Departments of 1 Environmental Medicine, 3 Radiation Oncology, and 2 Pediatrics, University of Rochester, Rochester, New York 14642
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ABSTRACT |
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High concentrations of
O2 inhibit epithelial cell
proliferation that resumes on recovery in room air. To determine
whether growth arrest is mediated by transforming growth
factor- (TGF-
), changes in cell proliferation
during exposure to hyperoxia were assessed in the mink lung epithelial
cell line Mv1Lu and the clonal variant R1B, which is deficient for the
type I TGF-
receptor. Mv1Lu cells treated with TGF-
accumulated
in the G1 phase of the cell cycle
as determined by propidium iodide staining, whereas proliferation of
R1B cells was unaffected by TGF-
. In contrast, hyperoxia inhibited
proliferation of both cell lines within 24 h of exposure through an
accumulation in the S phase. Mv1Lu cells treated with TGF-
and
exposed to hyperoxia accumulated in the G1 phase, suggesting that TGF-
can inhibit the S phase accumulation observed with hyperoxia alone.
Cyclin A was detected in cultures exposed to room air or growth
arrested by hyperoxia while decreasing in cells growth arrested in the
G1 phase by TGF-
. Finally,
hyperoxia failed to activate a TGF-
-dependent transcriptional
reporter in both Mv1Lu and R1B cells. These findings reveal that simple growth arrest by hyperoxia involves a defect in S phase progression that is independent of TGF-
signaling.
proliferation; transforming growth factor-
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INTRODUCTION |
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THERE HAS RECENTLY BEEN a greater appreciation for the role of reactive oxygen species (ROS) in regulating normal and abnormal disease processes. The lung is uniquely challenged by ROS because its large surface area is exposed to various environmental pollutants, inflammatory cells are recruited during infection, and supplemental O2 is used clinically to treat pulmonary disease. Studies (2, 7, 8) over the past 30 years in mice, rats, monkeys, and humans have described the effects of lethal (>90%) and sublethal (<85%) levels of O2 on the lung alveolus. The alveolus is composed of type I and II epithelial and microvascular endothelial cells. Endothelial and type I epithelial cells are rapidly killed when exposed to lethal levels of O2. Type II cells, which synthesize pulmonary surfactant, proliferate and differentiate into type I cells during recovery in room air (2, 30). Rapid proliferation of type II cells is crucial for both the survival and prevention of fibrosis (1). Similarly, sublethal levels of O2 kill endothelial cells. However, unlike during exposure to lethal levels of O2, type I epithelial cells survive and type II cells proliferate during the exposure (8). Because proliferation of type II cells plays a critical role in pulmonary repair processes, it is important to clarify molecular signals that regulate type II cell proliferation during and after oxidant stress.
Regulation of cell proliferation is complex and requires the interactions of intracellular and extracellular stimulatory and inhibitory signals that modulate activity of the cyclin-dependent kinases (Cdks). Cdk complexes are composed of a kinase subunit and a catalytic subunit termed cyclin because its expression cycles during the cell cycle (29). The G1 phase cyclins include cyclins D (D1, D2, and D3) and cyclin E, which bind different Cdks. Expression of cyclin A is observed in the S and early G2 phases and cyclin B in the G2/M phase. Active cyclin D and E complexes phosphorylate the retinoblastoma (Rb) gene product, resulting in release of the transcription factor E2F that increases transcription of S phase genes such as thymidine kinase. Growth inhibition is achieved through binding of small Cdk inhibitory proteins (CKIs) to cyclin-Cdk complexes, resulting in decreased kinase activity (29). Two families of CKIs have been identified and include the Cip/Kip family (p21, p27, and p57) and the Ink4 family (p15, p16, p18, and p19).
Antimitogens such as the cytokine transforming growth factor-
(TGF-
) inhibit cell cycle progression by altering the expression and
activities of CKIs. TGF-
first binds to the type II TGF-
receptor
(T
R-II), which then recruits and activates the kinase domain of the type I TGF-
receptor (T
R-I) through phosphorylation (13). The activated receptor complex phosphorylates various Smad
proteins that translocate to the nucleus and bind and transactivate downstream target genes. TGF-
inhibits cell cycle progression at the
G1/S phase boundary by increasing
the expression of p15 or p21 (9, 26). Cells arrested by TGF-
have
reduced levels of cyclin A associated with increased levels of
hypophosphorylated Rb gene product (27).
