Cyclin D1 antisense RNA destabilizes pRb and retards lung cancer cell growth

Barbara Driscoll, Lingtao Wu, Susan Buckley, Frederick L. Hall, Kathryn D. Anderson, and David Warburton

Department of Surgery and Cell and Developmental Biology Program, Childrens Hospital Los Angeles Research Institute, University of Southern California School of Medicine, Los Angeles, California 90027

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

To investigate the role of cyclin D1 in the regulation of lung cancer cell growth, we created five stably transfected cell lines carrying a cyclin D1 antisense construct. The transfected cells exhibited a marked decrease in the rate of cell growth, in contrast to the original lines (A549 and NCI-H441). The expression of several cell cycle-regulating proteins, including cyclin A, the cyclin-dependent kinases (cdk) 2 and cdk4, in addition to cyclin D1 itself, was markedly decreased. The expression of one cdk inhibitor, p21WAF1/CIP1, increased in the A549-derived cell lines. A specific target of cyclin D1 activity, the growth-suppressing product of the retinoblastoma gene, pRb, exhibited decreased expression and a decreased level of phosphorylation in the transfected cells. Decreased expression of pRb due to a significant increase in its turnover rate suggested that the stability of the protein may depend on phosphorylation by cyclin D1-dependent cdk activity. In addition to the impact on pRb stability, decreased expression of cyclin D1 induced susceptibility to cell death after withdrawal of exogenous growth factors in the antisense transfected cell lines, a response that was not observed in the original cancer cell lines. We conclude that abrogation of cyclin D1 overexpression in lung cancer cells disrupts several key pathways that are required for uncontrolled cell growth and induces those that lead to cell death after growth factor deprivation. Therefore, we speculate that use of antisense cyclin D1 expression in appropriate gene vectors could be a useful method for retarding lung cancer cell growth in accessible tumors such as those of the lung epithelium.

retinoblastoma protein; cell death

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

CELL CYCLE PROGRESSION is controlled by enzyme complexes comprised of cyclins, cyclin-dependent kinases (cdks), and cdk inhibitors. Entry into, and progression through, the G1 phase of the cell cycle is determined by the activity of complexes containing cyclins D1, D2, and D3, together with cdk4 and cdk6. The G1/S transition appears to be controlled by the actions of cyclins E and A and their catalytic partner cdk2. Progression through S phase, S/G2 transition, and progression through G2/M require the activity of cyclins A and B and cdk 1 and 2. In quiescent cells, high levels of cdk inhibitors p15INK4B, p16MTS1, p21WAF1/CIP1, and p27KIP1 prevent the formation of active complexes of cyclins and cdks by physically associating with cdks and inhibiting their catalytic activity, thereby preventing G1 phase progression and G1/S transition (reviewed in Refs. 9, 11, 12, and 25).

In normal cells, stimulation of entry into the cell cycle is induced by mitogens, several of which cause increased expression of the D-type cyclins (1, 2, 14, 24). Manipulation of the levels of expression of cyclin D family members in several different cell types has shown that these cyclins do, in fact, control progression through the G1 phase of the cell cycle (3, 14, 22, 27). Overexpression of exogenous cyclin D1 in normal cells results in acceleration of the cell cycle due to a decrease in length of the G1 phase (17, 20, 21). In cancerous cells exhibiting uncontrolled cell growth, cyclin D1 has been implicated as a protooncogene. Surveys of tumor cell lines have shown overexpression of cyclin D1 due to clonal rearrangement and/or amplification (5, 22). Although cyclin D1 is not intrinsically tumorigenic, it is able to cooperate with an activated ras oncogene and with the adenovirus E1A oncogene to transform primary rat fibroblasts (10, 14, 28).

The expression and activity of cyclin D1 are intimately involved with the expression and activity of the tumor suppressor retinoblastoma protein (pRb). The activity of pRb is controlled by both inactivating phosphorylations and physical association with growth-promoting factors of both cellular and viral origin. pRb must be phosphorylated for progression through G1 to occur, and cyclin D1, with its catalytic partner cdk4, can phosphorylate pRb in vitro (4, 7, 8, 13, 15). The transforming proteins of the oncogenic DNA tumor viruses, SV-40 and various types of adenoviruses and papilloma viruses, preferentially bind the underphosphorylated form of pRb, presumably for the purpose of sequestration, which allows cell cycle progression. Cyclin D1 physically associates with pRb in the same manner as these viral proteins, both in vitro and in vivo, through the binding of an LXCXE domain to the pocket region of pRb (6, 7, 13).

