toxicity of cocaine, perhaps by interfering with
cocaine metabolism and lengthening the half-life of cocaine,
would have to be considered. Another question to be
addressed is the half-life of the preventive drug used that
would be necessary to achieve a satisfactory level of patient
compliance; it is possible that a drug with a very long half-life,
e.g., a depot preparation, would be needed. Also, although the
mechanism that is described here appears to at least
contribute to the developmental toxicity of cocaine, it is
not necessarily the entire cause of cocaine’s adverse effects on
development. Thus, the long-term efficacy of cimetidine (or
alternative drugs) on brain development and function, and
efficacy in a second larger species, will also need to be
Limitations of This Study
During neocorticogenesis, the two germinal compartments
VZ and SVZ are composed of heterogeneous populations of
neural progenitor cells. It is difficult to isolate, define, and
examine specific sub-types of neural progenitor cells derived
from primary fetal cortex. It is also problematic to examine
regional cellular and molecular parameters in vivo in tissue
sections derived from embryonic rat brains at different ages
following intrauterine cocaine administration. The AF5
neural progenitor cell line used in this study may not have
properties identical to those of cortical VZ progenitors;
however, the AF5 cell line was able to reveal the molecular
mechanisms (e.g., free radical-induced ER stress), which are
involved in cocaine-induced proliferation inhibition of
neural progenitor cells. The AF5 cell model not only allowed
us to identify these molecular mechanisms, but also was
remarkably predictive of the in vivo findings. Nevertheless,
the expression and metabolic capacities of cytochrome P450
in neural progenitor cells of developing rodent and human
brains are not identical, which to same extent limits
extrapolation of our findings to cocaine-exposed human
A second issue involves the dose of cimetidine that is
required to prevent the effects of cocaine. The dose of
cimetidine used to block the histamine H2 receptor in
rodents is ;2.5–10 mg/kg ; however, doses of cimetidine
up to 100 mg/kg are used to inhibit the activity of cytochrome
P450 in rodents [68,69]. We therefore used 100 mg/kg
cimetidine for the present study. On a mg/kg basis, this dose
is about ten times higher than the human therapeutic dose.
Thus, it is not clear whether cimetidine, or any similar drug,
would be effective in preventing the adverse effects of
cocaine on neural progenitor cells in human cocaine abuse.
It will therefore be essential to determine whether clinically
relevant doses of cimetidine have the capacity to inhibit the
adverse effects of exposure of the human fetus to cocaine, as
well as whether there are other P450 inhibitors that are more
Understanding the molecular mechanisms by which in
utero cocaine exposure causes proliferation inhibition of
neural progenitor cells is important for developing preven-
tion and therapeutic strategies against long-lasting neuro-
logical and behavioral dysfunction caused by exposure of the
developing fetus to cocaine. Future research might be
focused on exploring the molecular and biochemical mech-
anisms involved in cellular functions such as differentiation
and migration changed by cocaine in neural progenitor cells,
determining the expression and metabolic capacity of
cytochrome P450s in various subtypes of cortical and
subcortical progenitor cells, and investigating whether
oxidative ER stress is involved in other disorders that have
been shown to be related to P450-dependent oxidative
metabolism of cocaine, such as immunosuppression [70–72],
hepatocyte injury [37,69,73,74], and cardiotoxicity .
Coronal brain sections of E15, E17, and E19 rat fetal brains stained
with cresyl violet illustrate cortical regions used for BrdU labeling
measurements (one-third and two-thirds of the cerebral cortex from
the superior sagittal sinus [SSS] to the caudal pole of the internal
capsule, red rectangles) for cocaine treatment at the early period of
neocortical neuronogenesis (E13–E15), the middle period (E15–E17),
and the late period of neocortical neuronogenesis (E17–E19). The
scale bar is 0.5 mm.
Found at doi:10.1371/journal.pmed.0050117.sg001 (4.9 MB TIF).
Cortical Regions Used for Measurements of BrdU
Figure S2. Time Course of Cocaine-Induced Proliferation Inhibition
in AF5 Cells
AF5 cells were treated with 100 lM cocaine every day during a
medium change for a total of 6 d. Data are presented as percentage of
control cell numbers at 0 h. Day 1: **, p¼0.001 compared to control;
days 3 and 6: ***, p , 0.001 compared to each control; n ¼ 4.
Found at doi:10.1371/journal.pmed.0050117.sg002 (698 KB TIF).
Cyclin A in AF5 Cells
AF5 cells were treated with 100 lM for 6 d, and cyclin A protein levels
were assessed by immunoblotting at day 1, 3, and 6.
Found at doi:10.1371/journal.pmed.0050117.sg003 (532 KB PPT).
