Pediatric Surgery, Developmental Biology, and Research Immunology/Bone Marrow Transplant Programs, Childrens Hospital Los Angeles Research Institute, Los Angeles, California 90027
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ABSTRACT |
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Apoptosis is a genetically controlled cellular response to developmental stimuli and environmental insult that culminates in cell death. Sublethal hyperoxic injury in rodents is characterized by a complex but reproducible pattern of lung injury and repair during which the alveolar surface is damaged, denuded, and finally repopulated by type 2 alveolar epithelial cells (AEC2). Postulating that apoptosis might occur in AEC2 after hyperoxic injury, we looked for the hallmarks of apoptosis in AEC2 from hyperoxic rats. A pattern of increased DNA end labeling, DNA laddering, and induction of p53, p21, and Bax proteins, strongly suggestive of apoptosis, was seen in AEC2 cultured from hyperoxic rats when compared with control AEC2. In contrast, significant apoptosis was not detected in freshly isolated AEC2 from oxygen-treated rats. Thus the basal culture conditions appeared to be insufficient to ensure the ex vivo survival of AEC2 damaged in vivo. The oxygen-induced DNA strand breaks were blocked by the addition of 20 ng/ml of keratinocyte growth factor (KGF) to the culture medium from the time of plating and were partly inhibited by Matrigel or a soluble extract of Matrigel. KGF treatment resulted in a partial reduction in the expression of the p21, p53, and Bax proteins but had no effect on DNA laddering. We conclude that sublethal doses of oxygen in vivo cause damage to AEC2, resulting in apoptosis in ex vivo culture, and that KGF can reduce the oxygen-induced DNA damage. We speculate that KGF plays a role as a survival factor in AEC2 by limiting apoptosis in the lung after acute hyperoxic injury.
Bax protein; p53 protein; p21 protein; deoxyribonucleic acid strand breaks; keratinocyte growth factor
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INTRODUCTION |
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RAT TYPE 2 ALVEOLAR EPITHELIAL CELLS (AEC2),
which are quiescent in the normal adult lung, respond to in vivo
hyperoxic lung injury by proliferating to repopulate the damaged
alveolar epithelial surface (8, 26). This proliferative response in
AEC2 is associated with the induction of cell cycle genes including A-
and D-type cyclins and proliferating cell nuclear antigen (PCNA),
activation of cyclin-dependent kinase activities, and downregulation of
autocrine transforming growth factor (TGF)-3 peptide secretion (1,
2).
Apoptosis is a genetically controlled cellular response to developmental or environmental stimuli that culminates in the death of a cell (30). Thus apoptosis is an important mechanism of negative selection that removes damaged cells that may be deleterious to the host. Because apoptosis has been reported in the lung after hyperoxia (12), we postulated that apoptosis may occur in AEC2 isolated from rats treated with sublethal doses of oxygen.
Keratinocyte growth factor (KGF) is a potent mitogen for AEC2 both in vivo and in vitro (18, 29). In addition, it has a strongly protective effect in the lung against a variety of injuries, including hyperoxia (17). We reasoned that if apoptosis occurred in AEC2 in response to hyperoxia, subsequent treatment with KGF might be protective.
We treated adult male rats with >90% humidified oxygen for 48 h and allowed the rats to recover in room air for various times as previously described (1). We then isolated AEC2 from the lungs of oxygen-treated and control rats, adding an extra differential adherence step to ensure maximal purity, and either fixed the cells immediately or cultured them for 24 h. In some experiments, the cells were cultured on a thin layer of Matrigel or with the addition of 20 ng/ml of KGF or a soluble extract of Matrigel added at the time of plating. We measured FITC-dUTP DNA end labeling by fluorescence-activated cell sorter (FACS) analysis of cells counterstained with propidium iodide (PI) because this gave us both quantitation of DNA strand breakage and cell cycle analysis. In parallel, we measured the expression of Bax, a protein that is elevated in apoptosis, and proteins more indirectly associated with apoptosis, p53 and p21, which are elevated when the cell stops at a cell cycle checkpoint to direct DNA repair or apoptosis, depending on the extent of the damage. Finally, we looked for DNA laddering, a phenomenon associated with the later stages of apoptosis. Significant apoptosis, as detected by FACS analysis of FITC-end-labeled DNA, a prediploid peak on FACS analysis of PI-stained cells, DNA laddering, and Western blotting of apoptotic proteins, was detected in cultured AEC2 from animals treated with oxygen for 48 h and allowed up to 24 h of recovery before death. The apoptosis resolved with recovery time in vivo. However, apoptosis was not detected in fresh isolates of AEC2 from oxygen-treated rats, suggesting that basal culture conditions could not sustain the damaged cells. If 20 ng/ml of KGF were added to the culture medium from the time of plating, DNA strand breakage could be blocked. Matrigel or a soluble extract of Matrigel was partially successful in blocking oxygen-induced DNA strand breaks, further suggesting that exogenous growth factors may prevent or ameliorate oxygen-mediated DNA damage.
