Am J Physiol Regul Integr Comp Physiol 288: R511–R521, 2005.
First published October 21, 2004; doi:10.1152/ajpregu.00520.2004.
Hypoxia induces major effects on cell cycle kinetics and
protein expression in Drosophila melanogaster embryos
R. M. Douglas,1 R. Farahani,1 P. Morcillo,1 A. Kanaan,1 T. Xu,3,4 and G. G. Haddad1,2
Departments of 1Pediatrics (Division of Respiratory Medicine) and 2Neuroscience, Albert Einstein College of
Medicine, New York, New York; and 3Department of Genetics, Boyer Center for Molecular Medicine and
4
The Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut
Submitted 3 August 2004; accepted in final form 13 October 2004
cyclin A; E2F1; oxygen deprivation; stabilization of expression
comprises several stages or phases that include
gap phase 1 (G1), DNA replication (S phase), gap phase 2
(G2), and mitosis (M phase) (49). Movement through the
various stages of the cell cycle is accomplished by two essential mechanisms: the sequential accumulation and degradation
of several cell cycle regulatory proteins, such as the cyclins,
and the sequential activation and inactivation of their cognate
partners, the cdks, and other protein complexes (35, 48, 84).
Therefore, there are two mechanisms of control of the cell
cycle: levels of protein expression and the phosphorylation
state of these proteins. The degradative pathways that operate
during the cell cycle include the Skp1, cullin (cdc53), and
F-box protein cdc4 (SCF) family of ubiquitin ligases at the
G1/S transition and the anaphase-promoting complex (APC) at
the G2/M transition, with some overlap. These families of
ubiquitin-conjugating ligases, in conjunction with the proteosome 26S or cyclosome, mediate the destruction of agents such
as cyclins A and B to permit the exit from metaphase (35, 82)
THE CELL CYCLE
Address for reprint requests and other correspondence: G. G. Haddad, Dept.
of Pediatrics, Albert Einstein College of Medicine of Yeshiva University, and
the Children’s Hospital at Montefiore, Rose F. Kennedy Center, Rm. 845, 1410
Pelham Parkway South, Bronx, NY 10461 (E-mail: [email protected]).
http://www.ajpregu.org
and the degradation of several G1/S regulatory proteins such as
E2F1, cyclin D, and cyclin E (36, 61), respectively.
Furthermore, the cell cycle is modulated by a series of
intrinsic and extrinsic factors. Intrinsic control mechanisms
include the oscillatory activity of protein complexes, such as
the cyclin/cdk, that determine the periodicity of the cell cycle
and checkpoint apparatus that can halt cell cycle activity in the
event of an endogenous stress, such as incomplete DNA
replication. The intrinsic factors also include mutations of
several cell cycle genes (35, 48, 84). Extrinsic stimuli that can
influence cell cycle progression include mitogenic factors, such
as growth factors, and a variety of environmental stresses (see
Refs. 22 and 42).
Environmental stresses can have direct effects on cell cycle
activity and therefore on cell division and growth of organs and
tissues, especially early in life. Ultraviolet (UV) and ionizing
(IR) radiation is known to cause DNA damage that leads to cell
cycle delay, cell cycle arrest, dysplasia, and/or cell death (8,
52, 60, 63). Other factors such as heat, overcrowding, and
desiccation also can lead to cell cycle arrest (17). Oxygen (O2)
deprivation or hypoxia is another such stress that appears to
have a direct effect on cell cycle activity and growth (18, 30).
Hypoxia can lead to either hypoplasia, as seen in the growth
retardation reported in response to in utero hypoxia (23), or
even hyperplasia, as demonstrated in the mouse pulmonary
vasculature (29), as well as in the Drosophila tracheal system
(34). Additionally, it has been demonstrated that hypoxia
induces multiple effects on early rapidly cycling embryos of
different species and can affect the cell cycle in a variety of
ways (7, 18, 24, 56, 79). It can cause the cycle to halt
completely, both reversibly (its activity resumes upon reoxygenation) and irreversibly (which leads to cell death) (2, 24, 81).
In the Drosophila embryo, the first 13 cell cycles are rapid
and under the control of the products loaded by the mother.
These early cycles exhibit only S and M phases. However, the
zygotic control starts at cell cycle 14, and, more importantly,
overexpression of String before cycle 14 will synchronize the
embryos to the premitotic stage of cycle 14 (16).
Recently, we (13) and others (7, 12, 39, 56, 81) have
demonstrated that hypoxia can lead to cell cycle arrest at either
metaphase or pre-S phase in early cycling (stages 1–13) embryos. The question arises as to which specific mechanisms are
employed by the cell in response to hypoxia to induce cell
cycle delay or arrest. To understand the mechanisms involved
in the hypoxia-induced metaphase and pre-S phase cell cycle
The costs of publication of this article were defrayed in part by the payment
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0363-6119/05 $8.00 Copyright © 2005 the American Physiological Society
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Douglas, R. M., R. Farahani, P. Morcillo, A. Kanaan, T. Xu,
and G. G. Haddad. Hypoxia induces major effects on cell cycle
kinetics and protein expression in Drosophila melanogaster embryos.
Am J Physiol Regul Integr Comp Physiol 288: R511–R521, 2005.
First published October 21, 2004; doi:10.1152/ajpregu.00520.2004.—
Hypoxia induces a stereotypic response in Drosophila melanogaster
embryos: depending on the time of hypoxia, embryos arrest cell cycle
activity either at metaphase or just before S phase. To understand the
mechanisms underlying hypoxia-induced arrest, two kinds of experiments were conducted. First, embryos carrying a kinesin-green fluorescent protein construct, which permits in vivo confocal microscopic visualization of the cell cycle, showed a dose-response relation
between O2 level and cell cycle length. For example, mild hypoxia
(PO2 ⬃55 Torr) had no apparent effect on cell cycle length, whereas
severe hypoxia (PO2 ⬃25–35 Torr) or anoxia (PO2 ⫽ 0 Torr) arrested
the cell cycle. Second, we utilized Drosophila embryos carrying a
heat shock promoter driving the string (cdc25) gene (HS-STG3),
which permits synchronization of embryos before the start of mitosis.