The introduction of constructs that code for sense and antisense versions of cell cycle control proteins into cells has been proposed as a basis for novel therapies that would modulate disease processes in which control of cell growth is required. Antisense cyclin D1 expression has been shown to be an effective inhibitor of growth in two cell types, osteosarcoma (25) and esophageal carcinoma (31). These studies were performed to assess the feasibility of the use of antisense expression vectors in a therapeutic manner, as well as to elucidate the role of cyclin D1 in certain types of tumor cells. In the current study, introduction of a cyclin D1 antisense construct into two human lung cancer cell lines, A549 and NCI-H441, resulted in decreased levels of endogenous cyclin D1 expression as well as in changes in the level of expression of several other key cell cycle-related proteins. In these cells, the rate of proliferation was markedly decreased, and the expression and phosphorylation of pRb, which before transfection appeared to be normal, was altered. This was shown to be due to a marked increase in the rate of turnover of the pRb protein, suggesting that basal phosphorylation of pRb by cyclin D1 may influence the stability of pRb. In addition to these effects, decreased expression of cyclin D1 in the transfected cells rendered them susceptible to the lethal effects of serum deprivation. These data support the conclusion that abrogation of cyclin D1 overexpression in lung epithelial tumor cells disrupts several key pathways that are required for uncontrolled cell growth and induces those that lead to cell death after growth factor deprivation.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell culture. The lung adenocarcinoma cell line NCI-H441 and lung carcinoma cell line A549 were obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in RPMI 1640 medium supplemented with antibiotics (GIBCO-BRL, Grand Island, NY) as follows: 10,000 U/100 ml penicillin, 10,000 µg/100 ml streptomycin, 4 mg/100 ml gentamicin, and 10% fetal bovine serum (FBS; Sigma) at 37°C in a 5% CO2 atmosphere.

Plasmids and transfection. Plasmids pcDNA3 and pcDNA3D1AS have been described elsewhere (25). For transfection, the manufacturer's instructions for lipofectamine (GIBCO-BRL) were followed. Briefly, A549 and NCI-H441 cells were plated at 20% confluence in six-well plates. After 4 h, cells were washed with OptiMEM (GIBCO-BRL) and then were overlaid with 1 ml OptiMEM containing 2 µg pcDNA3 or pcDNA3D1AS DNA that had been mixed with 12 µl of lipofectamine to form liposomes. Cells were incubated in the liposome mixture at 37°C for 16 h. At that time, cells were washed and reincubated in standard growth medium. Later (72 h), cells were subcultured 1:6 and were replated in medium supplemented with 500 µg/ml G418 (Sigma). Selection for G418-resistant cells was allowed to continue for 10 days. At that time, cells were plated sparsely in 100-mm dishes, and 1 wk later, individual colonies were transferred to 24-well plates.

Polymerase chain reaction assay for positive clones. Individual clones were tested for the presence of pcDNA3D1AS DNA by polymerase chain reaction (PCR) analysis. Primers corresponding to the Sp6 and T7 sequences flanking the cyclin D1 antisense insert of pcDNA3D1AS were used to analyze DNA from individual clones. To obtain template DNA, 106 cells from each clone were lysed in PCR lysis buffer [50 mM KCl, 10 mM tris(hydroxymethyl)aminomethane (Tris) base, pH 8.3, 2.5 mM MgCl2, 0.1 µg/ml gelatin, 0.45% Nonidet P-40 (NP-40), 0.45% Tween 20, and 6 µg/100 µl proteinase K] at 56°C for 1 h and then at 95°C for 10 min. Lysates were clarified briefly by centrifugation, and then 15 µl, corresponding to 1.5 × 105 cells and ~1 µg genomic DNA, were used for PCR. Agarose gel analysis was used to identify those colonies that contained amplifiable DNA that corresponded in size to the D1 antisense insert. This fragment could be amplified from the pcDNA3D1AS plasmid DNA using the same set of primers.

