1 Department of Surgery and Cell and Developmental Biology Program and 2 Department of Immunology/Bone Marrow Transplant, Childrens Hospital Los Angeles Research Institute, University of Southern California School of Medicine, Los Angeles, California 90027
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ABSTRACT |
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Cyclin D1 antisense (D1AS)-transfected lung epithelial cell lines were serum deprived and then analyzed for three hallmarks of apoptosis: appearance of single-strand DNA breaks, alteration of apoptosis-related protein expression, and induction of chromatin condensation. Single-strand DNA breaks appeared at significant levels 24 h after serum deprivation, whereas induction of chromatin condensation was observed after 72 h. The antioxidants dimethyl sulfoxide, ascorbate, and glutathione, as well as insulin-like growth factor-I, inhibited induction of DNA damage in this assay. Additionally, proliferating cell nuclear antigen expression is completely suppressed in the D1AS cells, indicating a mechanism to explain the reduced capacity for DNA repair. Increased expression of cyclin D1, which is a common lesion in lung cancer, may thus prevent induction of apoptosis in an oxidizing and growth factor-poor environment. Reducing cyclin D1 expression in lung cancer cells by expression of D1AS RNA disrupted these protective pathways.
antisense
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INTRODUCTION |
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AMPLIFIED EXPRESSION of cyclin D1 is a common molecular cell cycle control lesion in many epithelial cell cancers (reviewed in Refs. 6, 43). For example, levels of cyclin D1 expression are 4- to 100-fold higher in many non-small cell lung carcinomas than in normal bronchoepithelial cells (40). Manipulation of cyclin D1 expression reveals why this overexpression confers a growth advantage in tumor cells. Ectopically overexpressed cyclin D1 can accelerate the rate of the cell cycle of primary rat fibroblasts (38) and, in the same cells, cooperate with activated ras to induce transformation (21). Administration of cyclin D1 antisense (D1AS) RNA in a variety of tumor cell types results in the retardation of cell growth in culture and loss of tumorigenicity in vivo (2, 25, 41, 44, 56). Driscoll et al. (11) recently showed that in addition to causing a marked decrease in the growth rate of lung cancer epithelial cell lines A549 and NCI-H441, D1AS expression caused rapid death on the withdrawal of serum from culture medium, a response not observed in the parental lung cancer cell lines. In the present study, experiments were designed to determine whether this cell death could be attributed to apoptosis.
In adults, both proliferation and apoptosis are necessary cellular events that balance the requirement for growth, tissue replacement, and maintenance of stem cell populations with the elimination and replacement of aged or damaged cells. Apoptosis is the result of a tightly controlled series of steps that result in "active" cell death due to specific endonuclease cleavage of DNA (reviewed in Refs. 47, 52). This pathway is now recognized as a valid target for oncogenic events because its inhibition seems to be as effective in promoting uncontrolled cell growth as is the enhancement of proliferation pathways. Specific proteins that act in the apoptosis pathway include the cell cycle checkpoint protein p53 and a large family of apoptosis agonists or antagonists, such as Bax and Bcl-2, which act as mediators between cell surface signaling and the nuclear events that precede cell death.
Serum deprivation is a potent inducer of apoptosis in culture for many
different cell types (3, 50). Minimal factors in fetal bovine serum
required for cellular viability in culture include epidermal growth
factor (EGF) and insulin-like growth factor-I (IGF-I). IGF-I can
suppress apoptosis-related interleukin-1-converting enzyme proteinase activity and induce expression of the
apoptosis inhibitor Bcl-xl,
resulting in inhibition of serum deprivation-induced apoptosis in PC12
cells (23, 36). Keratinocyte growth factor (KGF) has both
growth-promoting and apoptosis-inhibiting effects in lung epithelial
cells; it can inhibit apoptosis in primary cultures of alveolar type II
epithelial cells isolated from hyperoxic rats (5) and can be used
prophylactically in vivo to minimize damage caused by treatment with
bleomycin (9).
Although specific cytokines protect certain cell types from the induction of apoptosis, other experiments have shown that serum deprivation-induced apoptosis may be due not to a loss of growth factors in the medium but to the elevated production of intracellular reactive oxygen intermediates (ROIs) coupled with decreased levels of intracellular and extracellular antioxidants. This effect was reversed by the administration of antioxidants to serumless medium (3, 54). A growing body of evidence supports the hypothesis that the intracellular redox state is a critical determinant for survival of environmental stress (37, 45). The apoptosis antagonist Bcl-2 may function by mediating an antioxidant pathway (22; reviewed in Ref. 48). Bcl-2 expression causes a shift in cellular oxidation-reduction potential (15), perhaps via its ability to induce the redistribution of glutathione to the nucleus (49).
An accumulation of evidence suggests that apoptosis may actually be an aborted attempt at proliferation, which fails due to a deficit in the essential factors, environmental and/or intracellular, required for proliferation (32). One very well characterized cell cycle-related protein, p53, plays a central role in the induction of cell death in the normal processes of tissue differentiation and in response to DNA damage (29, 31). Cyclin D1 and its catalytic partners cyclin-dependent kinase-4 and -6 are involved in the regulation of p53 growth-suppressing activity (8, 26), although what role they play in modifying the apoptosis-inducing activity of p53 is less clear. However, altered cyclin D1 expression could easily underlie a mechanism of p53-dependent apoptosis in antisense-transfected A549 cells, which express wild-type p53.
