Daniels MJ, Wang Y, Lee M, Venkitaraman AR.. Abnormal cytokinesis in cells deficient in the breast cancer susceptibility protein BRCA2. Science 306: 876-879

Abstract

Germ-line mutations inactivating BRCA2 predispose to cancer. BRCA2-deficient cells exhibit alterations in chromosome number (aneuploidy), as well as structurally aberrant chromosomes. Here, we show that BRCA2 deficiency impairs the completion of cell division by cytokinesis. BRCA2 inactivation in murine embryo fibroblasts (MEFs) and HeLa cells by targeted gene disruption or RNA interference delays and prevents cell cleavage. Impeded cell separation is accompanied by abnormalities in myosin II organization during the late stages in cytokinesis. BRCA2 may have a role in regulating these events, as it localizes to the cytokinetic midbody. Our findings thus link cytokinetic abnormalities to a hereditary cancer syndrome characterized by chromosomal instability and may help to explain why BRCA2-deficient tumors are frequently aneuploid.

Full text

DOI: 10.1126/science.1102574
, 876 (2004); 306Science
et al.Matthew J. Daniels,
Breast Cancer Susceptibility Protein BRCA2
Abnormal Cytokinesis in Cells Deficient in the
www.sciencemag.org (this information is current as of July 10, 2008 ):
The following resources related to this article are available online at
http://www.sciencemag.org/cgi/content/full/306/5697/876
version of this article at:
including high-resolution figures, can be found in the onlineUpdated information and services,
http://www.sciencemag.org/cgi/content/full/1102574/DC1
can be found at: Supporting Online Material
found at:
can berelated to this articleA list of selected additional articles on the Science Web sites
http://www.sciencemag.org/cgi/content/full/306/5697/876#related-content
http://www.sciencemag.org/cgi/content/full/306/5697/876#otherarticles
, 6 of which can be accessed for free: cites 18 articlesThis article
69 article(s) on the ISI Web of Science. cited byThis article has been
http://www.sciencemag.org/cgi/content/full/306/5697/876#otherarticles
19 articles hosted by HighWire Press; see: cited byThis article has been
http://www.sciencemag.org/cgi/collection/medicine
Medicine, Diseases
: subject collectionsThis article appears in the following
http://www.sciencemag.org/about/permissions.dtl
in whole or in part can be found at: this article
permission to reproduce of this article or about obtaining reprintsInformation about obtaining
registered trademark of AAAS.
is aScience2004 by the American Association for the Advancement of Science; all rights reserved. The title
CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005.
(print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by theScience
on July 10, 2008 www.sciencemag.orgDownloaded from

rium, each exchanging between the activated
and dormant state. Once the holoenzyme
has been formed, with both ThDPs bound in
place, this will be the state of the enzyme in
vivo at the start of each catalytic cycle, as it
is in our crystal structure.
In support of these ideas, we found that
limited proteolysis of the inactive E1 (NQ)
acidic tunnel mutant leads to almost com-
plete cleavage of the loops in both the active
sites (Fig. 3B). However, in the presence of
the deazaThDP, the E1 (NQ) behaves like
the wild-type E1, in which the active-site
loops are all protected from attack (Fig. 3B).
These observations suggest that the charge
state of ThDP is sufficient to control the
conformation of active-site loops, but also
that replacing acidic residues in the tunnel
severs communication between active sites
and dissipates the active-site asymmetry.
The involvement of the proton wire in
the activation of ThDP provides a molecu-
lar basis for the hysteretic properties of this
enzyme. It also resolves the puzzle of why
the first substrate, pyruvate, exhibits appar-
ently conflicting characteristics with respect
to ThDP activation. On the one hand, pyru-
vate induces positive cooperativity of ThDP
activation (28), yet several cocrystal struc-
tures of ThDP-dependent enzymes show that
substrate analogs are bound in only one of
the two active sites (17, 29). Substrate bind-
ing exclusively to only one of the active sites
is an extreme form of negative cooperativity
sometimes referred to as Bhalf of the sites_
reactivity[ and is common among many ping-
pong enzymes (30). These apparently con-
tradictory properties can be reconciled by
the molecular switch and proton-wire model,
which holds that the first ThDP is activated
by binding; in contrast, activation of the sec-
ond site is coupled to decarboxylation of
pyruvate in the first site (Fig. 4, A to D) (31).