Under conditions of anoxia, we induced a stabilization or an increase
in the expression of several G1/S (e.g., dE2F1, RBF2) and G2/M (e.g.,
cyclin A, cyclin B, dWee1) proteins. This study suggests that, in fruit
fly embryos, 1) there is a dose-dependent relationship between cell
cycle length and O2 levels in fruit fly embryos, and 2) stabilized cyclin
A and E2F1 are likely to be the mediators of hypoxia-induced arrest
at metaphase and pre-S phase.
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HYPOXIA AFFECTS CELL CYCLE AND PROTEINS IN FLY EMBRYOS
arrests observed in fruit fly embryos, we have asked two
questions. 1) Is there a hypoxic threshold for the arrest? 2)
What proteins have altered levels of expression that could
explain the hypoxia-induced arrest?
MATERIALS AND METHODS
Materials
Fly Stocks
Wild-type Canton-S strain containing the kinesin-green fluorescent
protein (K-GFP) construct (Ma⫹-⫹-GFP2l2 [II]) was obtained from
T. Xu (Yale Univ. School of Medicine, New Haven, CT). The heat
shock String fly strain [w, Df(w)67; HS-STG3/TM6B] was provided by
B. A. Edgar. The double-construct fly strain containing both K-GFP
and HS-STG (w; K-GFP/K-GFP; HS-STG/TM6B) was generated in
our laboratory.
Egg Collection and Preparation
Fly stocks were maintained at 25°C under standard conditions.
Embryos were collected on apple juice plates supplemented with a
small amount of yeast paste. Generally, adult fruit flies were anesthetized with 100% CO2, and, after a recovery period of 1–2 h, the first
collection was discarded to eliminate old eggs from the sample
population (16). To synchronize the eggs, two rapid 10-min collections were made and discarded. Subsequent collections for experimental purposes were made at 30 min, 1 h, 2 h, or 4 h after egg
deposition (AED). Embryos were rinsed from the agar plates into a
Nytex mesh holder with a solution containing 0.7% NaCl and 0.02%
Triton X-100 (TX-100; Sigma, St. Louis, MO). They were then
thoroughly rinsed with distilled deionized H2O (DDW). Embryos
were then dechorionated in 50% domestic bleach (sodium hypochlorite; Chlorox, Oakland, CA) in DDW for 2 min and again rinsed
thoroughly with DDW before any experimental manipulation.
Observing Living Embryos During Graded Hypoxia
Dechorionated K-GFP fly embryos were mounted onto coverslips,
and each coverslip was placed into a specially designed perfusion
chamber. The embryos were then enclosed within the perfusion
AJP-Regul Integr Comp Physiol • VOL
Immunoblot and Immunocytochemical Analysis on
Drosophila Embryos
Synchronization protocol. To generate a homogeneous population
of embryos, we utilized a fruit fly strain carrying a heat shock protein
70 promoter-String (yeast homolog, cdc25) construct (HS-STG3) that
permits embryonic cells, resting in G2, to be heat pulsed into an early
but well-synchronized mitosis and subsequent S phase. After several
precollections, as previously described, HS-STG3 embryos were collected on 100-mm apple juice plates over a 30-min period. The
HS-STG3 embryos were then aged for 130 min after egg deposition
(115–145 min AED) to generate embryos that had completed the first
13 cell cycle (nuclear) divisions and had entered the first G2 gap phase
of Drosophila embryogenesis (cycle 14, Fig. 1). During the aging
process, the embryos were dechorionated, returned to the agar plates,
and subsequently placed in an incubator at 37°C for 40 min. A heat
shock pulse of this duration induces global string mRNA and String
(STG) protein expression in the embryo and synchronously drives all
G2 cells into mitosis at cycle 14 within ⬃5–10 min of returning the
embryos to room temperature (RT). Mitosis is generally completed in
10 –20 min, and an S phase that lasts for 45 min immediately follows
this “premature” mitosis. The cells then return to G2 (cycle 14) and
remain there for 70 –170 min before resuming the normal developmental program (16). This was confirmed by in vivo observation of
the K-GFP/HS-STG3 double construct (data not shown).
Anoxic exposure. Synchronized, dechorionated HS-STG3 embryos
were placed into a specially designed anoxia chamber that allowed the
manipulation of the embryos while totally preserving the anoxic
environment. The chamber was continuously gassed with 100% N2
through a single port. Two small escape ports were located on the
other side of the chamber to allow for normalization of internal
chamber pressure. O2 concentration was monitored continuously with
a Clark-like O2 electrode (DO-051; Cameron Instrument, Aransas,
TX). After calibration of the Clark electrode with alternating 100% N2
and room air (21% O2) gases, the chamber was gassed with 100% N2
(0% O2) for ⬃30 min before insertion of the embryos submerged in
Shield’s and Sang medium in 15-ml centrifuge tubes into the chamber.
Tubing connected to the N2 source was also placed directly into the
15-ml centrifuge tubes containing the embryos to ensure total anoxia
in the microenvironment of the embryos. The PO2 in the anoxia
chamber was maintained at 0 Torr, as continuously measured with the
Clark O2 electrode, for the duration of the anoxic episode. After the
heat pulse, embryos were held at RT for 4.5 or 12.5 min before anoxic
exposure to recapitulate the arrests at metaphase (M) and pre-S phase
(S), respectively (Fig. 1). Heat-pulsed embryos were then subjected to
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Several antibodies were obtained from the Developmental Studies
Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the
University of Iowa, Department of Biological Sciences (Iowa City,
IA). These include monoclonal antibodies to ␤-tubulin (E7), cyclin A
(A12), and cyclin B (F2F4), which were developed by M.
Klymkowsky, C. F. Lehner, and P. H. O’Farrell, respectively. Rabbit
polyclonal antibodies to cyclin A (Rb270) and cyclin B (Rb271) and
a mouse monoclonal antibody to ␤-tubulin (Bx69) were also obtained
from David M. Glover (Cambridge University, Cambridge, UK).