The expression and activity of proliferating cell nuclear antigen (PCNA), another cell cycle and apoptosis pathway component, are tightly coordinated with those of cyclin D1. Quiescent cells express little or no PCNA, so analysis of this marker is an excellent prognosticator of the proliferative potential of any cell. In addition, PCNA plays a major role in DNA damage repair.
By analysis of several markers of apoptosis, including the induction of DNA strand breaks and chromatin condensation, as well as alteration of the expression of the apoptosis pathway proteins p53, Bax, and Bcl-2, we determined that expression of D1AS in lung cancer cells predisposes them to apoptosis induced by serum deprivation. We further observed that expression of PCNA is negligible in the D1AS cells in serum-supplemented medium and that removal of serum stimulates a very weak induction of PCNA expression. Apoptosis in this system can be blocked by the addition of antioxidants and IGF-I to the serumless medium. We conclude that cyclin D1 not only is a central component of the cell cycle machinery but also controls a pathway leading to apoptosis.
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METHODS |
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Cell culture. The lung carcinoma cell line A549 was obtained from the American Type Culture Collection (Manassas, VA). D1AS-transfected clone A549-5 and the empty vector A549-1 control cell line were created as previously described (11). Newly created cell lines A549-2 (empty vector transfected) and A549-43 and A549-46 (both D1AS transfected) were tested for cyclin D1 expression and susceptibility to death by serum deprivation before use in any further experiments. The cells were maintained in RPMI 1640 medium supplemented with antibiotics (10,000 U/100 ml of penicillin, 10,000 µg/100 ml of streptomycin, and 4 mg/100 ml of gentamicin; GIBCO BRL, Grand Island, NY), 10% fetal bovine serum (Sigma), and, in the case of the A549-V control cells and D1AS clones, 500 µg/ml of geneticin (G418) at 37°C in a 5% CO2 atmosphere. For serum deprivation, the cells were plated to give a density of 2 × 105/cm2 by the following day when the medium was removed and the cells were washed three times with PBS. The cells were then incubated in DMEM supplemented as above with the exception of serum. The cells were cultured under these conditions for the times indicated for each experiment. For analysis of terminal deoxytransferase (TdT) dUTP nick end labeling (TUNEL) inhibition by the addition of antioxidants and individual serum components to serumless medium, the reagents were added at the time of starvation at the following concentrations: 1% DMSO, 1 µM ascorbate, 1 mM GSH, 4% albumin, 20 ng/ml of IGF-1, 50 µg/ml of EGF, and 20 ng/ml of KGF.
TUNEL assay. The cells in culture for
in situ labeling or in suspension for fluorescence-activated cell
sorter (FACS) analysis were washed with PBS, then fixed on ice with 1%
paraformaldehyde in PBS for 15 min. The cells were washed with PBS and
incubated in 70% ethanol at 20°C for at least 24 h before
TUNEL analysis was performed according to the Apo-Direct kit
manufacturer's instructions (PharMingen, San Diego, CA). Briefly, the
fixed cells were washed, then incubated with TdT enzyme and substrate
(FITC-dUTP) for 1 h at 37°C. After being washed, the cells were
counterstained with a propidium iodide-RNase solution.
FACS analysis. Samples were analyzed with a Becton Dickinson FACScan equipped with a 488-nm argon laser as a light source and CellQuest software. Parameters for TUNEL were set with positive and negative control cells supplied with the Apo-Direct kit. For acquisition, a standard dual-parameter DNA doublet discrimination gating display template was created with the DNA area signal on the y-axis and the DNA width signal on the x-axis. For these experiments, 10,000 events were acquired, and the nonclumped cells were gated for analysis on a second dual-parameter display where DNA (linear red fluorescence) on the x-axis and FITC-dUTP (log green fluorescence) on the y-axis were used to analyze the cell cycle profile of the entire sample and to calculate the percentage of TUNEL-positive cells.
Visualization of chromatin condensation. The cells were plated, treated, fixed, and labeled in situ on LabTek Permanox chamber slides (Nalge Nunc, Naperville, IL), then viewed and photographed with a Nikon Diaphot-TMD inverted fluorescent microscope fitted with an FITC filter cube.
Gel electrophoresis and immunoblotting. SDS-PAGE and Western blotting were performed as previously described (11). Antibodies to cyclin D1 and PCNA were from Santa Cruz Biotechnology, whereas those for Bax, Bcl-2, and p53 were from Oncogene Sciences. Immunoreactive proteins were detected with the enhanced chemiluminescence system according to manufacturer's instructions (Amersham).
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RESULTS |
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In situ propidium iodide staining and TUNEL of
D1AS-transfected cells after 72 h of serum deprivation.
Driscoll et al. (11) showed that serum deprivation of D1AS-transfected
cells resulted in rapid cell death. The status of chromatin in these
cells was assessed as follows. A549 cells transfected with either empty vector or cyclin D1 expression plasmids were fixed in situ at 72 h
after removal of the serum. The cells were subjected to TUNEL followed
by propidiun iodide counterstaining (Fig.