As shown schematically in Fig. 4, the ac-
tivation and subsequent catalytic steps of this
Bslinky cycle[ are dependent on the push or
pull of a proton: while one site requires a
general acid, the other requires a general base,
and via the proton wire, they reciprocate their
catalytic needs. This mechanism also permits
the switching of active-site loops to coordi-
nate the uptake of substrates and release of
products, which is particularly important in
E1, because the specificity of lipoyl domain
recognition underlies the molecular mecha-
nism of substrate-channeling in the PDH
complex (12, 15).
In the homologous E1 from eukaryotes,
serine residues in the outer loop of the active
site are the targets of phosphorylation by a
specific kinase (EC 2.7.1.99), which regu-
lates the catalytic activity. Phosphorylation
at only one of the two active sites is suf-
ficient to inactivate the entire enzyme (32),
which demonstrates that coupling between the
two active sites is obligatory. Additionally,
kinetic evidence accumulated for a close
relative of E1, the yeast ThDP-dependent
pyruvate decarboxylase (EC 4.1.1.1) E(33, 34)
and references therein^, suggests the active
sites of the Bcatalytic dimer[ alternate. These
observations can readily be explained by the
dependence of E1 activity on the commu-
nication between active sites envisaged in
the molecular switch and proton-wire model
(Fig. 4E). It will be interesting to see how
far these proposals extend to other dimeric
ping-pong enzymes, particularly those re-
quiring an activated cofactor for catalysis.
References and Notes
1. N. K. Nagradova, FEBS Lett. 487, 327 (2001).
2. A. Schellenberger, Biochim. Biophys. Acta 1385, 177
(1998).
3. F. Horn, H. Bisswanger, J. Biol. Chem. 258, 6912 (1983).
4. R. Breslow, J. Am. Chem. Soc. 79, 1762 (1957).
5. A. Schellenberger, G. Hu¨bner, H. Neef, Methods
Enzymol. 279, 131 (1997).
6. F. Jordan et al., J. Am. Chem. Soc. 125, 12732 (2003).
7. Y. Lindqvist, G. Schneider, U. Ermler, M. Sundstrom,
EMBO J. 11, 2373 (1992).
8. D. Kern et al., Science 275, 67 (1997).
9. M.E.Caines,J.M.Elkins,K.S.Hewitson,C.J.Schofield,
J. Biol. Chem. 279, 5685 (2004).
10. M. Fries, H. I. Jung, R. N. Perham, Biochemistry 42,
6996 (2003).
11. N. Nemeria et al., Biochemistry 41, 15459 (2002).
12. R. N. Perham, Annu. Rev. Biochem. 69, 961 (2000).
13. I. A. Lessard, C. Fuller, R. N. Perham, Biochemistry 35,
16863 (1996).
14. H. J. Chauhan, G. J. Domingo, H. I. Jung, R. N. Perham,
Eur. J. Biochem. 267, 7158 (2000).
15. D. D. Jones, K. M. Stott, P. A. Reche, R. N. Perham,
J. Mol. Biol. 305, 49 (2001).
16. R.A.W.Frank,J.V.Pratap,X.Y.Pei,R.N.Perham,
B. Luisi, in preparation.
17. T. Nakai et al., J. Mol. Biol. 337, 1011 (2004).
18. A. Ævarsson, K. Seger, S. Turley, J. R. Sokatch, W. G. Hol,
Nature Struct. Biol. 6, 785 (1999).
19. A. Ævarsson et al., Structure Fold. Des. 8, 277 (2000).
20. E.M.Ciszak,L.G.Korotchkina,P.M.Dominiak,S.Sidhu,
M. S. Patel, J. Biol. Chem. 278, 21240 (2003).
21. P. Arjunan et al., Biochemistry 41, 5213 (2002).
22. M. Nikkola, Y. Lindqvist, G. Schneider, J. Mol. Biol.
238, 387 (1994).
23. Supporting data are available on Science Online.
24. M. Sundstrom, Y. Lindqvist, G. Schneider, FEBS Lett.
313, 229 (1992).
25. X. Huang, H. M. Holden, F. M. Raushel, Annu. Rev.
Biochem. 70, 149 (2001).
26. D. Hawksley, D. A. Griffin, F. J. Leeper, J. Chem. Soc.
[Perkin 1] 2001, 144 (2001).