Growth curves. Five clones were selected from the several obtained from each transfection for analysis of growth rate. These were A549-1, A549-5, A549-6, H441-HA2, and H441-HB2. These clones, as well as untransfected cells and cells pooled after transfection with the empty vector pcDNA3, were plated in duplicate in six-well plates at 5% confluence. The next day, cells were trypsinized and were counted in duplicate. Cells were then counted by the same method every other day for the next 8 days. A parallel trypan blue exclusion viability assay was performed for each cell count.

Immunohistochemical analysis. Cells were cultured in chambers on glass slides (Nunc) for 48 h. Slides were washed with PBS, then fixed in acid alcohol (5% acetic acid in absolute ethanol) on ice for 10 min. Slides were washed with phosphate-buffered saline (PBS) and then were blocked in PBS containing 5% horse serum and 1% bovine serum albumin (BSA). Primary antibody to cyclin D1 was a monoclonal antibody to PRAD-1 from Santa Cruz Biotechnology. The slides were incubated in anti-D1 antibody diluted at 1 µg/ml in 2% BSA and 1% normal goat serum (NGS) overnight at 4°C. Slides were washed in PBS containing 0.2% Triton X-100, and then the staining was developed using biotinylated secondary antibody and avidin-conjugated horseradish peroxidase (HRP) from the Vectastain ABC system (Vector Laboratories, Burlingame, CA), following the manufacturer's instructions. The HRP substrate used was diaminobenzidine at 0.8 mg/ml, which was enhanced with addition of nickel chloride and cobalt chloride at a concentration of 0.2% and catalyzed by addition of hydrogen peroxide at 0.03%.

Gel electrophoresis and immunoblotting. Cells were washed in ice-cold PBS and then lysed in EBC buffer [40 mM Tris base, pH 8.0, 150 mM NaCl, 0.5% NP-40, 10 mM beta -glycerol phosphate, 20 mM NaF, 0.1 mM NaVO4, 100 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 5 µg/ml pepstatin] on ice. Lysates were clarified by centrifugation, and then protein concentration was determined spectrophotometrically. Lysates were loaded at equal protein concentrations of 40 µg/lane and were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) using the Novex (San Diego, CA) system. After electrophoresis, proteins were electrophoretically transferred to an Immobilon-P membrane (Millipore, Bedford, MA). The membranes were blocked in PBS-T (PBS containing 0.1% Tween 20) containing 5% nonfat dry milk and 1% NGS overnight at 4°C. The membranes were washed in PBS-T and then were incubated at room temperature for 1 h in PBS-T containing 2% BSA and 1% NGS (PBS-T-BSA-NGS) to which primary antibodies had been added. Antibodies to cyclin D1, pRb, cyclin A, cdk2, cdk4, cdc2, and p21 (Santa Cruz Biotechnologies) were used at concentrations of 1-10 µg/ml. The monoclonal antibody to actin (ICN Biomedicals, Costa Mesa, CA) was used at a dilution of 1:20,000. Antiserum to cyclin B (BC-1) was a gift from Dr. Yuen-Kai Fung and was used at a dilution of 1:1,000. Membranes were washed in PBS-T and then were incubated at room temperature for 1 h in PBS-T-BSA-NGS containing HRP-conjugated secondary antibodies [goat anti-mouse immunoglobulin G (IgG) or goat anti-rabbit IgG at 1:20,000 (Sigma)]. After extensive washing in PBS-T, immunoreactive proteins were detected using the enhanced chemiluminescence system, according to the manufacturer's instructions (Amersham).