1). Fluorescence microscopy revealed few
significant visual changes in the serum-deprived empty
vector-transfected cells compared with cells grown in medium plus
serum. Although many of the D1AS-transfected cells had lifted off the
tissue culture dish by the 72-h time point, a significant proportion
(~20%) of the remaining attached cells exhibited nuclear blebbing,
indicating chromatin condensation characteristic of apoptosis. The
remaining attached cells contained intact nuclei and may have
represented the untransfected portion of the population. This pattern
reflected the results of Driscoll et al. (11), which showed that when
the medium was changed after the 72-h period of serum deprivation to
medium plus serum, the majority of the cells still attached exhibited a
level of expression of cyclin D1 comparable to that observed in the
empty vector-transfected cells, indicating no D1AS expression.
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DNA strand breaks are induced in D1AS-transfected
cells after serum deprivation. Cells, including the
original A549 cell line, two empty vector-transfected A549 cell lines
(A549-V1 and A549-V2), and three D1AS-transfected cell lines (A549-5,
A549-43, and A549-46), were assayed for three criteria as previously
described (11): cyclin D1 expression (Fig.
2A),
growth rate, and susceptibility to death by serum deprivation. Both
wild-type and empty vector-transfected cell lines exhibited normal
levels of cyclin D1 expression, normal growth rate, and no
susceptibility to death by serum deprivation. In contrast, all three
D1AS-transfected cell lines exhibited a marked reduction in cyclin D1
expression and growth rate, and all were vulnerable to the effects of
serum deprivation. To characterize this effect, all cell lines were
subjected to serum deprivation for 48 h. Before and after removal of
the serum, all cell lines were assayed for DNA strand breaks by TUNEL
with FITC-dUTP. The TUNEL assay was used here as a method for
determining the extent of DNA strand breakage, an early marker for
irreversible DNA damage, one of the hallmarks of apoptosis. Although
the D1AS-transfected cells exhibited a consistent slight elevation,
basal end labeling in medium plus serum was essentially at control
levels for all cell lines (data not shown). However, 48 h after removal
of the serum, cells expressing D1AS RNA exhibited significant levels of
end labeling, indicating substantial DNA damage (Fig.
2B). Neither the
original A549 nor the empty vector-transfected cells exhibited
significant increases in end labeling after serum deprivation. The low
level of DNA strand breakage in the empty vector-transfected cells
indicated that damage was not due to a nonspecific toxic effect of G418
because serum deprivation of both empty vector- and D1AS-transfected
cell lines was performed in the presence of identical concentrations of
G418 (500 µg/ml). Induction of DNA strand breakage and other markers
for apoptosis in the serum-deprived D1AS-transfected cells could,
therefore, be ascribed to decreased expression of cyclin D1.
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In our original serum deprivation experiments, the incidence of cell death was measured after 72 h of serum deprivation (11). For the present set of experiments, we were able to show a mean of 32% (±3%) of cells positive for DNA strand breaks in the D1AS-transfected cell lines at 48 h. However, we wished to determine how soon after removal of the serum significant levels of DNA damage could be observed. Therefore, the original A549 empty vector-transfected and D1AS-transfected cell lines were assayed for DNA strand breaks by TUNEL at intervals over a 48-h period (Fig. 2B). In the D1AS-transfected cells, we observed significant DNA damage by 24 h (P < 0.05), which increased markedly by 48 h (P < 0.01) compared with that in control cells.
Serum deprivation of D1AS-transfected cells alters
expression of proteins associated with induction of
apoptosis. Empty vector-transfected A549-V1 and
D1AS-transfected A549-43 cells were plated at 50 and 75% confluence,
respectively. Different starting densities were required to achieve
equal densities at the time of treatment for these two cell lines, with
widely varied growth rates. The next day, both cell lines had reached
~80% confluence and were subjected to 48 h of serum deprivation.
During that time period, an aliquot of each cell line was collected by
trypsinization, washed, and stored frozen at 0, 12, 24, and 48 h. The
cells were lysed, and equal quantities of protein from each sample were
analyzed by SDS-PAGE followed by Western blotting (Fig.
3B).
Although probing with an anti-actin antibody showed equal protein
loading, expression levels of other proteins changed significantly
during the period of serum deprivation.
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The levels of cyclin D1 expression were assessed to reconfirm the significant decrease in cyclin D1 protein expression due to the effect of D1AS expression. Interestingly, changes in cyclin D1 expression were noted during the serum deprivation period. Cyclin D1 levels in the A549-V cells rose at 12 h and remained elevated, a phenomenon that has been reported elsewhere (17, 35). However, in the D1AS-transfected cells, cyclin D1 expression peaked at 12 h, then declined to the pretreatment level.
The p53 expressed in A549 cells is wild type and, as such, is capable of responding to environmental stress by increased expression and activity to achieve checkpoint control of DNA damage. Although this response is observed in normal cells, tumor cells often develop mechanisms for overriding cell cycle checkpoint control by p53. Interestingly, the level of p53 expression in the A549-V cells increased by an insignificant amount during the serum deprivation period. However, in the D1AS-transfected cells, the level of p53 increased significantly by 12 h and peaked at 24 h at a level 10-fold higher than that observed at the 0-h time point. The initial increase occurred well before any significant TUNEL labeling was detected in the A549-43 cells.