27. W. Lattimer, W. Rodebush, J. Am. Chem. Soc. 42,
1419 (1920).
28. H. Bisswanger, U. Henning, Eur. J. Biochem. 24, 376
(1971).
29. G. Lu, D. Dobritzsch, S. Baumann, G. Schneider, S. Konig,
Eur. J. Biochem. 267, 861 (2000).
30. A. Levitzki, D. E. Koshland Jr., Curr. Top. Cell. Regul.
10, 1 (1976).
31. A. Szoke, W. G. Scott, J. Hajdu, FEBS Lett. 553, 18 (2003).
32. P. H. Sugden, P. J. Randle, Biochem. J. 173, 659 (1978).
33. E. A. Sergienko, F. Jordan, Biochemistry 41, 3952 (2002).
34. F. Jordan, Nat. Prod. Rep. 20, 184 (2003).
35. We are grateful to D. Hawksley and F. Leeper
(Chemical Laboratory, University of Cambridge) for
the generous gift of deazathiamine diphosphate and
advice. We thank L. Packman and co-workers from
the PNAC, Department of Biochemistry, University
of Cambridge, for the synthesis of oligonucleotides
and N-terminal sequencing and H. Dixon for stim-
ulating discussions and many helpful comments. This
work was supported by grants from the Biotechnol-
ogy and Biological Sciences Research Council (BBSRC)
and the Wellcome Trust. The coordinates and structure
factors of the E1-PSBD and E1 (NQ)-PSBD have been
deposited at the Protein Data Bank (accession code
1w85 and 1w88, respectively).
Supporting Online Material
www.sciencemag.org/cgi/content/full/306/5697/872/
DC1
Materials and Methods
SOM Text
Figs. S1 to S5
Tables S1 and S2
References and Notes
3 June 2004; accepted 14 September 2004
Abnormal Cytokinesis in Cells
Deficient in the Breast Cancer
Susceptibility Protein BRCA2
Matthew J. Daniels,
*
Yunmei Wang,
*
MiYoung Lee,
Ashok R. Venkitaraman.
Germ-line mutations inactivating BRCA2 predispose to cancer. BRCA2-deficient
cells exhibit alterations in chromosome number (aneuploidy), as well as
structurally aberrant chromosomes. Here, we show that BRCA2 deficiency
impairs the completion of cell division by cytokinesis. BRCA2 inactivation in
murine embryo fibroblasts (MEFs) and HeLa cells by targeted gene disruption or
RNA interference delays and prevents cell cleavage. Impeded cell separation is
accompanied by abnormalities in myosin II organization during the late stages
in cytokinesis. BRCA2 may have a role in regulating these events, as it localizes
to the cytokinetic midbody. Our findings thus link cytokinetic abnormalities to
a hereditary cancer syndrome characterized by chromosomal instability and
may help to explain why BRCA2-deficient tumors are frequently aneuploid.
Inherited mutations affecting the BRCA2
tumor suppressor predispose to breast, ovar-
ian, and other epithelial cancers with high
penetrance (1). BRCA2-deficient cells accu-
mulate gross chromosomal rearrangements,
including translocations and large deletions
29 OCTOBER 2004 VOL 306 SCIENCE www.sciencemag.org
876
R
EPORTS
on July 10, 2008 www.sciencemag.orgDownloaded from

during cell division (2–5), anomalies that are
attributed to the control by BRCA2 of the
RAD51 enzyme in reactions for DNA repair
and recombination, during the S phase of the
cell cycle (6). However, BRCA2 inactivation
also triggers alterations in chromosome
number (3–5) and abnormalities, such as
centrosome amplification (4), that might
arise from distinct roles during other cell-
cycle phases. For instance, inhibition of
BRCA2 by antibody microinjection is
reported to delay the transition from G
2
to
Mphase(7), a period when BRCA2 is
phosphorylated by the mitotic kinase, Plk1
(8, 9).