Metabolic labeling and immunoprecipitation. Dishes (100 mm) containing A549 or A549-5 cells at 75% confluence were washed with PBS and then were labeled using Trans35S-Label (ICN, Irvine, CA) at 500 µCi/ml in methionine-free Dulbecco's modified Eagle's medium (DMEM; GIBCO-BRL) supplemented with 3% dialyzed FBS for 4 h at 37°C. For pulse-chase experiments, labeled cells were washed thoroughly with PBS and then were incubated in growth medium. Cells were collected at the indicated time points by lysing in situ in EBC buffer on ice. pRb was immunoprecipitated from clarified cell lysates using a combination of monoclonal antibodies IF-8 (Santa Cruz Biotechnologies) and Ab-1 (Oncogene Sciences) and protein A agarose (Sigma). Immunoprecipitates were washed with EBC and then were resolved on a 6% SDS-PAGE gel. The gel was then fixed, dried, and exposed to X-ray film for autoradiography. Densitometric analysis of the band patterns was performed using a Hoeffer scanner and software (San Francisco, CA).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Reduced growth rate of cyclin D1 antisense transfected cells. After lipofectamine transfection and selection, colonies of A549 and NCI-H441 transfected cells were screened for the presence of pcDNA3D1AS DNA by PCR analysis. According to this screening method, original transfection efficiency was estimated to be ~10%. Three A549 clones (A549-1, A549-5, and A549-6) and two H441 clones (H441-HA2 and H441-HB2), positive for pcDNA3D1AS, were expanded and plated for determining growth rate (Fig. 1). The A549 and NCI-H441 pcDNA3D1AS positive clone growth rates were compared with those of the parental A549 and NCI-H441 cell lines and with pools of A549 and NCI-H441 cells transfected with the empty vector pcDNA3, which were grown in medium containing the same concentration of G418 (500 µg/ml) as the pcDNA3D1AS clones. Viability, as determined by a trypan blue exclusion assay, was determined for each cell count and consistently ranged from 4 to 8%. The growth rate results are reported in Fig. 1. Each point represents the mean ± SD of duplicate counts from two separate experiments (n = 4).


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Fig. 1.   Reduced growth rate of cyclin D1 antisense transfected cells. Cells were plated at a density of 1 × 104 cells/cm2 24 h before day 0 counts were recorded. Cells were plated and counted in duplicate, and points represent the means of these counts ± SD from 2 experiments (n = 4). Viability was determined using trypan blue exclusion during counting and in all cases ranged from 4 to 8%. A: 8-day growth curve for untransfected A549, empty vector transfected A549pcDNA3, and cyclin D1 antisense transfected clones A549-1, A549-3, and A549-5 (* P < 0.001). B: 8-day growth curve for untransfected NCI-H441, empty vector transfected H441pcDNA3, and cyclin D1 antisense transfected clones H441-HA2 and H441-HB2 (* P < 0.001).

Although the parental A549 cell line had a doubling time of ~36 h, the doubling times of the D1AS positive clones were extended an average of threefold to ~120 h (Fig. 1A). NCI-H441 cells have a doubling time of ~48 h, whereas the D1AS positive clone growth rates were extended twofold to ~96 h (Fig. 1B). In both cell lines, the growth rate of the D1AS transfected cells was significantly slower than that of either the untransfected or the empty vector transfected cells. In Fig. 1A, comparison of cell number at day 8 for A549 wild type and A549-5 gave a P value of <0.001 by Student's t-test. In Fig. 1B, comparison of cell number at day 8 for NCI-H441 wild type and H441-HB2 gave a P value of <0.001 by the same analysis. The same high G418 concentration was used to maintain both the empty vector and D1AS transfected cells; therefore, the reduction in growth rate of the pcDNA3D1AS positive clones could be attributed to the presence of the antisense construct and not to a nonspecific effect of the drug in the medium. Also, the slower growth rate could not be attributed to increased cell death, since a trypan blue exclusion viability assay performed in parallel showed that all cell counts included essentially equivalent numbers of dead cells.

Changes in levels of expression of cell cycle-related proteins in cyclin D1 antisense transfected cells. To determine the level of expression of endogenous cyclin D1 in response to antisense expression, cell lysates from wild-type A549 and NCI-H441 and two representative pcDNA3D1AS positive clones (A549-5 and H441-HB2) were resolved by SDS-PAGE and were Western blotted. Equal protein loading per lane (40 µg/lane) was confirmed by probing the 45-kDa region of the blot with an anti-actin antibody. Cyclin D1 levels were determined by probing the 30-kDa region of the same blot with an anti-cyclin D1 antibody. The level of cyclin D1 expression was decreased in A549-5 and H441-HB2 compared with expression in the untransfected cells (Fig. 2).