Many of the published reports (11, 17, 24, 32) describing the protective effect of KGF against damage to the lung itself or to lung epithelial cells involve the addition of KGF before the insult. Our results suggest that KGF may also play a role as a survival factor for AEC2 by limiting oxygen-induced DNA damage.
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METHODS |
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Oxygen treatment and recovery. Adult male Sprague-Dawley rats were exposed to short-term hyperoxia and were then allowed to recover in air exactly as previously described (1). Briefly, rats were placed in a 90 × 42 × 38-cm Plexiglas chamber, exposed to humidified >90% oxygen for 48 h, and either killed immediately or allowed to recover in room air. The 48-h oxygen exposure time induced lung damage with minimal mortality. Control rats were kept in room air. At the end of the exposure and/or recovery period, the rats were anesthetized with an intraperitoneal injection of pentobarbital sodium. After complete exsanguination by saline perfusion via the pulmonary artery, the lungs were lavaged to remove macrophages. The lavaged lungs were then used for AEC2 isolation and culture.
Isolation of AEC2. AEC2 were isolated
from lavaged lungs by elastase digestion followed by differential
adherence on IgG plates as described by Dobbs et al. (5). The cells
panned from the IgG plates were cultured for one more hour on plastic
in the presence of 10% FCS as an extra differential adherence step to
remove any remaining macrophages or fibroblasts, and the adherent cells
were discarded. This step was added because some of the fresh isolate was to be fixed without culturing. A portion of the freshly isolated AEC2 was fixed in ice-cold 1% paraformaldehyde for 15 min, washed, and
stored in 70% ethanol at 20°C until
analysis. The remainder of the
cells were plated at a density of 2 × 105
cells/cm2 in DMEM with 10% FCS
and allowed to attach for 24 h. The culture medium was then collected
from the cells, centrifuged, and frozen at
70°C. Any
unattached or floating cells were kept and pooled with the cells
recovered from the monolayer. The cells were then either lysed to
extract proteins for Western blotting, fixed in ice-cold 1%
paraformaldehyde, or stored at
20°C as a washed pellet for
subsequent DNA extraction. Cultures were immunostained to confirm
culture purity. More than 95% of the attached cells were vimentin
negative and surfactant protein C positive. The antibody to surfactant
protein C was kindly provided by Dr. Jeffrey Whitsett (Children's
Hospital Medical Center, Cincinnati, OH), and the antibody to vimentin
was from Sigma.
Treatment of AEC2 with KGF, Matrigel, or Matrigel
extract. Preliminary experiments had shown that maximal
DNA strand breakage occurred in cultured AEC2 isolated from rats
treated with oxygen for 48 h and allowed no in vivo recovery time.
Cells freshly isolated from these rats were plated in DMEM-10% FCS
with the addition of a thin layer (100 µg/cm2) of Matrigel (Becton
Dickinson, Bedford, MA) or 20 ng/ml of human recombinant KGF
(Peprotech, Rocky Park, NJ). Cells were also incubated in the presence
of a soluble extract of Matrigel (prepared in advance by incubating
DMEM containing 10% FCS with thinly coated Matrigel under identical
conditions used in the experiments) for 24 h. At the completion of the
experiment, any floating cells were collected and pooled with cells
recovered from the monolayer, and the cells were fixed for DNA end
labeling, lysed for Western blotting, or stored at 20°C as a
washed pellet for subsequent DNA extraction.