Additionally, a mouse monoclonal antibody to cyclin B was provided
by J. Raff (Wellcome/CRC Institute, Cambridge, UK), and a mouse
monoclonal antibody to ␤-tubulin was obtained from Accurate Biochemical and Scientific (Westbury, NY). Antibodies to E2F1 were
provided by T. Orr-Weaver (Whitehead Institute, MIT, Cambridge,
MA) and by M. Asano of Duke University Medical Center (Durham,
NC). The mouse monoclonal antibodies to E2F2 (Mei-8), RBF1
(DX-3, DX-5), RBF2 (DR6), and dDp (Yun-3, Yun-5) were kind gifts
from N. Dyson (Massachusetts General Hospital Cancer Center,
Harvard University, Boston, MA), and a rabbit polyclonal anti-dWee1
antibody was donated by S. Campbell (Univ. of Alberta, Edmonton,
Canada). Rabbit polyclonal antibodies to cdk1 (cdc2) and String
(cdc25) were generously provided by B. A. Edgar (Fred Hutchinson
Cancer Research Center, Seattle, WA), and the mouse monoclonal
antibody to Fizzy (cdc20) was a gift from I. Dawson (Molecular and
Cellular Biology, Yale University, New Haven, CT).
chamber with a circular Thermanox plastic coverslip (15-mm diameter; Nalge Nunc, Naperville, IL), immediately perfused with normoxic insect medium (Shields and Sang M3 Insect Medium, Sigma)
and viewed with a Bio-Rad MRC-1024 confocal microscope system
(Bio-Rad Laboratories, Hercules, CA). Images were captured every
20 – 40 s from K-GFP embryos for one or two cell cycles under
control normoxic conditions before switching to a hypoxic insect
medium. The hypoxic medium had been previously bubbled with
either 100% N2 (severe hypoxia, PO2 ⬃25–35 Torr), 5% O2 (mild
hypoxia, PO2 ⬃55– 60 Torr), or 2% O2 (moderate hypoxia, PO2
⬃40 – 45 Torr) for 2 h before the experiment and was continuously
bubbled with the same gas throughout the experiment. The design of
our perfusion chamber for in vivo confocal studies of embryos
permitted us to study a dose response under mild, moderate, and
severe hypoxia, as defined above. However, because of the openended design of the chamber, we could not reach anoxia (0 Torr) in
these studies. Perfusion with the hypoxic solution was maintained for
30 min to 2 h. After the hypoxic period, perfusion with normoxic
insect medium was reinitiated, and, on the resumption of cell cycle
activity, embryos were observed for an additional one or two cell
cycles during reoxygenation.
HYPOXIA AFFECTS CELL CYCLE AND PROTEINS IN FLY EMBRYOS
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anoxia for either 5 min (MA1 and IB1) or 30 min (MA2 and IB2) to
ascertain the effect of acute and protracted anoxia on protein expression (Fig. 1). At the end of the anoxic exposure, embryos were quickly
removed and frozen in liquid N2 for immunoblot analyses. The
appropriate controls were also run concurrently in medium maintained
in room air or bubbled with 21% O2 (C1–C6). These embryos were
then quickly frozen in liquid N2 for immunoblotting (n ⫽ 3).
Alternatively, 100 embryos were hand-counted before being placed
into the anoxia chamber on agar plates and subjected to the same
protocol (n ⫽ 3). Data were pooled from these six experiments
(n ⫽ 6).
Immunoblotting. For Western blotting, the embryos were homogenized with a glass-Teflon homogenizer (Thomas Scientific, Swedesboro, NJ) in Laemmli sample buffer (2% SDS, 10% glycerol, 67 mM
Tris, 20 mM EDTA, 20 mM EGTA, 0.1% ␤-mercaptoethanol, pH 6.7)
containing a cocktail of protease inhibitors. Protease inhibitors included a Complete tablet containing pronase, thermolysin, chymotrypsin, trypsin, and papain (Boehringer-Mannheim, Mannheim, Germany), a RIPA cocktail composed of pepstatin A (Sigma), leupeptin
(Roche, Mannheim, Germany), and aprotinin (Boehringer-Mannheim), as well as phenylmethylsulfonylfluoride (PMSF; Sigma). Embryo samples were then assayed for protein content (BCA Assay;
Pierce Chemical, Rockford, IL), and 10 –20 ␮g of total protein in a 4⫻
sample loading buffer (2% SDS, 10% glycerol, 67 mM Tris, 20 ␮M
EDTA, 20 ␮M EGTA, 0.1% ␤-mercaptoethanol, pH 6.7, with a
Complete tablet and PMSF) were loaded per lane for SDS-PAGE and
subsequent transfer to polyvinylidene difluoride (PVDF) membranes
(Immobilin-P, Millipore, Bedford, MA). Proteins were detected with
secondary antibodies conjugated to horseradish peroxidase (Jackson
ImmunoResearch Laboratories, Westgrove, PA, and Zymed Laboratories, San Francisco, CA) and the enhanced chemiluminescence
system (Amersham, Little Chalfont, UK). Scanning densitometry of
immunoblot films was performed on a Personal Densitometer SI
scanner (Molecular Dynamics, Sunnyvale, CA) and analyzed using
ImageQuaNT image analysis software (Molecular Dynamics).
AJP-Regul Integr Comp Physiol • VOL
Immunocytochemistry of Synchronized Embryos
To determine whether embryos utilized for immunoblot studies
were arrested at the desired stages, i.e., metaphase and pre-S, embryos
were subjected to the synchronization protocol and fixed for immunocytochemical processing. The detailed immunocytochemical procedure has been previously described (13). In brief, embryos were
aged for 130 min AED, heat pulsed for 40 min at 37°C, and held at RT
for 4.5 min (metaphase arrest) and 12.5 min (interphase/pre-S arrest)
before being exposed to anoxia for 30 min. After the anoxic exposure,
embryos were fixed inside the anoxia chamber with 2% paraformaldehyde in PBS and subsequently processed for the immunocytochemical detection of ␣-tubulin and propidium iodide.
Data Analysis
Data are reported as means ⫾ SE, and results were analyzed using
the Wilcoxon signed-rank test and one-way ANOVA. Differences in
the means were considered statistically significant when P ⬍ 0.05.