Molecular signals that regulate type II cell proliferation remain
unknown, in part, because type II cells rapidly lose their differentiated characteristics when cultured in vitro (28). Although
simian virus 40 (SV40) and other viral oncoproteins alter normal cell
proliferation by rearranging cyclin and Cdk partners, several
investigators have immortalized type II cells using SV40. The
proliferative response of SV40-immortalized rat type II cells (SV40T-T2) exposed to hyperoxia has been extensively studied. SV40T-T2
cells rapidly growth arrest when exposed to hyperoxia and resume
proliferation on recovery in room air (5). Hyperoxia increased mRNA
levels of TGF-1, T
R-I, and T
R-II (4, 6). Moreover, hyperoxia
increased the expression of p21 that bound and inhibited the activity
of cyclin E-Cdk 2 complexes. It was concluded that hyperoxia inhibited
proliferation through TGF-
signaling because increased cyclin E-Cdk2
activity was observed in cells exposed to both hyperoxia and
neutralizing antibodies to TGF-
. Although these studies suggested
that hyperoxia inhibits proliferation through TGF-
signaling, they
never demonstrated that administration of neutralizing antibodies
resulted in normal cell proliferation in the presence of hyperoxia. In
addition, it remains to be determined where in the cell cycle hyperoxia inhibits proliferation.
The present study investigates the effects of hyperoxia on
proliferation of the mink lung epithelial cell line Mv1Lu, which is
markedly growth arrested by TGF- (31). This cell line was chosen
because chemically induced mutant lines that are unresponsive to
TGF-
have also been identified (31). The R1B cell line is a member
of the R class of mutant Mv1Lu cells that have lost expression of
T
R-I. In the present study, we tested the hypothesis that hyperoxia
inhibits proliferation through TGF-
signaling by analyzing proliferation of Mv1Lu and R1B cells exposed to hyperoxia. Our findings
reveal that hyperoxia caused both cell lines to cease proliferation in
the S phase of the cell cycle independent of TGF-
signaling.
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MATERIALS AND METHODS |
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Cell culture. Mv1Lu (mink lung adenocarcinoma) cells were obtained from Dr. Anita Roberts (National Cancer Institute, National Institutes of Health, Bethesda, MD), and R1B (chemically induced mutant Mv1Lu) cells were obtained from Dr. Joan Massagué (Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY). The cells were incubated at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 50 U/ml of penicillin, and 50 µg/ml of streptomycin (GIBCO BRL). The cells were maintained in tissue culture flasks and routinely passaged every 3 days.
For exposures to hyperoxia or TGF-, the cells were trypsinized,
counted with a hemacytometer, and plated in 100-mm dishes at a density
of 5 × 105 overnight. The
medium was replenished the next morning, at which time the cells were
treated with 5 ng/ml of porcine TGF-
1 obtained from R&D Systems
(Minneapolis, MN) and/or exposed to hyperoxia in a Plexiglas box (Belco
Glass, Vineland, NJ). The box was sealed and flooded with 95%
O2-5%
CO2 for 15 min at a flow rate of 5 l/min. O2 concentrations were
monitored with a miniOXI analyzer from Catalyst Research (Owings Mills,
MD). The cells were harvested at various times with 0.25% trypsin,
counted with a hemacytometer, and stained for viability with 0.5%
trypan blue or 10 µg/ml of propidium iodide.
Flow cytometry. Cells were trypsinized, resuspended in their original medium, and centrifuged at 300 g. The medium was removed, and the cells were fixed in 75% ethanol for 24 h. The cells were resuspended in 1 ml of RNase (1 mg/ml) for 30 min, centrifuged, and resuspended in 0.5 ml of propidium iodide (10 µg/ml). The samples were analyzed on an Epics Profile (Coulter Electronics, Hialeah, FL) set to collect 10,000 events. DNA histograms were analyzed, and the percentages of G1, S, and G2/M phase cells were determined according to the mathematical model of Fried et al. (11). Terminal deoxynucleotidyltransferase dUTP nick end-labeling (TUNEL) staining was performed with the Apo-BRDU kit obtained from Phoenix Flow Systems (San Diego, CA), and fluorescent-positive cells were measured by flow cytometry. As a positive control for TUNEL staining, the cells were exposed to 5 Gy of 137Cs at a dose rate of 3.7 Gy/min and recovered in room air for 24 h.