Further analysis showed changes in the level of expression of two proteins intimately involved in promotion of and protection from apoptosis. Bax expression in the control cells was extremely low and was moderately induced during serum deprivation. Bax protein levels in the D1AS-transfected cells were already significantly higher than the control level at time 0 and then increased further, following the same time course as p53, which controls Bax expression (30). Expression of the apoptosis-inhibiting protein Bcl-2 was at equivalent levels for each cell line at time 0. This level was maintained in the control cells but dropped precipitously in the D1AS-transfected cells beginning at 12 h.
Expression of PCNA is altered in D1AS-transfected
cells. Analysis of PCNA expression was performed to
assess the proliferative and repair capabilities of the empty vector-
and D1AS-transfected cells under both serum-fed and serum-deprived
conditions (Fig. 4). Western blotting
showed that the empty vector-transfected cells expressed very high
levels of this protein, which was maintained throughout the starvation
period. Conversely, the D1AS-transfected cells expressed little or no
PCNA under serum-fed conditions. During the 48-h course of serum
deprivation, a weak induction of PCNA expression was observed. However,
this level was never more than one-third of the level observed in the
empty vector-transfected cells under any conditions. It is possible
that this response was contributed to by the minority untransfected
portion of the D1AS-transfected cells. Alternatively, the very weak
induction of cyclin D1 itself under these conditions, which coordinates PCNA expression, may account for both the poor induction of PCNA and
the loss of repair capability. The vigorous expression of PCNA under all conditions in the empty vector-transfected cells may
indicate why these cells, like the parental tumor cell line, are able
to withstand serum deprivation conditions for extended periods without incurring any substantial DNA damage.
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Selected serum components can block induction of DNA
damage due to serum deprivation in D1AS-transfected
cells. To determine whether adding back serum
components could prevent the induction of DNA damage in the
D1AS-transfected cells, KGF, IGF-I, and EGF were added to the medium at
the time the serum was removed. The concentrations used were equivalent
to those present in the medium supplemented with 10% fetal bovine
serum. After 24 h of serum deprivation, the cells were harvested and
analyzed with TUNEL (Fig. 5).
The addition of these components had no effect on the basal level of
TUNEL observed in either cell line in medium plus serum or in the
control cell line in medium without serum (data not shown). However,
addition of IGF-I resulted in a significant block in serum
deprivation-induced DNA damage in the D1AS-transfected cells. As for
other cytokines, neither EGF nor KGF successfully inhibited induction
of DNA damage in this system.
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Antioxidants can block induction of DNA damage due to
serum deprivation in D1AS-transfected cells. Recent
indirect evidence has shown that the DNA damage caused by serum
deprivation in a variety of cell types may be due not just to growth
factor deprivation but also to the effects of increased intracellular
ROIs (45). Supplementing the serumless medium with antioxidants, which
are either reducing agents or hydroxyl scavengers, may inhibit
ROI-induced damage. We therefore added the antioxidants ascorbate,
DMSO, and the reduced form of glutathione, GSH, to serum-free medium at the beginning of a 24-h period of serum deprivation, then analyzed for
DNA strand breaks by TUNEL (Fig. 6). None
of these reagents had any effect on the basal TUNEL-positive
percentages of the empty vector control cells under serum-fed or
serumless conditions. They showed a slight, although not significant,
effect on the D1AS-transfected cells in medium plus serum (data not
shown). However, all three antioxidants effectively blocked DNA damage induced by serum deprivation of D1AS-transfected cells.
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DISCUSSION |
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Previous work by Driscoll et al. (11) showed that expression of D1AS RNA in two different lung cancer cell lines resulted in the retardation of cell growth, alteration of expression of cell cycle-related proteins, particularly strong induction of p21, and decreased phosphorylation coupled with increased instability of the pRb tumor suppresser protein (11). In addition, they showed that a high percentage of cell death occurred in the D1AS-transfected cell lines on withdrawal of the serum for 72 h. A subsequent experiment showed that only those cells expressing high levels of cyclin D1 could survive this treatment. Similar patterns of decreased cyclin D1 expression leading to apoptosis have recently been described (24, 33, 55).
We therefore analyzed for the appearance of single-strand DNA breaks as measured with TUNEL for the condensation of chromatin and the alteration of apoptosis-related protein expression. All three hallmarks of apoptosis were observed in the serum-deprived D1AS-transfected cells and led us to conclude that specific induction of an apoptotic pathway had occurred.
The amplification of expression of cyclin D1 is so common in cancers of epithelial cell origin that its role in promoting tumor cell growth was recognized quickly and has been well characterized. In this study, we present evidence that cyclin D1 is not only a tumor cell growth promoter but may also function as a survival factor for tumor cells faced with adverse environmental conditions. Three observations led to this conclusion.