RESULTS
Effect of Graded Hypoxia on Drosophila Melanogaster
Embryonic Cell Cycle Kinetics
The exposure of the embryos to mild hypoxia (5% O2)
appears to have a limited impact on cell cycle duration and
kinetics during the first 13 cycles (see Fig. 2B, a–f) and looked
very similar to control embryos under normoxic conditions
(Fig. 2A, a–f). Furthermore, energids (nuclei and associated
cytoskeletal elements within the embryonic syncytium) within
embryos did not show any evidence of abnormalities or deranged movement of intracellular organelles or other structures. Formation of the spindle apparatus and centrosomes was
not impeded nor was cytokinesis or the formation of asters.
Subsequent cell cycles proceeded normally. Centrioles repli288 • FEBRUARY 2005 •
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Fig. 1. Synchronization of heat shock string (HS-STG3) embryos and hypoxia arrest paradigms. HS-STG3 embryos were collected during a 30-min period and
then dechorionated (see MATERIALS AND METHODS) and aged for 130 min [130 –160 min after egg deposition (AED)]. Embryos were then heat pulsed at 37°C
for 40 min. Heat-pulsed embryos initiate mitosis 5–7 min after returning to room temperature (RT) and tend to complete mitosis ⬃20 min after the heat pulse
(AHP). Therefore, HS-STG embryos were returned to RT for specific times before being exposed to normoxia (controls) or anoxia before N2 fixation. Metaphase
arrest paradigm: after 4.5 min at RT and just before the initiation of mitosis, embryos were exposed to anoxia for either 5 min (MA1) or 30 min (MA2) and
quickly fixed. Concomitant controls were fixed at 4.5 min (C1), 12.5 min (C2), and 34.5 min (C3) AHP. Pre-S phase arrest paradigm: after 12.5 min at RT or
AHP, when the embryos had progressed beyond metaphase, heat-pulsed embryos were subjected to anoxia for 5 min (IB1) or 30 min (IB2) before N2 fixation.
Equivalent controls were fixed 15 min (C4), 22 min (C5), or 45 min (C6) AHP while being maintained at RT. The normoxic period is indicated by the solid
area, and the anoxic period is indicated by the hatched area.
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HYPOXIA AFFECTS CELL CYCLE AND PROTEINS IN FLY EMBRYOS
cated and migrated toward the poles of nuclei for the next
round of DNA replication and mitosis. Switching to a normoxic perfusate after 5% O2 did not have an effect on cell
cycle activity. Moderate hypoxia (2% O2), by contrast, had a
AJP-Regul Integr Comp Physiol • VOL
dramatic effect in terms of kinetics and duration (Fig. 2B, g–l).
Within a few seconds of switching to a perfusate bubbled with
2% O2, energids simultaneously and synchronously slowed
their rate of cell cycle progression and thereby markedly
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Fig. 2. A and B: in vivo confocal imaging of a kinesin-green fluorescent protein (K-GFP) embryo exposed to graded hypoxia. Dechorionated K-GFP embryos
were placed in a perfusion chamber under normoxic conditions (21% O2). Several embryos were then exposed to mild (5% O2), moderate (2% O2), and severe
hypoxia (100% N2). Normoxia (A, a–f; n ⫽ 25): similar to 5% O2, it can be seen that embryos exposed to normoxic conditions required only ⬃12 min to complete
a cell cycle. Magnification ⫽ ⫻63. Severe hypoxia (A, g–l; n ⫽ 12): a K-GFP embryo was exposed to 100% N2 just after the metaphase (Met)-to-anaphase (Ana)
transition (g). Eight minutes after the induction of severe hypoxia, the embryo was seen to complete cytokinesis (CK), as previously reported, and arrest during
interphase (Inter; i). This arrest was maintained for up to 2 h, with some diminution in fluorescent intensity (i–j), and resumed cell cycle activity within ⬃20
min of reoxygenation (Re-Oxy). Magnification ⫽ ⫻63. Mild hypoxia (B, a–f; n ⫽ 4): K-GFP embryos were exposed to mild hypoxia (5% O2), which had no
apparent effect on cell cycle kinetics or embryonic morphology. Magnification, ⫻40. Moderate hypoxia (B, g–l; n ⫽ 4): in contrast to mild hypoxia, moderate
hypoxia (2% O2) had a dramatic effect on cell cycle progression. An embryo was seen that required ⬃65 min to progress from prophase (Pro; g) to telophase
(Telo; l). This demonstrates that cell cycle progression is slowed ⬃5-fold by exposure to 2% O2. Magnification ⫽ ⫻25 (zoom ⫽ ⫻1.6). Promet, prometaphase;
M, metaphase. C: cell cycle kinetics during graded hypoxia. A graphical representation of the response of K-GFP embryos exposed to normoxia (21% O2), mild
hypoxia (5% O2), moderate hypoxia (2% O2), and reoxygenation (21% O2) is shown. Whereas 5% O2 exposure elicited no change in cell cycle length (1,006 ⫾
26 s) compared with control (945 ⫾ 178 s), moderate hypoxia of 2% O2 markedly delayed the cell cycle (4,245 ⫾ 561 s) (*P ⬍ 0.05 vs. control). Reoxygenation
or reperfusion led to an almost immediate resumption of the control rate of cell cycle progression such that cell cycle length was again equivalent to control
(958 ⫾ 85 s).
HYPOXIA AFFECTS CELL CYCLE AND PROTEINS IN FLY EMBRYOS
Cell Cycle Regulatory Protein Expression During Anoxia in
Drosophila Embryos
Synchronization of Drosophila embryos for immunoblot studies. Embryos were aged, heat-pulsed, and maintained
at RT for 4.5 and 12.5 min before anoxic exposure to recapitulate the metaphase and interphase/pre-S arrests, respectively.