Flow cytometric method for measuring DNA end labeling in fixed cells. DNA strand breaks were measured in 1% paraformaldehyde-fixed AEC2 by labeling with fluorescent dUTP with an APO-DIRECT kit (PharMingen, San Diego, CA) according to the manufacturer's instructions. Fixed cells were incubated at 37°C for 1 h with terminal deoxynucleotidyltransferase (TdT) and FITC-labeled dUTP before being counterstained with PI. The stained cells were analyzed with a Becton Dickinson FACScan equipped with a 488-nm argon laser. Nonclumped cells were gated with PI staining, and the gated cells are displayed two ways: 1) as DNA area (linear red fluorescence) on the x-axis versus cell number on the y-axis, giving cell cycle analysis; and 2) as DNA area on the x-axis versus FITC-dUTP (log green fluorescence) on the y-axis. A horizontal gate was then applied to this display to discriminate between apoptotic (FITC-staining) and nonapoptotic cells. This was done with control positive and negative cells supplied with the kit, which were end labeled in parallel with the test samples. The AEC2 were then analyzed, and the percentage of gated cells that were FITC positive was compared for each treatment group. No FITC fluorescence was seen in either control or oxygen-treated AEC2 that had been incubated in the labeling reaction mixture in the absence of TdT. Cell cycle analysis of PI-stained cells was also performed. Cellquest (Becton Dickinson) and Modfit LT 2.0 (Verity House, ME) software were used for the analyses.
Western blotting of proteins. Western analysis was performed on cell lysates as described by Bui et al. (2), with 20-40 µg protein/lane depending on the sensitivity of the antibody used. Equal loading was confirmed by blotting with an antibody to actin. Proteins of interest were detected with horseradish peroxidase-linked secondary antibodies and the enhanced chemiluminescence system following the manufacturer's instructions (Amersham, Arlington Heights, IL). The antibody to Bax was from Oncogene Science (Uniondale, NY). The antibodies to Bcl2, p21, PCNA, and p53 were from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody to actin was from ICN (Irvine, CA). Secondary antibodies were from Sigma (St. Louis, MO).
Detection of DNA laddering by gel electrophoresis. DNA was extracted from 24-h cultures of control and hyperoxic AEC2 with a QIAamp kit according to the manufacturer's instructions (Qiagen, Valencia, CA). Approximately 1 µg of DNA from each sample was analyzed by electrophoresis through a 1% agarose gel in parallel with a 100-bp DNA ladder (GIBCO BRL, Gaithersburg, MD). The gel was then examined under ultraviolet (UV) light for the presence of nucleosomal size fragments, or ladders, with ethidium bromide staining for visualization.
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RESULTS |
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DNA strand breakage as indicated by FITC-dUTP end labeling and a pre-G1 peak in PI labeling occurs in cultured AECs from rats treated with oxygen for 48 h. FACS analysis of 24-h cultures of AEC2 isolated from oxygen-treated rats showed FITC-dUTP end labeling (Fig. 1B) that was not present in the control cultures (Fig. 1A). Parallel FACS analysis of PI-stained cells showed a prediploid DNA peak present in AEC2 from oxygen-treated animals (Fig. 1D), representing fragmented DNA. This peak was absent in the control cultures (Fig. 1C).
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Significant DNA strand breakage as indicated by FITC-dUTP end labeling in cultured AEC2 from hyperoxic rats decreases with recovery time in vivo. When fresh isolates of oxygen-treated and recovered cells were fixed immediately after isolation, end labeled with FITC-dUTP, and analyzed by FACS, no significant end labeling was detected. Yet, when aliquots of these cells were cultured for 24 h on plastic, significant end labeling was seen in 24-h cultures of AEC2 from rats treated with oxygen for 48 h and allowed no recovery time (P < 0.001 compared with control rats, Student's t-test; n = 7-10 cultures). After 24-h in vivo recovery, the end labeling seen in cultured AEC2 was still significantly different from the control rats (P < 0.05; Fig. 2). The oxygen-induced DNA strand breakage decreased with recovery time in vivo and preceded the peak of oxygen-induced proliferation, which occurs in our hyperoxia model after 48 h of recovery (1, 2). Surprisingly, DNA damage as measured by end labeling did not correlate with the severity of the damage in vivo; AEC2 cultured from rats that suffered a more pronounced response to oxygen by developing pulmonary edema or pneumothorax showed a DNA labeling profile similar to that from rats with less discernible damage. There was a large animal-to-animal variation in the extent of oxygen-induced DNA end labeling. FACS analysis of PI-stained AEC2 showed that the FITC-labeled cells were in both the G1 and G2 phases, consistent with p53-mediated apoptosis. Control AEC2, whether freshly isolated or cultured, showed ~5% end labeling.