To investigate possible functions of
BRCA2 during mitosis, we monitored cell di-
vision by serial time-lapse imaging in murine
embryo fibroblast (MEF) cultures homozy-
gous for a targeted mutation (Brca2
Tr
)that
truncates and inactivates Brca2 (3, 10). MEF
cultures isolated from littermate embryos
with the Brca2
þ/þ
or Brca2
Tr/þ
genotype at
an identical passage in culture served as
controls. A frequency distribution is shown
in Fig. 1A for the time taken for cells to
progress from anaphase onset (when chromo-
some segregation becomes visible), to com-
plete daughter cell separation. Although in
Brca2
þ/þ
cells (controls), this takes about 35
min, it is slightly prolonged (median, 45 min)
in Brca2
Tr/þ
cells and is severely extended in
Brca2
Tr/Tr
cells (median, 90 min). Binucleate
cells, the product of incomplete cell division,
occur frequently after Brca2 inactivation
(Fig. 1B). A representative series of time-
lapse images (fig. S1) shows delayed pro-
gression through cytokinesis, culminating in
nuclear division without cell separation, and
generating a binucleate cell. The frequency of
binucleates increases during the passage of
Brca2
Tr/Tr
cells in culture (supporting online
University of Cambridge, Cancer Research UK, De-
partment of Oncology and the Medical Research
Council (MRC) Cancer Cell Unit, Hutchison/MRC
Research Centre, Hills Road, Cambridge CB2 2XZ, UK.
*These authors contributed equally to this work.
.To whom correspondence should be addressed.
E-mail: arv22@cam.ac.uk
Fig. 1. BRCA2 inactivation delays and prevents
cytokinesis. (A) Frequency distribution of the
time taken after anaphase to complete cytoki-
nesis. Brca2
Tr/Tr
MEFs during their second pas-
sage in culture were compared with Brca2
Tr/þ
and Brca2
þ/þ
cells from litter-mate embryos.
Mitosis was monitored in living cells by serial
time-lapse imaging (12). Live cells were visual-
ized by bright-field microscopy every 5 min
from anaphase onset until completion of cell
separation, or for up to 6 hours. The percentage
of cells on the vertical axis is plotted against
time taken to complete cytokinesis. Cells that
failed to complete cytokinesis within 6 hours are
enumerated under ‘‘Fail to divide.’’ Results
shown are typical of at least three independent
experiments, using three distinct MEF cultures for each genotype. (B) Cells that failed to
divide after 6 hours were considered in two groups: the percentage of those that remain in
mitosis without completing cytokinesis (‘‘Not completed cytokinesis’’) and those that complete
nuclear division but not cytokinesis (‘‘Binucleate cells, no cytokinesis’’). (C) BRCA2 depletion by
siRNA delays cytokinesis and provokes abnormal divisions. HeLa cells treated with control or
BRCA2 siRNAs (12) (fig. S2) were monitored by serial time-lapse imaging as above.
ab
cd
A
n= 79
n= 103
n= 123
n= 105
B
Abscission CleavageEnlarged
Control
siRNA
BRCA2
siRNA
Myosin II
Tubulin
DNA
C
Abscission
Myosin II
Tubulin
DNA
Capan-1
Enlarged
Cleavage Abscission
Control siRNA
BRCA2 siRNA
Abnormal myosin II
organization (%)
60
30
0
Fig. 2. Myosin II mislocalization after BRCA2 inactivation.
(A) Myosin II distribution during cleavage and abscission in
HeLa cells treated with control or BRCA2 siRNAs. Repre-
sentative images of cells co-stained (12) with DAPI and
antibodies against myosin II or tubulin are shown. Arrows
mark myosin II accumulation at the site of furrow
formation in (a). The areas boxed in white in (c) and (d)
show the midbody and are enlarged below. (B) Frequency of
abnormal myosin II organization in cells undergoing cleav-
age or abscission steps during cytokinesis. The number of
stained cells analyzed in each sample is indicated by n.(C)
Abnormal abscission in Capan-1 cells. Staining for DNA,
myosin II, and tubulin was performed as described above. A representative image is shown. The
area boxed in white is enlarged to the right. Scale bars, 5 6m.
www.sciencemag.org SCIENCE VOL 306 29 OCTOBER 2004
877
R
EPORTS
on July 10, 2008 www.sciencemag.orgDownloaded from

text), but is not significantly changed by
different culture densities (from 0.4 to 1.2 
10
5
cells per ml) or by the addition of
Matrigel extracellular matrix (11).
Cytokinetic abnormalities also apparently
occur in vivo. Binucleation is more than 30
times as frequent (2.7 T 0.7%) in cells
freshly isolated from day 13 to 14 mutant
embryos than in wild-type controls (0.08 T
0.01%). Indeed, although some Brca2
Tr/Tr
embryos survive (with growth retardation
and developmental defects) until later stages
in development, up to 50% are lost early
during embryogenesis (10).