They were then fixed and processed for the immunocytochemical localization of ␣-tubulin and propidium iodide to detect the
plasma membrane and chromatin material of cells/energids in
the embryos, respectively, to determine the cell cycle stage at
which the embryos arrested. Embryos held at RT for 4.5 min
after the heat pulse (AHP) before the anoxic exposure were
seen to be arrested at metaphase (data not shown), whereas
embryos held for 12.5 min at RT AHP before anoxia were
arrested at interphase (see Fig. 3B). All of the cells/energids in
these embryos were at the same cell cycle stage with condensed, centrally located chromosomes predictive of interphase. Control embryos (Fig. 3A) were also in interphase as
predicted, since they would have completed a full cell cycle
and returned to the interrupted G2 after 42.5 min AHP.
Pre-S phase arrest and G1/S proteins. To look for the
mechanisms underlying the pre-S phase cell cycle arrest, we
examined several G1/S proteins by Western blotting to determine their levels in response to low O2 (Fig. 4A). dE2F1,
which is the major positive regulator of the initiation of S phase
and DNA replication, was detected as a single band of 90 –95
kDa. dE2F1 significantly increased over control levels by
⬃45% after 5 min of anoxia (Fig. 4B, B1 vs. C5) in embryos
that were arrested during pre-S phase. At the end of the anoxic
period (30 min), dE2F1 levels did not change or declined
somewhat but were still greater over its equivalent control (Fig.
4B, B2 vs. C6). A similar response pattern was seen for dE2F2
(data not shown) during the pre-S phase arrest.
RBF proteins (RBF1, RBF2) are considered negative regulators of the G1/S transition in Drosophila (14). The responses
AJP-Regul Integr Comp Physiol • VOL
of RBF1 (data not shown) and RBF2 (Fig. 4C) were similar to
the dE2F1 anoxic response (Fig. 4B). RBF2 appears as a band
of ⬃83 kDa on Western blot. The expression of RBF2 was
significantly increased (60%) over comparable controls (Fig.
4C, B1 vs. C5) and then declined over the anoxic period but not
below control levels (Fig. 4C, B2 vs. C6). In summary, G1/S
regulatory proteins examined in this study demonstrated, in
general, an increase in expression during the early stages of
anoxia but subsequently declined over the next one-half hour
of anoxic exposure.
Pre-S phase arrest and G2/M proteins. Cyclins A and B are
necessary for the G2/M transition, as well as progress through
the initial stages of mitosis. However, recent studies using
microarray analyses have indicated that cell cycle regulatory
proteins that have traditionally been ascribed specific roles in
cell cycle progression may very well regulate other stages in
the cell cycle (see Ref. 47). We therefore examined the response of proteins known to control the G2/M transition to
determine their role at the G1/S transition, i.e., in the pre-S
phase arrest paradigm. Cyclin A is detected as a band of ⬃59
kDa, and cyclin B and dWee1 kinase are detected as single
bands of ⬃69 kDa and ⬃110 kDa, respectively (Fig. 5).
During the pre-S phase arrest, cyclin A, cyclin B, and the
dWee1 kinase either stayed close to baseline or increased
significantly over control (Fig. 5, B1 vs. C5). Similar to the
expression profile of dE2F1 (Fig. 4B) and RBF2 (Fig. 4C)
during the pre-S phase arrest, the expression of cyclin A and
dWee1 kinase declined but remained above comparable controls (Fig. 5, B and D, B2 vs. C6) during the anoxic exposure.
On the other hand, protein expression levels of cyclin B
continued to increase during anoxia such that cyclin B expression in embryos was significantly greater (40%) than that seen
in control embryos (Fig. 5C, B2 vs. C6) at the end of the
30-min anoxic exposure.
Metaphase arrest and G2/M proteins. Regulation of the
G2/M transition and progress through mitosis are controlled by
a relatively complex hierarchy of cell cycle proteins, including
the major positive regulators cyclins A and B and the histone
H1 kinase (cdc2 or cdk1 in yeast), as well as the negative and
positive regulators of cdk1 activity, such as dWee1 and String
(cdc25 in yeast), respectively. During an anoxia-induced metaphase arrest, the expression of several cell cycle regulatory
proteins such as cdk1, dDp, dE2F2, Fizzy (cdc20 in yeast),
RBF1, and String was either unaltered or slightly increased
compared with relevant controls (data not shown). Interestingly, the cyclins A and B (Fig. 6) also demonstrated stabilization in expression (Fig. 6, B and C, C2 vs. A1) during a
metaphase arrest. Even though these changes were not significant, it is known that cyclins A and B must be degraded during
the metaphase-to-anaphase transition of mitosis. Therefore, it
is apparent that cyclins A and B are not degraded during
anoxia, since control and anoxic embryos (Fig. 6, B and C, C2
vs. A1 and A2 vs. C3) have equivalent expression levels
throughout the anoxic exposure.
Metaphase arrest and G1/S proteins. The examination of
G1/S proteins during the metaphase arrest also revealed that, in
general, cell cycle protein expression was stabilized during
anoxia. For example, the expression of dE2F1 during anoxia
was equivalent to control throughout anoxia (data not shown).
On the other hand, the expression of RBF2 appeared to be
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protracted cell cycle length. Cell cycle duration, which is
normally ⬃950 s, increased by approximately fivefold and
extended to ⬃4,300 s (70 min), with no bias to any particular
cell cycle stage (Fig. 2C). The morphology of the subcellular
organelles and structures, i.e., spindle apparatus and centrosomes, appeared to be unaffected, as during 5% O2 or normoxic exposure. A complete cessation of cell cycle activity did
not occur. Therefore, embryos slowed their cell cycle activity
tremendously, with no other abnormalities observed. Switching back to a normoxic perfusate allowed the embryos to
rapidly resume their original cell cycle program. A complete
cell cycle arrest occurred only when very low O2 concentrations (severe hypoxia, ⬍0.5%) were used in these embryos
(Fig. 2A, g–l). Depending on the time of initiation of severe
hypoxia, embryos either arrested at metaphase or arrested just
before the S phase. Embryos that were exposed to severe
hypoxia just before metaphase halted their cell cycle activity at
metaphase with chromosomes aligned along the metaphase
plate and a fully developed spindle apparatus (data not shown).
On the other hand, embryos that were exposed just after
metaphase or during or past anaphase, migrated through the
rest of the cycle and arrested in a hypoxia-induced pre-S phase
(Fig. 2A, g–l). Furthermore, the normoxia and hypoxia treatments gave the same result as before (13).