First, we evaluated the expression of PCNA. This protein is a major component of the DNA repair pathway and its activity and expression are intimately linked with those of cyclin D1 (53). In the present study, PCNA levels are high in control tumor cells and remain so throughout the serum deprivation period. In contrast, PCNA is expressed at extremely low levels in the D1AS-transfected cells and is induced, on serum starvation, at levels significantly below those observed in control cells. During cell cycle progression, cyclin D1 expression precedes that of PCNA, and because the PCNA promoter sequence contains recognition sequences for E2F-1 (27), expression of the pRb kinase activity of cyclin D1 is required for the release of E2F-1 and induction of PCNA expression. Once expressed, PCNA facilitates the function of DNA repair enzymes (1, 4, 20). In normal cells, DNA damage that induces p53 expression results in increased expression of p21, which interacts directly with PCNA to selectively inhibit its proliferation-stimulating function. In this situation, the DNA repair function of PCNA remains intact, and thus p53 exerts its checkpoint control function (7, 28). However, overexpressed p21 is, by itself, a potent mediator of serum deprivation-induced apoptosis (13), and excess production of p21 coupled with a deficiency in PCNA repair function can cause persistent DNA damage because excess p21 can block repair until a sufficient quantity of PCNA is expressed (34). In this scenario, limited quantities of cyclin D1, causing insufficient expression of PCNA coupled with excess expression of p21 disallows repair of damaged DNA. We found exactly this situation in the D1AS-transfected cells, which express much higher than control levels of p21 (11) and much lower than control levels of PCNA. This combination directly affects the ability of the D1AS-transfected cells to survive serum deprivation, whereas the opposite combination of low p21 and high cyclin D1 and PCNA expression is probably a key to survival for the parental tumor cell lines under the same conditions.
Our second observation was that stimulation of a specific signal transduction pathway by IGF-I could block DNA damage induced by removal of serum from the D1AS-transfected cells. This result is similar to that observed in serum-deprived PC12 cells where IGF-I administration inhibits apoptosis, apparently by suppressing interleukin-1-converting enzyme proteinase activity and inducing expression of the apoptosis-inhibiting protein Bcl-xl (23, 36). Additionally, in 3T3 cells, IGF-I can inhibit etoposide-induced apoptosis (42). The relationship between IGF-I and cyclin D1 is quite direct because IGF-I can induce cyclin D1 expression in breast epithelial and a variety of other cells (12) and has a direct stimulatory effect on the cyclin D1 promoter (18). Whether this stimulation can overcome the effect of D1AS RNA expression in the D1AS-transfected cells used in our study is currently under investigation. Control cells used in this assay showed no such requirement for IGF-I supplementation, and the expression of cyclin D1 in those cells under normal conditions is at a high level and increases on serum deprivation. In several cell types, ectopic overexpression of cyclin D1 induces apoptosis (19, 46). However, in the A549 cells, this increased expression is coupled with survival, indicating that the empty vector-transfected A549 cells may not respond to the conflicting signals of a growth factor-poor environment coupled with increased expression of cell cycle progression factors by dying but by becoming quiescent. Indeed, we have maintained untransfected A549 cells under serum-deprived conditions for 11 days and observed no significant reduction in cell number.
A third observation supporting a role for cyclin D1 in an apoptosis pathway was that reduction of cyclin D1 expression in lung tumor cells appeared to inhibit DNA repair pathways that respond to the damaging effects of ROIs produced under serum-poor conditions. It is now recognized that in addition to alteration of expression of oncogenes and tumor suppressor proteins, aggressive and drug-resistant tumor cells also display changes in the expression of cellular proteins involved in detoxification, including antioxidants (10, 14). Although the empty vector-transfected A549 cells used in these experiments resisted the oxidizing effects of serum deprivation, the D1AS-transfected cells required the addition of antioxidants to the serumless medium for survival. The role of Bcl-2 is no doubt critical here because, with a direct effect on the redox state of the cell and an ability to direct glutathione to the nucleus, it provides needed protection from intracellular ROI-mediated damage (15, 49). Reduction in cyclin D1 expression that then causes, either directly or indirectly, a reduction in the expression of Bcl-2 may play the key role in the D1AS-transfected cell response to serum deprivation. Interestingly, cyclin D1 and Bcl-2 have implied functional homology by hydropathic sequence analysis (39), and it may be that they perform overlapping functions in the A549 response to serum deprivation conditions. In support of this concept, ectopic expression of both Bcl-2 and cyclin D1 can enhance survival and delay the onset of apoptosis of 32D clone 3 cells exposed to ionizing radiation (16). Decreased expression of both, as observed in the D1AS-transfected cells, is apparently fatal.
Our experimental results on lung cancer epithelial cells illuminate one theory on the nature of the cancer cell versus normal cell response to adverse environmental conditions. It has been suggested that in terminally differentiated cells overexpression of growth-promoting proteins coupled with an inability to replicate DNA are the conflicting signals that precipitate apoptosis. Conversely, those cells that retain the ability to self-renew via the intact progression of the cell cycle under any condition will escape apoptosis. Amplified expression of cyclin D1 obviously confers a growth advantage, and recent evidence has shown that this change in phenotype is an early event in the development of cancer (51). The A549 cells used in this experiment express wild-type versions of pRb, p53, and p21 while carrying a deletion of p16 and amplified expression of cyclin D1. As such, they are a good model system for studying the interactions of pathways involved in this early oncogenic event. Our data strongly suggest that abrogation of cyclin D1 overexpression in lung epithelial tumor cells not only retards cell growth but predisposes them to serum deprivation-induced apoptosis.
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ACKNOWLEDGEMENTS |
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This work was supported by an American Lung Association Young Investigator Award (to B. Driscoll) and National Heart, Lung, and Blood Institute Grants HL-44060, HL-60231 (both to D. Warburton) and HL-54850 (to K. Weinberg).