Western blot analysis. The cells were
harvested at 4°C by scraping in 50 mM Tris, pH 7.4, 150 mM sodium
chloride, 2 mM EDTA, 25 mM sodium fluoride, 25 mM -glycerol
phosphate, 0.1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride,
0.2% Triton X-100, 0.3% Nonidet P-40, 10 µg/ml of leupeptin, 10 µg/ml of pepstatin, and 10 µg/ml of aprotinin. The cell lysates
were cleared by centrifugation, and protein concentrations were
determined with a modified Lowry assay (Bio-Rad, Hercules, CA) with
bovine serum albumin as a standard. The lysates were boiled in 2×
Laemmli buffer (1× is 62.5 mM Tris, pH 6.8, 2% SDS, 10%
glycerol, 0.025% bromphenol blue, and 5%
-mercaptoethanol). Proteins (10 µg/ml) were separated by size on polyacrylamide-SDS gels
and transferred to nitrocellulose. The membranes were blocked in PBS
containing 5% nonfat dry milk overnight at 4°C before incubation with an anti-cyclin A antibody (Santa Cruz Biotechnology, Santa Cruz,
CA) at a 1:1,000 dilution for 1 h at room temperature.
Nonspecific interactions were removed by washing in PBS containing
0.05% Tween 20 before the blots were incubated in goat anti-rabbit
peroxidase-conjugated secondary antibody at 1:5,000 (Jackson
ImmunoResearch Laboratories, West Grove, PA). The blots were
extensively washed again, and the conjugates were visualized with
chemiluminescence (Amersham, Arlington Heights, IL) by exposure to
Kodak Bio-Max film. The blots were reblotted with anti-
-actin
antibody (Sigma, St. Louis MO) at a 1:5,000 dilution as a loading control.
TGF--inducible luciferase reporter
assays. The cells were transfected with the TGF-
inducible reporter p3TP-Lux with calcium phosphate as previously
described (21). The p3TP-Lux reporter contains three
12-O-tetradecanoylphorbol 13-acetate (TPA) response elements
(TRE) from the human collagenase promoter and the TGF-
responsive
element from the plasminogen activator inhibitor-1 promoter ligated
upstream to the adenovirus E4 minimal promoter (31). Transfection
efficiencies were normalized with the pRL-TK vector (Promega, Madison,
WI) that expresses the Renilla
luciferase gene. Luciferase assays were performed with the
dual-luciferase reporter assay system (Promega) and measured with a
luminometer from Tropix (Bedford, MA).
Statistical analyses. Values are expressed as means ± SD. Group means were compared by ANOVA with Fisher's procedure post hoc analysis with StatView software for Macintosh, with P < 0.05 being considered significant.
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RESULTS |
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Effects of TGF- on cell cycle
distribution of Mv1Lu and R1B cells. Mv1Lu and R1B
cells were incubated for 24 h in the absence and presence of 5 ng/ml of
TGF-
1 and harvested, and genomic DNA was stained with propidium
iodide. Flow cytometric analysis revealed that asynchronous cultures of
Mv1Lu cells cultured in room air contained cells in all phases of the
cell cycle. Approximately 55% of the cells were in the
G1 [diploid (2n) of DNA where n
is haploid] phase, 30% in the S phase, and 15% in
the G2 (4n) phase (Table
1). Mv1Lu cells treated with TGF-
accumulated in the G1 phase of the
cell cycle, with a significant decrease in the percentage of cells in
the S and G2 phases. In fact,
nearly 90% of TGF-
-treated cells accumulated in the
G1 phase. Although asynchronous cultures of R1B cells had a similar cell cycle distribution as Mv1Lu
cells when cultured in room air, they were unaffected by exposure to
TGF-
(Table 1). These findings confirm a previous study (31) that
demonstrated that the Mv1Lu clonal variant R1B cell line is
unresponsive to TGF-
.
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Hyperoxia inhibits Mv1Lu and R1B
proliferation. Subconfluent cultures of Mv1Lu and R1B
cells were exposed to room air or hyperoxia to determine whether
hyperoxia inhibited their proliferation. The cells were harvested every
24 h and counted with a hemacytometer. Mv1Lu and R1B cultures had an
increasing number of cells over time when incubated under normoxic
conditions (Fig. 1). In contrast, the cell
number did not increase in cultures exposed to hyperoxia. The effects
of hyperoxia on total cell number were distinguishable within the first
24 h of exposure.