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HYPOXIA AFFECTS CELL CYCLE AND PROTEINS IN FLY EMBRYOS
lower in anoxia-exposed embryos compared with controls at
both anoxia time points examined (data not shown).
DISCUSSION
Our data indicate that changes in the level of O2 to which D.
melanogaster embryos are exposed have a major impact on cell
cycle progression. Moderate hypoxia (40 – 45 Torr, 2% O2)
slowed cell cycle progression almost fivefold compared with
control or mild hypoxia (55– 60 Torr, 5% O2), whereas severe
hypoxia (25–35 Torr, 100% N2) resulted in total cell cycle
arrest at two points: metaphase and pre-S phase. In our current
studies, we have also investigated whether, during these arrests, analysis of protein levels can be useful for understanding
the basis of the hypoxia-induced phenomena.
Effect of Low O2 on Cell Cycle Progression
The mechanisms that are potentially involved in slowing the
cell cycle in response to a hypoxic stress are currently not
known, although a drop in ATP levels is one possibility (24).
Depletion of ATP stores during hypoxia may slow the cell
AJP-Regul Integr Comp Physiol • VOL
cycle of D. melanogaster embryos by decreasing overall metabolic activity. Indeed, it has been hypothesized that a reduction of the metabolic rate will decrease protein and RNA
synthesis and metabolism, thus slowing down the cell cycle (7
and reviewed in Ref. 74). Decreased protein synthesis in
hypoxia has been observed in a variety of species, including
marine snails, fish, turtles, and humans (see 39). Although this
may still be true in Drosophila embryos, the decreased protein
levels are not homogenous. For example, we noted an
increase in cyclin B levels during the anoxic period, indicating that active protein synthesis or a possible decrease in
protein degradation occurs during anoxia in Drosophila
embryos.
O’Farrell and colleagues (12, 81) have suggested another
possibility: nitric oxide, rather than ATP, plays a critical role in
hypoxia-induced arrest in Drosophila. Yet other investigators
believe that phosphorylation/dephosphorylation, and, in particular, phosphorylation of histone in Caenorhabditis elegans
(56) and eIF-2␣ in Littorina littorea (39), is a requisite component of the hypoxia checkpoint. Therefore, it is possible that
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Fig. 3. Cell cycle stage during metaphase and interphase arrests. To determine the cell cycle stage at which embryos
arrested during the metaphase and interphase arrest paradigms,
embryos were held at RT for 4.5 and 12.5 min AHP before
exposure to anoxia. After anoxia, embryos were fixed and
processed for the immunocytochemical localization of ␣-tubulin (green) and propidium iodide (red) to detect the plasma
membrane and chromatin material, respectively. Control embryos that were heat pulsed and held at RT for 42.5 min
apparently completed the hs-string-induced cell cycle and returned to the interrupted interphase, G2 (A). Embryos that were
heat pulsed and exposed to anoxia for 12.5 min AHP can be
seen to be in interphase, as demonstrated by the condensed
prophase-like chromatin structure (B). All of the cells/energids
within the embryo are at the same cell cycle stage. Magnification ⫽ ⫻40 for A and B. Magnification ⫽ ⫻264 for insets
(middle; indicated by white arrows).
HYPOXIA AFFECTS CELL CYCLE AND PROTEINS IN FLY EMBRYOS
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several cellular mechanisms, acting in a concerted manner, are
involved in the hypoxia-induced slowing and arrest of cell
cycle activity.
Role of Cyclin A and B Proteins in Transducing an AnoxiaInduced Arrest
Because we did not observe a decrease in cyclin A and
cyclin B levels during both metaphase and pre-S phase anoxiainduced arrests, the question arises whether the stabilized
cyclin A or cyclin B can induce arrest. In general, G1 arrest can
occur in response to DNA damage elicited by ionizing radiation and UV radiation (1), whereas G2 arrest can be induced by
the spindle checkpoint and/or DNA damage (see Ref. 8).
Additionally, mutations in cyclins A and B have been shown to
induce temporary or permanent arrest (41, 48, 51).
In Drosophila, cyclin A is normally degraded before the
metaphase-to-anaphase transition, cyclin B is degraded at the
metaphase-to-anaphase transition, and cyclin B3 is degraded
during anaphase (11, 41, 59 and reviewed in Ref. 75) by the
mitotic ubiquitin ligase system, the APC (82). Because the
degradation of cyclin A and other proteins is required for cells
to progress beyond metaphase (62, 82), the lack of reduction of
cyclin A levels (e.g., inhibition of cyclin A proteolysis) may
induce an arrest at the metaphase-to-anaphase transition (9,
77). To date, there is significant evidence that stabilized cyclin
A can induce a metaphase arrest. Rimmington et al. (66) and
Sigrist et al. (69) were among the first to report that stable
cyclins A, B, and B3 result in a delay at metaphase, early
anaphase arrest, and a late anaphase arrest, respectively. More
recently, it has been demonstrated that nondegradable cyclin A
induces an arrest at metaphase in Drosophila and mammalian
AJP-Regul Integr Comp Physiol • VOL
cells (21, 33, 59) and an arrest at metaphase with condensed
chromosomes in Xenopus embryos (45). Stable cyclin B and
cyclin B3 induce arrests during anaphase B (58) and in late
anaphase with deep cytokinetic furrows (59), respectively.
Furthermore, Su and Jaklevic (76) have recently reported
that DNA damage-induced stabilization of cyclin A is responsible for the DNA damage-induced cell cycle arrest at G2 in
Drosophila gastrula cells and that cells lacking cyclin A do not
delay in metaphase in response to DNA damage. In addition,
overexpression of XDRP1, a Xenopus dsk2-related protein
important in the spindle checkpoint pathway that leads to
inhibition of the degradation of cyclin A (but not cyclin B),
blocks embryonic cleavage cycles (20). Therefore, Parry and
O’Farrell (59) suggest that cyclin A-like cyclins act to inhibit
the metaphase-to-anaphase transition in diverse species and
that degradation of these cyclins is required for progress
through the cell cycle.