Time Course of Cocaine-Induced Down-Regulation of
Figure S4. Data from Figure 4 Shown in Scattergram Form
(A) Scattergram of Figure 4D.
(B) Scattergram of Figure 4E.
(C) Scattergram of Figure 4G.
(D) Scattergram of Figure 4H.
Found at doi:10.1371/journal.pmed.0050117.sg004 (78 KB PPT).
Figure S5. BrdU Incorporation in the Developing Rat Neocortex for
the Middle and Late Neuronogenesis Periods
The VZ and SVZ of cocaine-exposed fetuses during middle (E15–E17)
and late (E17–E19) periods of neocortical neuronogenesis are shown.
Immunoreactivity for BrdU (red) and Ki67 (green) are shown for E17
and E19 fetal brains. Cocaine decreased the number of BrdU-labeled
progenitor cells in VZ at the middle of the neurogenesis period.
Images for the early period of neuronogenesis are shown in Figure
4F. The scale bar is 20 lm.
Found at doi:10.1371/journal.pmed.0050117.sg005 (530 KB PDF).
Cocaine for 30 Min
AF5 cells were treated with cocaine on the basis of the concentrations
curve measured in fetal brain following cocaine injections (Figure
4C). AF5 cells were treated with 30 lM cocaine for 30 min, and ATF4
protein level was measured by immunoblotting. The expression of
ATF4 was normalized to a-tubulin. **, p¼0.006 compared to control,
n ¼ 4.
ATF4 Expression for AF5 Cells Treated with 30 lM
PLoS Medicine | www.plosmedicine.org June 2008 | Volume 5 | Issue 6 | e1171001
Cocaine and Neural Progenitor Proliferation
Found at doi:10.1371/journal.pmed.0050117.sg006 (1.3 MB PDF).
Figure S7. Data from Figure 7 Shown in Scattergram Form
(A) Scattergram of Figure 7A.
(B) Scattergram of Figure 7B.
(C) Scattergram of Figure 7C.
(D) Scattergram of Figure 7D.
Found at doi:10.1371/journal.pmed.0050117.sg007 (58 KB PPT).
istration of Cocaine and Cimetidine
Pregnant rats at the early period of neurogenesis (E13–E15) were
pretreated with 100 mg/kg of cimetidine IP 1 h before receiving 20
mg/kg of cocaine using the regimen described in Figure 4A.
(A and B) Effects of cimetidine on cell death in the developing rat
neocortical VZ and SVZ of cocaine-exposed fetuses. Apoptotic index
[(number of condensed nuclei/number of total nuclei) 3 100%] was
calculated in the regions used for quantitative evaluation of the BrdU
labeling described in the methods. n ¼ six samples from three
(C and D) Effects of cimetidine on cell density in the developing rat
neocortical VZ and SVZ of cocaine-exposed fetuses. Cell density
([number of total nuclei/rectangle area with 160 lm-width] 3 100%)
was calculated in the regions used for quantitative evaluation of the
BrdU labeling described in the methods. n ¼ six samples from three
(E) Effects of cimetidine on the mortality of cocaine-exposed fetuses.
Perinatal mortality was calculated by (number of dead fetuses/
number of total fetuses)3100%. n¼four to five samples from four to
five pregnant dams. None of the differences were statistically
Found at doi:10.1371/journal.pmed.0050117.sg008 (937 KB TIF).
Developmental Indicators Following Prenatal Admin-
Table S1. Comparison of p-Values Obtained by t-Tests and Mann-
Whitney U Tests
Found at doi:10.1371/journal.pmed.0050117.st001 (61 KB DOC).
The authors thank P.W. Hinds (Harvard Medical School, Boston,
Massachusetts, United States of America) for providing the pRc/CMV-
CycA plasmid. We thank Diane Teichberg, William H. Wood III (DNA
Array Unit, NIA IRP), and Ann William (NIH/NHLBI) for their expert
assistance. We also thank Cindy Ambriz for preparing the manu-
Author contributions. CTL, TH, and WJF conceived and designed
the study. CTL, JC, SYT, and JFS performed the experiments
involving cell proliferation and cytotoxicity assays, transfection,
immunocytochemistry, and in vivo experiments. CTL and JC
performed the quantitative real-time RT-PCR. SLE and RA
performed the Western blot analysis and helped with in vivo
experiments. JS prepared the human primary CNS cells. KGB
performed the microarray analysis. CTL, TH, and WJF interpreted
the data. RHL and MAH performed the measurements of cocaine
concentrations in fetal brain. CTL and WJF drafted the manuscript.