Cytokinesis is also delayed or prevented
when BRCA2 is depleted by RNA interfer-
ence using short, interfering (si)RNAs (12)
(fig. S2). The period from anaphase onset to
completion of cell division is significantly
extended in HeLa cells treated with BRCA2
siRNA (median, 112 min) when compared
with controls (median, 60 min); again, this
delay is associated with failure to divide
(Fig. 1C).
Assembly and activation of the actomyosin
contractile ring are key events during cyto-
kinesis (13, 14), brought about by the or-
ganization of actin and type II myosin in
the ingressing cleavage furrow. During cleav-
age (fig. S3), myosin II normally concen-
trates at the site of furrow formation EFig.
2A, arrows in (a)^. This is not seen in more
than 50% of cells treated with BRCA2
siRNA EFig. 2, A (b) and B^. Moreover,
during the abscission of daughter cells in
telophase, the normal accumulation of myo-
sin II as a band at each cell edge EFig. 2A
(c), enlarged image^ is undetectable in many
cells after BRCA2 depletion EFig. 2, A (d)
and B^.
A significant accumulation of Brca2
Tr/Tr
MEFs in abscission suggests delayed pro-
gression through late stages in cytokinesis.
Brca2
Tr/Tr
MEFs in abscission compose 64 T
10% of all cells in stages from anaphase
onset to cell separation (n = 158 in three ex-
periments), compared with 36 T 7% of wild-
type controls E(n =86),P =0.04bythe
two-tailed t test^. Cells treated with BRCA2
siRNA also tend to accumulate in abscis-
sion E56 T 6% of all cells from anaphase
onset to cell separation (n 0 191 cells in
three experiments), compared with 47 T 3%
of control siRNA E(n 0 193), P 0 0.1^.
Incomplete BRCA2 depletion by siRNA (fig.
S2) may explain why this effect is not more
pronounced.
Similar abnormalities in cytokinesis oc-
cur in an epithelial cancer cell line, Capan-1,
isolated from a patient carrying the non-
functional BRCA2 6174delT mutation (15).
Asynchronous cultures contain many binu-
cleate cells Emean frequency, 12 T 2% (n 0
500)^, and show abnormal myosin II orga-
nization and midbody morphology during
abscission (Fig. 2C).
The intracellular localization of BRCA2
during cytokinesis is consistent with its
possible participation in these events. A key
group of proteins implicated in cytokinesis
(16–19), including the inner centromere
protein (INCENP), Aurora B kinase, and
survivin, localize to central structures dur-
ing cell separation. BRCA2 follows a sim-
ilar but not identical pattern. In human cells
labeled with the DNA dye DAPI (4¶,6¶-
diamidino-2-phenylindole) and with anti-
bodies against BRCA2 (12) and Aurora B,
BRCA2 colocalizes with Aurora B in cen-
tral structures during the elongation stage of
cytokinesis (arrow in Fig. 3D), and notably,
both proteins accumulate in the midbody
during late cleavage and abscission (Fig. 3,
B and C).
Collectively, our findings suggest that
BRCA2 may regulate the fidelity of late
stages in cytokinesis but is not an essential
component of the machinery for cell separa-
tion. Thus, BRCA2-deficient cells experience
considerable delays in cytokinesis, but many,
nevertheless, go on to complete cell division.
Abnormal organization of myosin II in the
contractile ring occurs during cleavage and
abscission. BRCA2 may have a role in reg-
ulating these events, as it migrates from cen-
tral structures during the elongation phase of
cytokinesis to the cytokinetic midbody during
cleavage and abscission.
BRCA2 is required for the repair of DNA
double-strand breaks by recombination (20),
and BRCA2-deficient cells spontaneously
acquire DNA breaks and structurally aber-
rant chromosomes during the S phase (3–5).
Elongation Cleavage Abscission
MergeBRCA2DNA AuroraB
A B C
DEF
G H I
J K L
Fig. 3. Localization of BRCA2 to cytokinetic structures. The panels show typical HeLa cells at the
elongation, late cleavage, and abscission steps in cytokinesis. Staining was with DAPI and mouse
monoclonal antibodies against Aurora B and BRCA2 (12) (fig. S4). Arrows mark BRCA2 staining in
(D). In the Merge images (A to C), co-localization of the red and green channels appears as yellow-
green areas. (D to L) Individual images from which Merge panels were assembled. Scale bars, 5 6m.