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Changes in total cell number in the presence of hyperoxia could be due
to decreased cell proliferation or increased cell death. Although Mv1Lu
and R1B cells remain attached to their plates for the first 72 h of
exposure, trypan blue dye exclusion was used to quantitatively measure
cell viability. More than 90% of the cells exposed to hyperoxia for up
to 72 h continued to possess good membrane integrity based on their
ability to exclude dye (Fig.
2A).
Similarly, R1B cells maintained membrane integrity over this time
period (data not shown). Although membrane integrity remains a
reasonable method to determine the viability of necrotic cells, it does
not adequately detect apoptotic cells (25). TUNEL staining was used to
assess apoptosis in Mv1Lu cells exposed to hyperoxia. Minimal
TUNEL-positive cells were observed in cultures exposed to room air or
hyperoxia (Fig. 2B). Hyperoxia also
does not induce DNA laddering in Mv1Lu cells (data not shown). As a positive control for apoptotic cell death, Mv1Lu cells were exposed to
5 Gy of ionizing radiation. In contrast to the effects of hyperoxia, nearly 90% of Mv1Lu cells exposed to radiation became TUNEL positive. TUNEL staining was not measured in R1B cells because this finding and a
previous study (16) have shown that hyperoxia kills cells in vitro
through necrosis. Collectively, our findings suggest that the effects
of hyperoxia on cell number were not due to increased cell death.
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Mv1Lu and R1B cells were exposed to room air or hyperoxia for 24 h and
analyzed by flow cytometry to determine whether hyperoxia altered their
cell cycle distribution. Asynchronous cultures of Mv1Lu and R1B cells
cultured in room air had cells in all phases of the cell cycle (Fig.
3). In contrast, cultures of both cell lines exposed to hyperoxia for 24 h had a marked increase in the percentage of cells in the S phase associated with a decrease in the
percentage of cells in the G1
phase. In fact, the percentage of cells in the S phase nearly doubled
after 24 h of exposure (Table 1). Continued exposure to hyperoxia for
48 and 72 h resulted in a continued increase in the percentage of cells
in the S phase (Fig. 4).
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Differential effects on the cell cycle by
TGF- and hyperoxia. Asynchronous
cultures of Mv1Lu cells were then exposed to hyperoxia in the absence
and presence of 5 ng/ml of TGF-
1 to determine whether TGF-
could
maintain a G1 phase growth arrest
in the presence of hyperoxia. Changes in the percentage of Mv1Lu cells
in the S phase were time dependent as noticed by the continual increase in the number of cells with a DNA content of >2n and <4n (Fig. 4,
left). Approximately 80% of the
cells were in the S phase based on propidium iodide staining after 72 h
of hyperoxia. In contrast, cells exposed to hyperoxia and treated with
TGF-
accumulated in the G1
phase and remained predominantly in the
G1 phase even after 72 h of
hyperoxia (Fig. 4, right). Although
the percentage of cells retained in the
G1 phase with TGF-
treatment
decreased from 90% after 24 h to ~70% after 72 h, it was still
significantly greater than the percentage of cells exposed to hyperoxia
alone. TGF-
inhibited S phase entry of cells exposed to hyperoxia
for 3 days when the medium was replenished every 24 h as well as when the cells were treated once at the beginning of the exposure (data not shown).
The expression of cyclin A was also determined as further evidence that
TGF- and hyperoxia inhibit proliferation through distinct
mechanisms. Cyclin A is expressed by cells in the S and early
G2 phases and is decreased in
Mv1Lu cells treated with TGF-
(27, 29). Mv1Lu cells were exposed to
room air, TGF-
, hyperoxia, or TGF-
and hyperoxia for 24 h. Cyclin
A was readily detected in asynchronous cultures growing under normoxic
conditions and in cultures growth arrested by hyperoxia (Fig.
5). In contrast, cyclin A abundance was
decreased to nearly undetectable levels in cultures exposed to TGF-
or TGF-
and hyperoxia. Thus cyclin A was detected in asynchronous
cultures exposed to room air or arrested by hyperoxia but not in
cultures arrested by TGF-
.
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Effects of hyperoxia on a
TGF--dependent transcriptional
reporter. These observations demonstrate that TGF-
inhibits proliferation in the G1
phase, whereas hyperoxia causes cells to accumulate in the S phase.