Although our results indicate that both cyclins A and B are
stabilized during anoxia, the morphological characteristics of
our embryos, which display a metaphase arrest profile with
condensed chromosomes on the metaphase plate (12, 13), leads
us to believe that cyclin A, rather than cyclin B, is the major
player in the metaphase arrest, since stable cyclin B would
induce an arrest at the end of anaphase, not at metaphase.
Clearly, this does not preclude stable cyclin B from participating in the extended arrest. Because cyclin B is stabilized by the
spindle checkpoint but cyclin A is not (11, 80), the present
study indicates that anoxia may activate both the spindle
checkpoint as well as a separate checkpoint subserved by
cyclin A.
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Fig. 4. Effect of anoxia on G1/S protein
expression during the pre-S phase arrest. A:
synchronized HS-STG3 embryos were exposed to anoxia (100% N2) for 5 min (IB1)
and 30 min (IB2) AHP of 40 min at 37°C
and resting at RT for 12.5 min. Pre-S phasearrested HS-STG3 embryos were then immediately fixed in liquid N2 and subsequently probed for cell cycle protein expression. Concomitant controls were also run to
match the time AHP of our anoxia-exposed
embryos. Control embryos that were maintained at RT throughout the experiment were
therefore fixed after 15 min (C4), 22 min
(C5), and 45 min (C6) AHP (see Fig. 1).
Western blots of these embryos demonstrated increases in expression of E2F1 (B)
and RBF2 (C) during the first 5 min of
anoxia. As seen in Fig. 5, B and C, the
differences among control and anoxic embryos were significant (paired symbols indicate significant difference between bars:
*P ⬍ 0.05 and #P ⬍ 0.05) when expression
at 5 min of anoxia was compared with equivalent controls (B1 vs. C5 and C6). Both
E2F1 and RBF2 declined to but not below
control levels after 30 min of anoxia (B2).
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HYPOXIA AFFECTS CELL CYCLE AND PROTEINS IN FLY EMBRYOS
The difference between our previous study (13), in which
we report a lower level of cyclin B with anoxia, and the
present, in which we report a stable or increased cyclin B,
is due primarily to 1) the cyclical nature of cyclin B
during mitosis and 2) the time at which the embryos
were fixed in relation to the cyclical nature of cyclin B.
Cyclin B is higher in control embryos that have not passed
mitosis than in anoxic embryos that have progressed beyond
mitosis, whereas anoxic embryos that have passed mitosis
and arrested in interphase have similar or higher levels of
cyclin B than control embryos at a similar or more advanced
stage.
Fig. 6. Effect of anoxia on G2/M protein
expression during the metaphase arrest. A:
synchronized HS-STG3 embryos were exposed to anoxia (100% N2) for 5 min (MA1)
and 30 min (MA2) AHP of 40 min at 37°C
and resting at RT for 4.5 min. Metaphasearrested HS-STG3 embryos were then immediately fixed in liquid N2 and subsequently probed for cell cycle protein expression. Concomitant controls were also run to
match the time AHP of our anoxia-exposed
embryos. Control embryos that were maintained at RT throughout the experiment were
therefore fixed after 5 min (C1), 12.5 min
(C2), and 34.5 min (C3) AHP. Western blots
of these embryos demonstrated no change in
the expression of cyclin A (B) or cyclin B
(C) after 5 min and 30 min of anoxic exposure (A1 vs. C2 and A2 vs. C3). However,
expression of cyclins A and B was maintained at control levels and did not diminish
below control levels during anoxia (A2 vs.
C3).
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Fig. 5. Effect of anoxia on G2/M protein
expression during the pre-S phase arrest. A:
synchronized HS-STG3 embryos were exposed to anoxia (100% N2) for 5 min (IB1)
and 30 min (IB2) AHP of 40 min at 37°C
and resting at RT for 12.5 min. Pre-S phasearrested HS-STG3 embryos were then immediately fixed in liquid N2 and subsequently probed for cell cycle protein expression. Concomitant controls were also run to
match the time AHP of our anoxia-exposed
embryos. Control embryos that were maintained at RT throughout the experiment were
therefore fixed after 15 min (C4), 22 min
(C5), and 45 min (C6) AHP. Western blots
of these embryos demonstrated no change in
the expression of cyclin A (B) after 5 min
and 30 min of anoxic exposure. However,
expression of cyclin B (C; B2 vs. C6) and
dWee1 (D; B1 vs. C5 and C6) was significantly increased (paired symbols indicate
significant difference between bars: *P ⬍
0.05 and #P ⬍ 0.05) over control after 30
min of anoxia. Interestingly, the expression
of cyclin B (C) appeared to increase during
anoxia.
HYPOXIA AFFECTS CELL CYCLE AND PROTEINS IN FLY EMBRYOS
It is conceivable also that cyclin A could induce arrest of cell
cycle activity by other mechanisms. Cyclin A has been reported to inactivate cdh1, or Fizzy-related, which is responsible
for the activation of the APC via phosphorylation and dissociation of cdh1 from the APC (55, 70) and which permits
egress from metaphase. The other intriguing possibility is that
cyclin A can also participate in a G1/S arrest via its role in S
phase progression by suppression of E2F1 activity (37). For
example, coexpression of cyclin A/cdk1, with an absolute
requirement for cyclin A and the simultaneous accumulation of
E2F, has been demonstrated to induce an S phase arrest,
possibly by blocking E2F in Drosophila larval cells (26, 27,
46). We propose that stabilization of cyclin A is necessary and
sufficient to induce a metaphase arrest during anoxia.
In mammalian cells, the transcription factor E2F1 is important for the initiation of the S phase in moving from the
quiescent (G0, G1) to the proliferative state (reviewed in Ref.
15). E2F regulates genes involved in the initiation of DNA
replication (cdc6), cell cycle activity (cdc25A, cyclins A and
E), and growth (bMyb, p107, E2F1). Although there are six
E2F family members (E2F1– 6) in mammals, only two members have been described in Drosophila, dE2F1 and dE2F2
(reviewed in Ref. 15; 47, 68, 78).