29 OCTOBER 2004 VOL 306 SCIENCE www.sciencemag.org
878
R
EPORTS
on July 10, 2008 www.sciencemag.orgDownloaded from

Attempts to segregate malformed chromo-
somes, if they lag on the central spindle,
might prolong or prevent cytokinesis. But it
is difficult to explain delayed abscission,
abnormal myosin II organization, or the
mitotic localization pattern of BRCA2 on
this basis.
Besides structurally aberrant chromo-
somes, primary cultures of BRCA2-deficient
cells accumulate with 4N and greater DNA
content during successive passage, consistent
with exit from mitosis without cytokinesis (3).
Also, aneuploidy often occurs in cancers from
BRCA2 mutation carriers (2) and might, as in
sporadic cancers, predict poor clinical out-
comes. Our findings suggest that both these
phenotypes may arise from the inactivation of
previously unrecognized functions of BRCA2
in cytokinesis, which is a possible link
between cytokinetic abnormalities and the
pathogenesis of a human genetic disease as-
sociated with chromosomal instability and
cancer predisposition.
References and Notes
1. K. N. Nathanson, R. Wooster, B. L. Weber, Nature
Med. 7, 552 (2001).
2. S. Gretarsdottir et al., Cancer Res. 58, 859 (1998).
3. K. J. Patel et al., Mol. Cell 1, 347 (1998).
4. A. Tutt et al., Curr. Biol. 9, 1107 (1999).
5. V. P. C. C. Yu et al., Genes Dev. 14, 1400 (2000).
6. A. R. Venkitaraman, Cell 108, 171 (2002).
7. L. Y. Marmorstein et al., Cell 104, 247 (2001).
8. H. R. Lin, N. S. Ting, J. Qin, W. H. Lee, J. Biol. Chem.
278, 35979 (2003).
9. M. Lee, M. J. Daniels, A. R. Venkitaraman, Oncogene
23, 865 (2004).
10. L. S. Friedman et al., Cancer Res. 58, 1338 (1998).
11. Y. Wang, unpublished results.
12. Materials and methods are available as supporting
material on Science Online.
13. M. Glotzer, Annu. Rev. Cell Dev. Biol. 17, 351 (2001).
14. J. M. Scholey, I. Brust-Mascher, A. Mogilner, Nature
422, 746 (2003).
15. M. Goggins et al., Cancer Res. 56, 5360 (1996).
16. S. Kaitna, M. Mendoza, V. Jantsch-Plunger, M. Glotzer,
Curr. Biol. 10, 1172 (2000).
17. A. F. Severson, D. R. Hamill, J. C. Carter, J. Schumacher,
B. Bowerman, Curr. Biol. 10, 1162 (2000).
18. S. P. Wheatley, A. Carvalho, P. Vagnarelli, W. C.
Earnshaw, Curr. Biol. 11, 886 (2001).
19. R. R. Adams, H. Maiato, W. C. Earnshaw, M. Carmena,
J. Cell Biol. 153, 865 (2001).
20. M. E. Moynahan, A. J. Pierce, M. Jasin, Mol. Cell 7,
263 (2001).
21. We thank J. Pines and L.K. Ferrigno (Cambridge) for
helpful comments on this paper. M.J.D. received an
AstraZeneca studentship through the Cambridge
University M.B., Ph.D. program. Work in A.R.V.’s
laboratory is supported by Cancer Research UK and
the Medical Research Council.
Supporting Online Material
www.sciencemag.org/cgi/content/full/1102574/DC1
Materials and Methods
SOM Text
Figs. S1 to S4
References and Notes
9 July 2004; accepted 7 September 2004
Published online 16 September 2004;
10.1126/science.1102574
Include this information when citing this paper.