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DNA strand breakage as measured by end labeling of AEC2 cultured from oxygen-treated rats is inhibited by KGF and partially inhibited by Matrigel or a soluble extract of Matrigel. The addition of 20 ng/ml of KGF to the oxygen-treated AEC2 from the initial plating significantly inhibited the in vivo oxygen-induced DNA end labeling (Fig. 3). Growth on Matrigel partially blocked the end labeling. Incubation with a soluble extract of Matrigel had the same effect as growth on wells coated with Matrigel, suggesting that it was the soluble components of Matrigel rather than the attachment to it per se that was causing the amelioration of DNA strand breakage.
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Expression of apoptosis-associated p53, p21, and Bax proteins is increased in cultured AEC2 from oxygen-treated rats. This increase in expression is partially blocked by 20 ng/ml of KGF. The oxygen-induced DNA strand breaks associated with cell cycle blocks in the G1 and G2 phases suggested the possibility of p53-mediated apoptosis. p53 responds to DNA damage by arresting the cell cycle in either the G1 or G2 phase to allow DNA repair to take place. This arrest is mediated by activation of the G1/G2 cyclin-dependent kinase inhibitor p21WAF1/CIP1 and may result in apoptosis if the cell is unable to repair the DNA damage. The ratio of Bax to Bcl2 is elevated under these circumstances because Bax expression increases while Bcl2 expression decreases. Oxygen-treated AEC2 cultured with and without 20 ng/ml of KGF were lysed for Western analysis of p53 as well as p21, Bax, and Bcl2, proteins regulated through p53 under certain apoptotic conditions. Oxygen treatment induced the expression of p53, Bax, and p21, whereas 20 ng/ml of KGF partially offset the induction (Fig. 4). The latter results were less dramatic than the end labeling, suggesting that either the partial reduction of p53 and its partners is sufficient to remove the damage signal or the DNA strand breaks were indicative of other DNA damage in addition to apoptosis. No difference in Bcl2 expression was detected under any condition tested; however, because the available antibodies reacted poorly with rat Bcl2, subtle decreases may have gone undetected (data not shown).
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Gel electrophoresis shows DNA laddering in AEC2 cultured from hyperoxic rats. A characteristic ladderlike DNA degradation pattern is seen in DNA isolated from AEC2 cultured from hyperoxic rats (Fig. 5). This DNA fragmentation is not seen in control AEC2. As with the end labeling, there is noticeable animal-to-animal variation in the extent of laddering. Treatment of the cells with 20 ng/ml of KGF from the time of plating has no effect on DNA laddering.
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PCNA expression is strongly induced when AEC2 from oxygen-treated
rats are cultured with KGF. PCNA is the auxiliary protein of DNA
polymerase-, which is required for both leading-strand DNA
replication and DNA repair and is synthesized in the early G1 and S phases of the cell cycle.
PCNA expression is increased when AEC2 from oxygen-treated rats are
treated with 20 ng/ml of KGF from the time of plating (Fig.
6). This increased PCNA expression is
associated with decreased DNA damage as shown by a reduction in end
labeling, suggesting that PCNA-mediated DNA repair may be occurring
under these conditions.
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DISCUSSION |
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Sublethal hyperoxic lung injury in rodents is characterized by a
complex yet reproducible pattern of injury and repair. After short-term
hyperoxic treatment, the normally quiescent AEC2 respond to the
resultant damage by proliferating to repopulate the denuded alveolar
epithelium during the recovery process in vivo (8, 26). This
oxygen-induced proliferative response of AEC2 is also seen in vitro and
in cultures of cells isolated from oxygen-treated and recovered rats
and is characterized by induction of key cell cycle genes, increased
[3H]thymidine and
bromodeoxyuridine uptake, and downregulation of autocrine TGF-
peptide secretion (1, 2). Thus AEC2 can and do respond in vitro to
signals initiated while in vivo.