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FOOTNOTES |
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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: D. Warburton, Dept. of Surgery, Childrens Hospital Los Angeles Research Institute, Smith Research Tower, MS 35, 4650 Sunset Blvd., Los Angeles, CA 90027 (E-mail: dwarburton{at}chla.usc.edu).
Received 18 August 1998; accepted in final form 12 January 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aboussekhra, A.,
M. Biggerstaff,
M. K. Shivji,
J. A. Vilpo,
V. Moncollin,
V. N. Podust,
M. Protic,
U. Hubscher,
J. M. Egly,
and
R. D. Wood.
Mammalian DNA nucleotide excision repair reconstituted with purified protein components.
Cell
80:
859-868,
1995[Medline].
2.
Arber, N.,
Y. Doki,
E. K. Han,
A. Sgambato,
P. Zhou,
N. H. Kim,
T. Doherty,
M. G. Klein,
P. R. Holt,
and
I. B. Weinstein.
Antisense to cyclin D1 inhibits the growth and tumorigenicity of human colon cancer cells.
Cancer Res.
57:
1569-1574,
1997[Abstract].
3.
Atabay, C.,
C. M. Cagnoli,
E. Kharlamov,
M. D. Ikanomovic,
and
H. Manev.
Removal of serum from primary cultures of cerebellar granule neurons induces oxidative stress and DNA fragmentation: protection with antioxidants and glutamate receptor antagonists.
J. Neurosci. Res.
43:
465-475,
1996[Medline].
4.
Ayyagari, R.,
K. J. Impellezzeri,
B. L. Yoder,
S. L. Gary,
and
P. M. Burgers.
A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and repair.
Mol. Cell. Biol.
15:
4420-4429,
1995[Abstract].
5.
Buckley, S.,
L. Barskey,
B. Driscoll,
K. D. Anderson,
K. Weinberg,
and
D. Warburton.
Apoptosis and DNA damage in type 2 alveolar epithelial cells cultured from hyperoxic rats.
Am. J. Physiol.
274 (Lung Cell. Mol. Physiol. 18):
L714-L720,
1998
6.
Cordon-Cardo, C.
Mutations of cell cycle regulators. Biological and clinical implications for human neoplasia.
Am. J. Pathol.
147:
545-560,
1995[Abstract].
7.
Cox, L. S.,
and
D. P. Lane.
Tumor suppressors, kinases and clamps: how p53 regulates the cell cycle in response to DNA damage.
Bioessays
17:
501-508,
1994.
8.
Del Sal, G.,
M. Murphy,
E. M. Ruaro,
D. Lazarevic,
A. J. Levine,
and
C. Schneider.
Cyclin D1 and p21/waf1 are both involved in p53 growth suppression.
Oncogene
12:
177-185,
1996[Medline].
9.
Deterding, R. R.,
T. Yanos,
A. M. Havill,
C. R. Jacoby,
J. M. Shannon,
W. S. Simonet,
and
R. J. Mason.
Keratinocyte growth factor prevents bleomycin lung injury in rats (Abstract).
Am. J. Respir. Crit. Care Med.
151:
A198,
1995.
10.
Dive, C.
Avoidance of apoptosis as a mechanism of drug resistance.
J. Intern. Med. Suppl.
740:
139-145,
1997[Medline].
11.
Driscoll, B.,
L. Wu,
S. Buckley,
F. L. Hall,
K. D. Anderson,
and
D. Warburton.
Cyclin D1 antisense RNA destabilizes pRb and retards lung cancer cell growth.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L941-L949,
1997
12.
Dufourny, B.,
J. Alblas,
H. A. van Teefelen,
F. M. van Schaik,
B. van der Burg,
P. H. Steenbergh,
and
J. S. Sussenbach.
Mitogenic signaling of insulin-like growth factor I in MCF-7 human breast cancer cells requires phosphatidylinositol 3-kinase and is independent of mitogen-activated protein kinase.
J. Biol. Chem.
272:
31163-31171,
1997
13.
Duutaroy, A.,
J.-F. Qian,
J. S. Smith,
and
E. Wang.
Up-regulated p21CIP1 expression is part of the regulation quantitatively controlling serum deprivation-induced apoptosis.
J. Cell. Biochem.
64:
434-446,
1997[Medline].
14.
El-Deiry, W. S.
Role of oncogenes in resistance and killing by cancer therapeutic agents.
Curr. Opin. Oncol.
9:
79-87,
1997[Medline].
15.
Ellerby, L. M.,
H. M. Ellerby,
S. M. Park,
A. L. Holleran,
A. N. Murphy,
G. Fiskum,
D. J. Kane,
M. P. Testa,
C. Kayalar,
and
D. E. Bredesen.
Shift of the cellular oxidation-reduction potential in neural cells expressing Bcl-2.
J. Neurochem.
67:
1259-1267,
1996[Medline].
16.
Epperly, M.,
L. Berry,
A. Halloran,
and
J. S. Greenberger.
Inhibition of G1-phase arrest induced by ionizing radiation in hematopoietic cells by overexpression of genes involved in the G1/S-phase transition.
Radiat. Res.
143:
245-254,
1995[Medline].
17.
Freeman, R. S.,
S. Estus,
and
E. M. Johnson, Jr.
Analysis of cell cycle-related gene expression in postmitotic neurons: selective induction of cyclin D1 during programmed cell death.
Neuron
12:
343-355,
1994[Medline].
18.