CTL, TH, TPS, HMG, and WJF performed critical revision of the
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Cocaine and Neural Progenitor Proliferation
Editors’ Summary Download full-text
Background. Every year, cocaine abuse by mothers during pregnancy
exposes thousands of unborn infants (fetuses) to this powerful and
addictive stimulant. Maternal cocaine abuse during early pregnancy
increases the risk of miscarriage; its use during late pregnancy slows the
baby’s growth and can trigger premature labor. Babies exposed to
cocaine shortly before birth are often irritable and have disturbed sleep
patterns. They can also be very sensitive to sound and touch and
consequently hard to comfort. These problems usually resolve sponta-
neously within the first few weeks of life but some permanent birth
defects are also associated with frequent cocaine abuse during
pregnancy. In particular, babies exposed to cocaine before birth
sometimes have small heads—an abnormality that generally indicates
a small brain—and, although they usually have normal intelligence, the
development of their thinking skills and language is often delayed, and
they can have behavioral problems.
Why Was This Study Done? Exposure to cocaine before birth clearly
interferes with some aspects of brain development. More specifically, it
reduces the number and position of neurons (the cells that transmit
information in the form of electrical impulses around the body) within
the brain. All neurons develop from neural progenitor cells, and previous
research suggests that cocaine exposure before birth inhibits the
proliferation of these cells in the developing brain. It would be useful
to understand exactly how cocaine affects neural progenitor cells,
because it might then be possible to prevent the drug’s adverse effects
on brain development. In this study, therefore, the researchers
investigate the molecular mechanism that underlies cocaine’s effect on
neural progenitor cells.
What Did the Researchers Do and Find? When the researchers
investigated the effects of cocaine on AF5 cells (rat neural progenitor
cells that grow indefinitely in the laboratory), they found that
concentrations of cocaine similar to those measured in fetal brains after
maternal drug exposure inhibited the proliferation of AF5 cells by
blocking the ‘‘G1-to-S transition.’’ This is a stage that cells have to pass
through between each round of cell division (the production of two
daughter cells from one parent cell). Next, the researchers showed that
cocaine-treated AF5 cells made much less cyclin A2, a protein that
controls the G1-to-S transition, than untreated cells. Cocaine also
decreased cyclin A2 levels in neural progenitor cells freshly isolated
from human fetal brains and in fetal rat brains exposed to the drug while
in their mother’s womb. Treatment of AF5 cells with a cyclin A2
expression vector (a piece of DNA that directs the production of cyclin
A2) counteracted the down-regulation of cyclin A2 and restored AF5
proliferation in the presence of cocaine. Other experiments indicate that
the reduction of cyclin A2 by cocaine in AF5 cells involves the
accumulation of ‘‘reactive oxygen species,’’ by-products of the break-
down of cocaine by a protein that is a member of a family of proteins
called cytochrome P450. Finally, treatment of pregnant rats with
cimetidine (which inhibits the action of cytochrome P450) counteracted
both the inhibition of neural progenitor cell proliferation and the cyclin
A2 down-regulation that cocaine exposure induced in the brains of their
What Do These Findings Mean? These findings show that the cocaine-
induced inhibition of neural progenitor cell proliferation involves, at least
in part, interfering with the production (that is, causing down-regulation)
of cyclin A2. They also show that this down-regulation is induced by the
breakdown of cocaine by cytochrome P450, and that in both a rat cell
line and in fetal rats, the cytochrome P450 inhibitor cimetidine (a drug
that is already used clinically for stomach problems) can block the
adverse effects of cocaine on the proliferation of neural progenitor cells.
These findings need to be confirmed in animals more closely related to
people than rats, and the long-term effects of cimetidine need to be
investigated, in particular its effects on cocaine toxicity. Nevertheless
these results raise the possibility that giving cimetidine or other drugs
with similar effects to pregnant women who are addicted to cocaine
might prevent some of the harm that their drug habit does to their
unborn children, although it is not clear whether there is a dosage of
cimetidine that might be both safe and adequate for this purpose.
Additional Information. Please access these Web sites via the online
version of this summary at http://dx.doi.org/10.1371/journal.pmed.
? A PLoS Medicine Perspective article by Steven Hyman further discusses
? The US National Institute on Drug Abuse provides a fact sheet on
cocaine (in English and Spanish)
? The UK charity Release provides information and advice to the public
and professionals about the law and drugs, including information
? MedlinePlus also provides a list of links to information about cocaine
(in English and Spanish)
? The March of Dimes Foundation, a US nonprofit organization for the
improvement of child health, provides information about illicit drug
use during pregnancy (in English and Spanish)
? The Organization of Teratology Information Specialists also provides a
fact sheet on cocaine and pregnancy (in English, Spanish, and French)
PLoS Medicine | www.plosmedicine.org June 2008 | Volume 5 | Issue 6 | e1171004
Cocaine and Neural Progenitor Proliferation