Apoptosis, or programmed cell death, occurs as a response to developmental or environmental stimuli and is seen during embryogenesis, metamorphosis, and the turnover of senescent cells (30). Death by apoptosis involves characteristic hallmarks: cytoplasmic condensation, loss of plasma membrane microvilli, and nuclear condensation and segmentation. Apoptosis may be mediated by environmental insult, such as irradiation (16), or simply by the lack of a survival factor, such as neurotropins in the nervous system (15). Apoptosis has been reported in the lung after hyperoxic injury as demonstrated by in situ end labeling of DNA in fixed sections of a hyperoxic lung. In contrast, no apoptosis was detected in A549 cells after oxygen treatment, indicating that the signals induced in the lung that mediated the apoptosis in vivo were absent in vitro (12). Fibroblast-conditioned medium, derived from both fibrotic human and paraquat-treated rat lung fibroblast cultures, induced apoptosis in cultured rat AEC2 (28).
Apoptosis is usually detected by DNA end labeling [TdT-mediated dUTP nick end labeling (TUNEL)] or DNA laddering, although neither of these methods is an exclusive measure of apoptosis (9). The complex model of sublethal oxygen injury in the rat lung almost certainly involves multiple responses to cell damage, including apoptosis, necrosis, and DNA repair. We chose to measure TUNEL by FACS analysis of cells counterstained with PI because this gives a quantitative estimate of DNA strand breakage, confirms morphological nuclear changes, and also provides cell cycle analysis. In addition, we looked at DNA laddering, which is less quantitative but gives information as to the size of DNA fragments. Finally, we examined the expression of Bax, a protein intimately associated with apoptosis, and of p21 and p53, proteins that are elevated during the cell cycle blocks preceding apoptosis.
In our study, cultured AEC2 isolated from oxygen-treated rats with an in vivo recovery time of up to 24 h showed significant apoptosis as detected by DNA end labeling, a prediploid DNA peak on FACS analysis of PI staining, DNA laddering, and increased Bax, p53, and p21 expression compared with AEC2 cultured from control rats breathing room air. However, if AEC2 isolated from oxygen-treated rats were fixed immediately after isolation, no significant apoptosis was detected. This suggests that culture conditions were not conducive to the survival of the oxygen-damaged cells and that one or more "survival factor," possibly an antioxidant, growth factor, or hormone, was absent from our culture medium. Damaged AEC2, however, were repaired in vivo because apoptosis detected in vitro receded with the animal's recovery time.
The exact nature of the damage to AEC2 caused by hyperoxia, which results in subsequent DNA strand breakage in vitro, remains to be established. Using UV-irradiated Chinese hamster ovary (CHO) cell lines with differing DNA repair capacities, Orren et al. (16) have shown that persistent DNA damage due to UV irradiation (and not activation of signal transduction pathways resulting from general cellular stress) is the cause of prolonged delays in cell cycle progression that eventually leads to apoptosis in CHO cells. The signal(s) resulting in the apoptosis seen in AEC2 cultured from oxygen-treated rats appears to originate in the lung and is not due to a soluble factor produced by cultured oxygen-damaged AEC2 because supernatants collected from apoptotic cells did not render normal cultured AEC2 cells apoptotic (data not shown). Uhal et al. (27) have recently reported apoptosis located in epithelium adjacent to cells expressing smooth muscle actin in sections of fibrotic human lung. This suggests that myofibroblasts in the mesenchyme may be one of the mediators of apoptosis associated with fibrosis in vivo. However, immunostaining of our oxygen-treated AEC2 cultures with a monoclonal antibody to smooth muscle actin has proved negative, suggesting that myofibroblasts do not play a role in this model of in vitro apoptosis.