Furlanetto, R. W.,
S. E Harwell,
and
K. K. Frick.
Insulin-like growth factor-I induces cyclin-D1 expression in MG63 human osteosarcoma cells in vitro.
Mol. Endocrinol.
8:
510-517,
1994[Abstract].
19.
Han, E. K.,
M. Begemann,
A. Sgambato,
J. W. Soh,
Y. Doki,
W. Q. Xing,
W. Liu,
and
I. B. Weinstein.
Increased expression of cyclin D1 in a murine mammary epithelial cell line induces p27kip1, inhibits growth, and enhances apoptosis.
Cell Growth Differ.
7:
699-710,
1996[Abstract].
20.
Henderson, D. S.,
and
D. M. Glover.
Chromosome fragmentation resulting from an inability to repair transposase-induced DNA double-strand breaks in PCNA mutants of Drosophila.
Mutagenesis
13:
57-60,
1998[Abstract].
21.
Hinds, P. W.,
S. F. Dowdy,
E. N. Eaton,
A. Arnold,
and
R. A. Weinberg.
Function of a human cyclin gene as an oncogene.
Proc. Natl. Acad. Sci. USA
91:
709-713,
1994[Abstract].
22.
Hockenbery, D. M.,
Z. N. Oltvai,
X. M. Yin,
C. L. Milliman,
and
S. J. Korsmayer.
Bcl-2 functions in an antioxidant pathway to prevent apoptosis.
Cell
75:
241-251,
1993[Medline].
23.
Jung, Y.-K.,
M. Miura,
and
J. Yuan.
Suppression of interleukin-1-converting enzyme-mediated cell death by insulin-like growth factor.
J. Biol. Chem.
271:
5112-5117,
1996
24.
Kaneko, Y.,
and
A. Tsukamoto.
Apoptosis and nuclear levels of p53 protein and proliferating cell nuclear antigen in human hepatoma cells cultured with tumor promoters.
Cancer Lett.
91:
11-17,
1995[Medline].
25.
Kornmann, M.,
N. Arber,
and
M. Korc.
Inhibition of basal and mitogen-stimulated pancreatic cancer cell growth by cyclin D1 antisense is associated with loss of tumorigenicity and potentiation of cytotoxicity to cisplatinum.
J. Clin. Invest.
101:
344-352,
1998
26.
Latham, K. M.,
S. W. Eastman,
A. Wong,
and
P. W. Hinds.
Inhibition of p53-mediated growth arrest by overexpression of cyclin-dependent kinases.
Mol. Cell. Biol.
16:
4445-4455,
1996[Abstract].
27.
Lee, H. H.,
W. H. Chiang,
S. H. Chiang,
Y. C. Liu,
J. Hwang,
and
S. Y. Ng.
Regulation of cyclin D1, DNA topoisomerase I, and proliferating cell nuclear antigen promoters during the cell cycle.
Gene Expr.
4:
95-109,
1995[Medline].
28.
Li, R.,
G. J. Hannon,
D. Beach,
and
B. Stillman.
Subcellular distribution of p21 and PCNA in normal and repair-deficient cells following DNA damage.
Curr. Biol.
6:
189-199,
1996[Medline].
29.
Lowe, S. W.,
T. Jacks,
D. E. Housman,
and
H. E. Ruley.
Abrogation of oncogene-associated apoptosis allows transformation of p53-deficient cells.
Proc. Natl. Acad. Sci. USA
91:
2026-2030,
1994[Abstract].
30.
Merchant, A. K.,
T. L. Loney,
and
J. Maybaum.
Expression of wild-type p53 stimulates an increase in both Bax and Bcl-xL protein content in HT29 cells.
Oncogene
13:
2631-2637,
1996[Medline].
31.
Merritt, A. J.,
C. S. Potten,
C. J. Kemp,
J. A. Hickman,
A. Balmain,
and
D. P. Lane.
The role of p53 in spontaneous and radiation-induced apoptosis in the gastrointestinal tract of normal and p53-deficient mice.
Cancer Res.
54:
614-617,
1994[Abstract].
32.
Nuydens, R.,
M. de Jong,
G. Van Den Kieboom,
C. Heers,
G. Dispersyn,
F. Cornelissen,
R. Nuyens,
M. Borgers,
and
H. Geerts.
Okadaic acid-induced apoptosis in neuronal cells: evidence for an abortive mitotic attempt.
J. Neurochem.
70:
1124-1133,
1998[Medline].
33.
Otori, K.,
Y. Yano,
N. Takada,
C. C. Lee,
S. Hayashi,
S. Otani,
and
S. Fukushima.
Reversibility and apoptosis in rat urinary bladder papillomatosis induced by uracil.
Carcinogenesis
18:
1485-1489,
1997[Abstract].
34.
Pan, Z. Q.,
J. T. Reardon,
L. Li,
H. Flores-Rozas,
R. Legerski,
A. Sancar,
and
J. Hurwitz.
Inhibition of nucleotide excision repair by the cyclin-dependent kinase inhibitor p21.
J. Biol. Chem.
270:
22008-22016,
1995
35.
Pandey, S.,
and
E. Wang.
Cells en route to apoptosis are characterized by the upregulation of c-fos, c-myc, c-jun, cdc2, and RB phosphorylation, resembling events of early cell cycle traverse.