Furthermore, they suggest that TGF-
signaling can overcome the
effects of hyperoxia because cells treated with TGF-
and hyperoxia
accumulate in the G1, not in the
S, phase. To confirm that TGF-
and hyperoxia signal through distinct
pathways, the TGF-
-responsive luciferase reporter gene p3TP-Lux was
used to measure TGF-
-dependent transcriptional responses (31). Mv1Lu cells were transfected with this plasmid and exposed to room air, TGF-
, hyperoxia, or TGF-
and hyperoxia for 24 h. Room air-exposed cells had minimal luciferase activity that was markedly induced by
treatment with TGF-
(Fig. 6). Cells
exposed to hyperoxia alone also had minimal luciferase activity that
was not significantly different from that in room air-exposed cells. In
contrast, cells exposed to both TGF-
and hyperoxia had induced
luciferase activity, consistent with the ability of TGF-
to signal
even in O2-exposed cultures. As a
control for reporter specificity, R1B cells were transfected and
exposed to room air, TGF-
, hyperoxia, or TGF-
and hyperoxia.
Minimal luciferase activity was detected in room air-exposed cultures
and was not induced by TGF-
or hyperoxia (Fig. 6).
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DISCUSSION |
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The present study extends previous observations on the growth-arresting
activities of hyperoxia in other cell lines by showing that hyperoxia
caused Mv1Lu and R1B cells to accumulate in the S phase of the cell
cycle. These changes were independent of TGF- signaling because
hyperoxia inhibited proliferation of the TGF-
-unresponsive cell line
R1B. Mv1Lu cells exposed to room air or hyperoxia expressed cyclin A,
whereas cells treated with TGF-
accumulated in the G1 phase and had reduced levels of
cyclin A. Moreover, Mv1Lu cells exposed to both TGF-
and hyperoxia
accumulated in the G1 phase, with
decreased expression of cyclin A, suggesting that TGF-
signaling can
overcome the growth-arresting activities of simple hyperoxia. This was
confirmed by demonstrating functional activity of a TGF-
-dependent transcriptional reporter in Mv1Lu cells cultured in room air or hyperoxia. However, hyperoxia by itself was unable to activate TGF-
-dependent transcription. Collectively, these observations suggest that TGF-
does not participate in the growth-arresting activities of hyperoxia. However, TGF-
may modify the cellular response to hyperoxia by preventing S phase entry.
Previous studies with rat SV40T-T2 cells demonstrated that their
proliferation was inhibited by hyperoxia. Although growth arrest was
associated with increased mRNA expression of histone and thymidine
kinase mRNAs, they were not efficiently translated, which could account
for the failure of these cells to proliferate (5). In addition,
hyperoxia increased mRNA levels of TGF-1, T
R-I, and T
R-II (4,
6). Hyperoxia also increased p21, which inhibited the kinase activity
of cyclin E-Cdk2 complexes (6). The authors concluded that TGF-
participates in mediating the growth-arresting activities of hyperoxia
because SV40T-T2 cells cultured with a neutralizing antibody to TGF-
had a modest increase in cyclin E-Cdk2 activity. Unfortunately, these
studies never measured cell cycle progression or determined whether
addition of the neutralizing TGF-
antibody resulted in an increase
in total cell number. Based on the present findings, it is possible that blocking TGF-
activity resulted in more cells exiting the G1 phase and entering the S and/or
G2/M phases where they arrested. Alternatively, hyperoxia may inhibit cell proliferation by different mechanisms that are cell-type dependent. For example, the effects of
hyperoxia on SV40T-T2 cells may be unique to these cells because SV40
and other DNA tumor proteins are known to rearrange cyclin and Cdk
partners (32). In contrast, there is no evidence that the Mv1Lu cells,
which are derived from the fetal mink lung, express viral genes (14).
The present study reveals that Mv1Lu cells exposed to hyperoxia
accumulate predominantly in the S phase of the cell cycle. Although
G1 and
G2 cell cycle checkpoints in
response to DNA damage have been studied extensively, less is known
about the existence of an S phase checkpoint. Cells exposed to ionizing
radiation growth arrest in the G1
phase through the DNA damage-dependent accumulation of the tumor
suppressor p53, which transcriptionally increases p21 (10, 17, 20). In
addition to blocking S phase entry, p21 also binds proliferating cell
nuclear antigen, which participates in both DNA replication and repair
(18). Additional studies in yeast have identified DNA polymerase- as
a DNA damage sensor that may integrate DNA replication and repair (19).