E2F1 accumulates in late G1 phase of the cell cycle. It is
then rapidly degraded in S/G2 phases of the cell cycle via
interaction with the F-box protein p45SKP2, a component of the
ubiquitin ligase SCFSKP2 (reviewed in Ref. 36; 46, 61). dE2F1,
like E2F1, -2, and -3 in mammals, activates, and dE2F2, akin
to mammalian E2F4, -5, and perhaps -6, represses gene transcription, while dE2F1 and dE2F2 antagonize each other (4, 6,
19, 68, 78). Surprisingly, in the present study, anoxia induced
an increase in the expression of dE2F1 during the pre-S phase
block. Although the anoxia-induced increase in dE2F1 protein
expression was unexpected, it is not unparalleled: O’Connor
and Lu (54) have reported that hypoxia of 24-h duration
induced an increase in the expression of E2F1 that was independent from both p53 and pRb. Additionally, other stresses
such as UV, IR, and DNA-damaging agents such as cisplatin
and etoposide are also able to increase the levels of E2F1 in a
variety of tumor cells (27, 31, 44, 54). Although it is known
that the overexpression of E2F1 in normal cells leads to S
phase entry and apoptosis (reviewed in Ref. 10), this was not
observed in our study. However, it has been noted that E2F1
can also function as a repressor during the cell cycle (4, 6, 19).
Several studies implicate E2F1, and possibly -2 and -3, in
repressing gene activity to permit exit from the cell cycle (6,
67). For example, Zhang et al. (83) believe that active repression by E2F/Rb and not inactivation of E2F is responsible for
the G1 arrest triggered by contact inhibition, p16INK4a, and
transforming growth factor (TGF)-␤. Additionally, He et al.
(27) propose that E2F actually suppresses the onset of S phase
in cells arrested by ␥-irradiation. A direct role for E2F1 in the
DNA damage checkpoint-induced cell cycle arrest is also
suggested by its higher levels after DNA damage via protein
stabilization mediated by ATM-induced phosphorylation (44,
54). Furthermore, a stabilized form of E2F1 has been demonstrated to induce an S phase delay and apoptosis (38). However, although E2F has an essential role in cell cycle arrest in
AJP-Regul Integr Comp Physiol • VOL
several cell lines (83), this stress-induced E2F1 does not appear
to be transcriptionally active but yet plays a role in DNA
damage-induced G1 arrest (54).
Recently, E2F1 has been shown, via microarray analysis, to
possibly play a nontraditional role at G2/M (4, 32, 63, 65, 71).
These studies implicate E2F in mitosis, chromosome segregation, the spindle checkpoint, DNA repair, chromatin assembly/
condensation, apoptosis, differentiation, and development (32;
reviewed in Ref. 47). For example, Polager and Ginsberg (63)
have demonstrated that E2F1 is essential for maintaining a
genotoxin (doxorubicin)-mediated G2 arrest by repressing critical mitotic regulators such as stathmin (oncoprotein 18) and
aurora and Ipl-1-like midbody-associated protein kinase.
Therefore, anoxia-induced E2F1 protein may participate, in
addition to the pre-S phase arrest, in the arrest at metaphase.
The retinoblastoma protein (pRb) family members are critical regulators of cell cycle activity and play a predominant
role in the regulation of E2F activity (3, 14, 42). Our study
indicates that RBF1 is stabilized (data not shown) and that
RBF2 is elevated during the anoxia-induced pre-S phase arrest.
RBF1 is known to participate in a DNA damage-induced G1/S
arrest (25, 43), as well as during TGF-␤- and p16ARF-induced
arrests (83). RBF2 is a newly described protein in Drosophila
that has homology to human pRb and other pocket proteins
such as RBF1, p130, and p107. In conjunction with dE2F2,
RBF2 has been proposed to have global repressor function,
binding to and inhibiting several promoter sites in dE2F1responsive genes, antagonizing dE2F1/dDp transactivation,
and, when overexpressed, blocking S phase entry in vivo (50,
72, 73). Stevaux et al. (72) indicated that RBF2 can function as
a repressor, either alone or in combination with the E2F family
(19). Therefore, we raise the question as to whether the
increase in RBF2 noted in these studies could participate in an
anoxia-induced cell cycle arrest at G1/S.
Role of Other Proteins Such as dWee1 Kinase in AnoxiaInduced Arrests
The dWee1 and dMyt1 kinases are known to be involved in
regulation of the G2/M transition in Drosophila via their
inhibitory phosphorylations on the mitotic activator cdc25
(String) (5, 28, 57). dWee1 and dMyt1 are active during S
phase to prevent a premature mitosis, can be activated during
the DNA damage checkpoint to arrest cells in G2 (40; reviewed
in Ref. 53), and have been suggested to block apoptosis via
interactions with p53 (64). However, in our study, an increase
in the expression of dWee1 was seen during the pre-S phase
arrest in HS-STG3 embryos. More experiments are required to
elucidate the possible activities of dWee1 at points in the cell
cycle other than at the G2/M transition.
Perspectives
In summary, several stresses are capable of invoking the
checkpoint apparatus, whether it is DNA damage, failure of
DNA replication, or spindle damage. However, it appears that
hypoxia stimulates cell cycle arrest via redundant, known
checkpoint pathways but also by unique and independent
pathways. For example, whereas the spindle checkpoint induces degradation of cyclin A, hypoxia induces stabilization of
cyclin A; therefore, multiple pathways are likely to be invoked
during hypoxia (60). Analysis of the interactions of cell cycle
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Role of dE2F1 and RBF2 in Anoxia-Induced Arrests
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HYPOXIA AFFECTS CELL CYCLE AND PROTEINS IN FLY EMBRYOS
proteins during hypoxia should begin to shed light on pathways
that are exclusively activated by hypoxia compared with those
activated by other stresses such as DNA damage.
ACKNOWLEDGMENTS
We thank Shelagh Campbell, Iian Dawson, Nick Dyson, Bruce A. Edgar,
Haig Kesheshian, and Amanda Purdy for insightful discussions related to this
project and Ralph Garcia and Joe Zavilowitz for superb technical assistance.
GRANTS
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