J. Cell. Biochem.
58:
135-150,
1995[Medline].
36.
Parrizas, M.,
A. R. Saltiel,
and
D. LeRoith.
Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 3'-kinase and mitogen-activated protein kinase pathways.
J. Biol. Chem.
272:
154-161,
1997
37.
Powis, G.,
M. Briehl,
and
J. Oblong.
Redox signaling and the control of cell growth and death.
Pharmacol. Ther.
68:
149-173,
1995[Medline].
38.
Quelle, D. E.,
R. A. Ashmun,
S. A. Shurtleff,
J.-Y. Kato,
D. Bar-Sagi,
M. F. Roussel,
and
C. J. Sherr.
Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts.
Genes Dev.
7:
1559-1571,
1993[Abstract].
39.
Radulescu, R. T.
The "LXCXE" hydropathic superfamily of ligands for retinoblastoma protein: a proposal.
Med. Hypotheses
44:
28-31,
1995[Medline].
40.
Schauer, I. E.,
S. Siriwrdana,
T. A. Langan,
and
R. A Scalfani.
Cyclin D1 overexpression vs. retinoblastoma inactivation: implications for growth control evasion in non-small cell and small cell lung cancer.
Proc. Natl. Acad. Sci. USA
91:
7827-7831,
1994[Abstract].
41.
Schrump, D. S.,
A. Chen,
and
U. Consoli.
Inhibition of lung cancer proliferation by antisense cyclin D1.
Cancer Gene Ther.
3:
131-135,
1996[Medline].
42.
Sell, C.,
R. Baserga,
and
R. Rubin.
Insulin-like growth factor I (IGF-I) and the IGF-I receptor prevent etoposide-induced apoptosis.
Cancer Res.
55:
303-306,
1995[Abstract].
43.
Sherr, C. J.
Cancer cell cycles.
Science
274:
1672-1677,
1996
44.
Skotzko, M.,
L. Wu,
W. F. Anderson,
E. M. Gordon,
and
F. H. Hall.
Retroviral vector-mediated gene transfer of antisense cyclin G1 (CYCG1) inhibits proliferation of human osteogenic sarcoma cells.
Cancer Res.
55:
5493-5498,
1995[Abstract].
45.
Slater, A. F.,
C. Stefan,
I. Nobel,
D. J. van den Dobblesteen,
and
S. Orrenius.
Signaling mechanisms and oxidative stress in apoptosis.
Toxicol. Lett.
82-83:
149-153,
1995.
46.
Sofer-Levi, Y.,
and
D. Resnitzky.
Apoptosis induced by ectopic expression of cyclin D1 but not cyclin E.
Oncogene
13:
2431-2437,
1996[Medline].
47.
Stewart, B. W.
Mechanisms of apoptosis: integration of genetic, biochemical, and cellular indicators.
J. Natl. Cancer Inst.
86:
1286-1296,
1994[Abstract].
48.
Strasser, A.,
D. C. Huang,
and
D. L. Vaux.
The role of the bcl-2/ced-9 gene family in cancer and general implications of defects in cell death control for tumorigenesis and resistance to chemotherapy.
Biochim. Biophys. Acta
1333:
F151-F178,
1997[Medline].
49.
Voehringer, D. W.,
D. J. McConkey,
T. J. McDonnell,
S. Brisbay,
and
R. E. Meyn.
Bcl-2 expression causes redistribution of glutathione to the nucleus.
Proc. Natl. Acad. Sci. USA
95:
2956-2960,
1998
50.
Wang, H.-G.,
J. A. Millan,
A. D. Cox,
C. J. Der,
U. R. Rapp,
T. Beck,
H. Zha,
and
J. C. Reed.
R-ras promotes apoptosis caused by growth factor deprivation via a bcl-2 suppressible mechanism.
J. Cell Biol.
129:
1103-1114,
1995[Abstract].
51.
Weinstein, I. B.
Relevance of cyclin D1 and other molecular markers to cancer chemoprevention.
J. Cell. Biochem.
25:
23-28,
1996.
52.
White, E.
Life, death, and the pursuit of apoptosis.
Genes Dev.
10:
1-15,
1996[Medline].
53.
Xiong, Y.,
H. Zhang,
and
D. Beach.
D type cyclins associate with multiple protein kinases and the DNA replication and repair factor PCNA.
Cell
71:
505-514,
1992[Medline].
54.
Xu, Y.,
Q. Nguyen,
D. C. Lo,
and
M. J. Czaja.
c-myc-Dependent hepatoma cell apoptosis results from oxidative stress and not a deficiency of growth factors.
J. Cell. Physiol.
170:
192-199,
1997[Medline].
55.
Yamauchi, Y.,
A. Tanaka,
K. Yamaguchi,
M. Kobayashi,
S. Shimamura,
and
F. Hanaoka.
Apoptosis was promoted at a non-permissive temperature in DNA replication-defective temperature-sensitive mutants of mouse FM3A cells.
Exp. Cell Res.
238:
317-323,
1998[Medline].
56.
Zhou, P.,
W. Jiang,
Y. Zhang,
S. M. Kahn,
L. Schieren,
R. M. Santella,
and
I. B. Weinstein.
Antisense to cyclin D1 inhibits growth and reverses the transformed phenotype of human esophageal cells.
Oncogene
11:
571-580,
1995[Medline].