Ionizing radiation also induces a
G2 phase checkpoint that is
controlled by RAD53 and other related kinases (29). In contrast,
ultraviolet B radiation, which creates DNA adducts, prolonged
G1 and S phase progression in
neonatal rat keratinocytes (24). Similarly, alkylating agents slow S
phase progression in Saccharomyces
cerevisiae (23). Because DNA replication was dependent
on the MEC1 and RAD53 genes, the authors concluded that alkylating
agents inhibited proliferation through activation of a checkpoint
control rather than through failure to bypass DNA lesions.
Checkpoints were defined as places in the cell cycle where progression was dependent on completion of the previous phases (12). Failure to complete the previous phase resulted in a transient growth arrest during which the cells were able to conclude incomplete biochemical processes. Although hyperoxia alone is not cytotoxic, it is converted to genotoxic ROS that would be predicted to elicit the classic G1 phase checkpoint involving p53 and p21 or the G2 phase checkpoint involving RAD53 (20, 29). The present finding that cells exposed to hyperoxia fail to progress through the S phase is consistent with an inability to appropriately replicate DNA. Further studies are needed to determine whether this is due to a global shutdown in cellular function or a novel DNA damage checkpoint.
The present study found that TGF- could maintain the
G1 phase checkpoint in cells
exposed to hyperoxia, thereby preventing the entry and subsequent
arrest in the S phase caused by hyperoxia alone. It is widely believed
that the cells arrested in the G1 or G2 phase will have a greater
capacity to survive genotoxic stress compared with cells in the S phase
when DNA replication is occurring (29). However, yeast cells arrested
in the S phase with the alkylating agent methylmethane sulfonate have
enhanced survival when challenged with ionizing radiation (23). The
authors suggest that methylmethane sulfonate may protect against
radiation-induced DNA damage because DNA repair events are coupled with
replication. This hypothesis is consistent with the observation that
DNA polymerase-
and p21 coordinate DNA repair and replication (18,
19). The concept that resistance to genotoxins may be coupled to the
cell cycle is also consistent with a recent study (15) where confluent (growth-arrested) cultures of small-airway epithelial cells were more
resistant to hyperoxic injury than subconfluent (proliferating) cultures. Although these studies use different cell types and DNA-damaging agents, they suggest that cells in different phases of the
cell cycle are not equally injured by genotoxins. Thus cells arrested
in the G1 phase by TGF-
may
have an altered ability to survive exposure to hyperoxia than cells
arrested in the S and G2 phases by
hyperoxia alone. Because TGF-
decreases antioxidant enzyme
expression and enhances the cytotoxicity of hydrogen peroxide in A549
cells, future experiments must take into account the multitude of
biological activities associated with this cytokine in addition to its
ability to simply inhibit proliferation (3).
In summary, the findings in this study reveal that hyperoxia induced
Mv1Lu and R1B cells to accumulate in the S phase independent of TGF-
signaling. TGF-
induced a G1
phase growth arrest that blocked entry into and subsequent arrest in
the S phase caused by hyperoxia. These changes in cell cycle
progression were not associated with decreased cell viability. It is
worth noting that TGF-
expression increased within 1-3 h of
exposure to hyperoxia in pulmonary epithelial cells of adult mice (22).
Although the role that TGF-
plays in hyperoxic lung injury remains
to be determined, this finding is interesting because it suggests that
TGF-
may prevent S phase entry of pulmonary cells exposed to
hyperoxia in vivo.
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ACKNOWLEDGEMENTS |
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We thank Anita Roberts for the Mv1Lu cell line and Joan Massagué for the R1B cell line and p3TP-Lux plasmid.
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FOOTNOTES |
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This work was funded by the American Lung Association; American Heart Association Beginning Grant-in-Aid 9860004T; and National Heart, Blood, and Lung Institute Grant HL-58774 (to M. A. O'Reilly). Additional support was provided by National Cancer Institute Grants CA-73725 and CA-11198 (to P. C. Keng).
R. C. Rancourt was supported by National Institute of Environmental Health Sciences Training Grant ES-07026.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. A. O'Reilly, Dept. of Pediatrics (Neonatology), Box 777, Children's Hospital at Strong, The Univ. of Rochester, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: oreillym{at}envmed.rochester.edu).
Received 28 October 1998; accepted in final form 3 August 1999.
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