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Fig. 2.   Changes in levels of expression of cell cycle-related proteins in cyclin D1 antisense transfected cells. Western blot analysis of cyclin D1 and cell cycle-related proteins was performed using the enhanced chemiluminescence (ECL) detection system on 40 µg of protein from cell lysates of asynchronous cultures. For all blots, lane 1 contains results from untransfected cells, whereas lane 2 contains results from a representative cyclin D1 antisense transfected cell line. A: untransfected A549 and cyclin D1 antisense transfected clone A549-5. B: untransfected NCI-H441 and cyclin D1 antisense transfected clone H441-HB2. C: further comparison of the A549 cells, with the cyclin D1 antisense transfected clone A549-5 showing a slight increase in the expression of cyclin E, essentially no change in the expression of cyclin B, and a marked increase in the expression of the cyclin-dependent kinase (cdk) inhibitor p21. For all antibodies, expression levels for empty vector transfected cell lines were similar to those of the parental lines (data not shown). In this experiment, A549-5 and H441-HB2 were used as representative examples of expression levels that were similar in all other cyclin D1 antisense transfected cell lines. D1 antisense transfected cells exhibited significant changes (P values ranging from <0.05 to <0.0001) in levels of protein expression for cyclin D1, cyclin D2, cdk4, cyclin A, cdk2, cyclin E, and p21 as measured by densitometric scanning. No significant changes were observed for cyclin B or for the internal loading control (actin).

Because cyclin D1 expression and activity are central factors in cell cycle control, we were interested to see what effect decreased expression of cyclin D1 would have on other proteins, the expression of which is involved in cell cycle control. Although some altered expression may be due to the marked reduction in growth rate of the D1AS transfected cells, others may be directly affected by decreased levels of cyclin D1 itself. Therefore, we used Western blot analysis of the antisense transfected clones to observe any ancillary changes in cell cycle-related protein expression in these cells (Fig. 2). All blots were analyzed using anti-actin blotting as an internal loading control. Another member of the D family of cyclins, cyclin D2, showed decreased expression in the antisense transfected cells. This result was in agreement with previous experiments that showed that cyclin D2 expression was a significant marker for the proliferative vs. nonproliferative phenotype of lung epithelial cells (30). Expression of the cyclin D1/D2 catalytic partner cdk4 was also decreased in the antisense transfected cells, as was the expression of cyclin A and cdk2, the enzyme complex believed to be required for G1/S transition. One noticeable change in cdk2 was that the slower migrating phosphorylated form of the kinase was much more strongly expressed in the proliferating untransfected cells than in the antisense transfected cells. Because of limited amounts of lysate available for the NCI-H441 transfected clones, three other protein levels were analyzed in the A549 D1AS clone only. In these cells, the G1/S phase transition cyclin, cyclin E, was slightly decreased in the antisense transfected clone, whereas expression of the S phase-specific cyclin, cyclin B, was essentially unaffected by the slower growth rate of the transfected cells, indicating, perhaps, that the antisense cyclin D1 exerted its most potent effects on cells that were in G1 or transiting G1 into early S phase. Another cell cycle-regulating protein level influenced by D1AS transfection was the cdk inhibitor p21WAF1/CIP1. In the cyclin D1-deficient A549-5 cells, an increase in the expression of p21WAF1/CIP1 was observed. Expression of p21 in NCI-H441 could not be detected in either transfected or untransfected cells.

Changes in pRb phosphorylation and stability in cyclin D1 antisense transfected cells. A549 and NCI-H441 both express an apparently normal form of pRb. In these asynchronous populations, pRb exists in both a hypophosphorylated form (Fig. 3A, "pRb"), corresponding to a protein with a basal level of phosphorylation, and multiple hyperphosphorylated forms (Fig. 3A, "ppRb"), which, due to specific phosphorylations, have both altered conformations and a slower migration pattern in SDS-PAGE (Driscoll, unpublished observations). These forms appear as indistinct smears (Fig. 3A), or, occasionally, as distinct bands (Fig. 3B). A marked decrease in the level of expression of pRb, along with a decrease in the phosphorylation level, was noted in both representative antisense transfected cell lines (Fig. 3A) but not in the empty vector pools (data not shown).