The oxygen-treated AEC2 also had increased expression of p53, p21, and Bax proteins. The tumor suppresser gene p53 induces apoptosis in a dose-dependent manner after DNA breakage. It arrests the cell in either the G1 or G2 phase to allow DNA repair to take place via activation of the G1/G2 cyclin-dependent kinase inhibitor p21CIP1/WAF1. If successful repairs are not effected, p53 induces apoptosis, thereby preventing the propagation of genetic defects to successive generations of cells (3, 7). The ratio of Bax to Bcl2 determines survival or death for many cells after an apoptotic stimulus such as the removal of growth factors (14). Bax expression may also be stimulated as a result of p53 activity. Although no data proving a causative role for these proteins is presented in this paper, the increased expression in the context of DNA strand breakage and laddering is consistent with p53-mediated apoptosis.
The DNA strand breaks seen in cultured, oxygen-treated AEC2 could be
prevented by the addition of 20 ng/ml of KGF to the medium at the time
of plating. Matrigel and a soluble extract of Matrigel could also block
the DNA strand breaks but less efficiently than KGF. Growth factors are
logical candidates as mediators of lung repair. Growth factors such as
KGF are present in the lung but are absent (or at reduced
concentrations) in basal culture medium. KGF, as well as being a potent
mitogen for AEC2, has been shown to have a protective effect against
lung injury, whether induced by various chemicals (4, 11, 13, 31, 32)
or oxygen (17). Our data demonstrating inhibition of oxygen-induced DNA damage in cultured AEC2 by in vitro KGF treatment is consistent with a
role for KGF in DNA damage repair as previously suggested by Takeoka et
al. (24), who demonstrated that preincubation with KGF can facilitate
the repair of radiation-induced DNA damage in A549 cells. Using
specific DNA polymerase blockers, they demonstrated that KGF
facilitates nuclear excision repair through DNA polymerase-, -
,
and -
. PCNA is the auxiliary protein of DNA polymerase-
(21),
which is required for both leading-strand DNA replication and DNA
repair (20, 23, 25). Our observation that KGF amelioration of
oxygen-induced DNA end labeling in AEC2 is associated with increased
expression of PCNA suggests that repair of oxygen damage may proceed
via similar mechanisms, possibly by moving the cells into the cell
cycle to the next checkpoint where DNA strand breaks can be repaired.
Polunovsky et al. (19) has shown in endothelial cells that
susceptibility to tumor necrosis factor-
-mediated apoptosis varies
according to the cell cycle stage at the time of apoptotic insult. KGF
has also been shown to augment DNA repair by several other mechanisms;
H2O2-induced
DNA damage in A549 cells was shown to be ameliorated by KGF through
signal transduction pathways in addition to DNA polymerase-dependent
pathways (31). Growth factors are already implicated in upstream events
preceding apoptosis; Sachsenmaier et al. (22) have shown that growth
factor receptors play a role in the early response of HeLa cells to
short-wavelength UV light-induced damage. The
short-wavelength UV response was inhibited at the transcriptional level
by growth factor prestimulation, by suramin, an inhibitor of receptor
activation, or by expression of a dominant negative epidermal growth
factor-receptor mutant. The preventive effect of KGF on the cell damage
varied according to the parameter measured: DNA end labeling was
completely inhibited; Bax, p21, and p53 protein expression was partly
inhibited; and DNA laddering was unaffected. This suggests a repair
specificity and/or a critical timing window for the protective
effects of KGF.
We are now currently investigating the nature of the DNA damage in oxygen-treated AEC2 to determine the specific protective effects of KGF. We speculate that other growth factors may also be protective against oxygen-induced DNA fragmentation in cultured cells; for example, basic fibroblast growth factor has been shown to ameliorate radiation-induced apoptosis in the pulmonary epithelium (10). Our observation that Matrigel, which contains many growth factors including epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, and basic fibroblast growth factor, can partially inhibit oxygen-induced DNA strand breaks in AEC2 adds support to the speculation that growth factors may be key to the prevention of apoptosis.
A protective role for KGF against a variety of environmental insults is already well documented in many systems. Our data now suggest a possible additional therapeutic role for KGF in limiting oxygen-induced DNA damage and subsequent apoptosis in the alveolar epithelium during lung repair.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-44060 and HL-44977 to D. Warburton and HL-54850 to K. Weinberg.
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FOOTNOTES |
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Address for reprint requests: D. Warburton, Childrens Hospital Los Angeles Research Institute, 4650 Sunset Blvd. (MS 35), Los Angeles, CA 90027.
Received 8 September 1997; accepted in final form 29 January 1998.
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