Early-Life Blockade of the 5-HT
Transporter Alters Emotional
Behavior in Adult Mice
Mark S. Ansorge,
1,2,3
Mingming Zhou,
2,3
Alena Lira,
2,3
Rene
´
Hen,
2,4
Jay A. Gingrich
2,3
*
Reduced serotonin transporter (5-HTT) expression is associated with abnormal
affective and anxiety-like symptoms in humans and rodents, but the
mechanism of this effect is unknown. Transient inhibition of 5-HTT during
early development with fluoxetine, a commonly used serotonin selective
reuptake inhibitor, produced abnormal emotional behaviors in adult mice. This
effect mimicked the behavioral phenotype of mice genetically deficient in
5-HTT expression. These findings indicate a critical role of serotonin in the
maturation of brain systems that modulate emotional function in the adult
and suggest a developmental mechanism to explain how low-expressing 5-HTT
promoter alleles increase vulnerability to psychiatric disorders.
5-HTT appears to be a critical regulator of
emotional function. It is the primary molecular
target for many antidepressants, especially the
serotonin selective reuptake inhibitors (SSRIs),
which are used as a first-line treatment for a
number of psychiatric conditions (1). SSRIs
increase serotonergic tone, and this effect is
thought to mediate their therapeutic actions.
A genetic variant that reduces expression
of 5-HTT has been associated with elevated
levels of neuroticism, anxiety-like traits, and
depressive symptoms in some (2–4) but not
all studies (5). A study including environ-
mental factors in its analysis demonstrated
that individuals with one or two copies of the
low-expressing 5-HTT allele are more prone
to depression and suicidality only after
childhood or adulthood stressors (6). Such a
gene-environment interaction may explain
the variability between studies.
Mice lacking the 5-HTT gene (5-HTT
j/j
)
also exhibit increased depression- and anxiety-
related behaviors (7, 8). The emotional and
behavioral abnormalities produced by genet-
ically reduced 5-HTT function are paradoxical
because in a mature organism, long-term
treatments with SSRI antidepressants also
produce a reduction of 5-HTT function, yet
these agents act to ameliorate anxiety- and
depression-related symptoms.
Because 5-HT acts as a trophic factor
modulating developmental processes such as
neuronal division, differentiation, migration,
and synaptogenesis (9), we hypothesized that
the divergent effects of adult pharmacologic
and lifelong genetic inhibition of 5-HTT
function may be explained by events occur-
ring during early brain maturation (10). Thus,
we investigated whether we could mimic the
effect of genetic 5-HTT disruption by briefly
inhibiting 5-HTT function between postnatal
days 4 and 21 (P4 and P21) with the use of
the SSRI fluoxetine (FLX) in mice.
Mice heterozygous for the 5-HTT muta-
tion (5-HTT
þ/
j
) were crossed to produce a
Mendelian mix of 5-HTT
þ/þ
, 5-HTT
þ/
j
, and
5-HTT
j/j
offspring (11). Mixed litters were
randomly assigned to either saline or FLX
(10 mg/kg, intraperitoneally) treatments be-
ginning on P4 and lasting until P21. This
design allowed us to directly compare the
behavioral effects of transient pharmacolog-
ical 5-HTT inhibition and constitutive dis-
ruption of the 5-HTT gene.
We chose to use FLX to pharmacologi-
cally block 5-HTT function because of its
common use in humans and its extended
half-life. Our dosing regimen produced
therapeutically relevant blood levels (FLX:
360 T 123 ng/ml; norfluoxetine: 708 T 168
ng/ml) and had no gross effects on viability
or growth (fig. S1, A and B). Although FLX
has high selectivity for 5-HTT, it is reported
to exhibit weak activity at other transporter
and receptor sites (12). The specificity of FLX
wasmonitoredin5-HTT
j/j
mice because
these mice allowed us to distinguish between
5-HTT–mediated and 5-HTT–independent
effects of FLX.
Starting at 12 weeks of age (9 weeks after
the last injection of FLX), we tested mice in the
open field and in the elevated plus-maze. In
comparison to saline-treated pups, postnatal-
FLX (PN-FLX) treatment decreased explorato-
ry behavior in both 5-HTT
þ/þ
and 5-HTT
þ/
j
1
Sackler Institute for Developmental Psychobiology,
2
Department of Psychiatry, Columbia University
College of Physicians and Surgeons, New York, NY
10032, USA.
3
Department of Developmental Psycho-
biology,
4
Department of Neurobiology and Behavior,
New York State Psychiatric Institute, New York, NY
10032, USA.
*To whom correspondence should be addressed.
E-mail: jag46@columbia.edu
www.sciencemag.org SCIENCE VOL 306 29 OCTOBER 2004
879
R
EPORTS
on July 10, 2008 www.sciencemag.orgDownloaded from