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Fig. 3.   Alteration of pRb phosphorylation levels and protein stability in cyclin D1 antisense transfected cells. A: Western blot analysis of pRb expression and phosphorylation levels was performed using the ECL detection system on protein extracted from asynchronous cultures. pRb extracted from these cells exhibited varying levels of both underphosphorylated (pRb) and hyperphosphorylated (ppRb) forms of the protein. Lane 1: untransfected A549; lane 2: cyclin D1 antisense transfected clone A549-5; lane 3: untransfected NCI-H441; lane 4: cyclin D1 antisense transfected clone H441-HB2. All lanes contained 40 µg of protein, and equal loading was determined by probing the 45-kDa molecular mass area of the same filter with an anti-actin antibody. For this experiment, equivalent expression levels of pRb were observed for both parental and empty vector transfected cell lines (data not shown). However, a significant decrease in both protein level and phosphorylation was observed in the cyclin D1 antisense transfected cell lines as determined by densitometric scanning. B: pulse-chase labeling was performed to determine the stability of the pRb expressed in untransfected, empty vector transfected, and cyclin D1 antisense transfected A549 cells. Duplicate dishes for each cell line were pulse labeled with [35S]methionine and then were washed extensively with phosphate-buffered saline (PBS) and reincubated in normal growth medium. pRb was extracted at the indicated time points (hours) after labeling. Equivalent amounts of protein (100 µg) were used for immunoprecipitation. Expression levels and stability of pRb immunoprecipitated from empty vector transfected cell lines were similar to those of the parental lines (data not shown). In this experiment, A549-5 was used as a representative example of expression level and stability of pRb, both of which were observed to be similar in all other cyclin D1 antisense transfected cell lines. C: protein levels of [35S]methionine-labeled pRb after a chase period in standard medium were determined by densitometric scanning of the pRb bands on film. Protein quantity during the chase period is represented as densitometric band area at 0, 4, and 8 h after [35S]methionine labeling of A549 wild type and D1AS clone A549-5. For each point, n = 2, and values represent the means of the densitometric band areas ± SD. Significant differences in protein levels were observed between pRb extracted at 0 h (phi  P < 0.001), reflecting differences in levels observed in A. In addition, there was a significant decrease in pRb protein level over the chase period (* P < 0.003) for the D1AS A549-5 cells, whereas the change in labeled protein levels in the A549 wild-type cells was not significant for the same period.

pRb activity has been implicated in the expression of D-type cyclins and formation of cyclin/cdk complexes (19, 22), whereas cyclin D1 has a well-documented effect on pRb phosphorylation. However, the effect of altered cyclin D1 expression on levels of pRb protein expression is less well understood. To determine if the decrease in pRb protein levels was due to an increased turnover rate, A549 cells and the D1 antisense transfected clone A549-5 were pulse labeled with [35S]methionine and then were chased over a period of 8 h (Fig. 3B). The half-life of pRb has previously been determined to be 10-12 h (16). pRb was immunoprecipitated from each cell line at 0-, 4-, and 8-h time points during the chase period. Cells from each time point were lysed, and protein concentrations were determined so that pRb could be immunoprecipitated from equal amounts of protein at each time point. In the A549 cells, both hyperphosphorylated and hypophosphorylated forms of pRb exhibited stability over a period of 8 h, with only a slight reduction in intensity in the hyperphosphorylated form by the 8-h period. However, pRb extracted from A549-5 was much less stable, and both hyperphosphorylated and hypophosphorylated forms began to degrade during the first 4 h of the chase period. The majority of the labeled pRb from A549-5 was degraded by the 8-h time point. This result was quantitated by densitometric scanning of the film image produced by the radioactive proteins, and the results are summarized in Fig. 3C. As was the case with the Western blot analysis of protein levels in Fig. 3A, the amount of pRb immunoprecipitated from the A549 cyclin D1 antisense transfected clones was significantly lower than the amount extracted from the wild-type cell lysate (P < 0.001 by Student's t-test; used to compare densitometric band area of pRb from A549 wild type at time 0 with densitometric band area of pRb from A549-5 at time 0, n = 2). Although there was no significant change over time in the level of labeled pRb extracted from the wild-type A549 cells, the same 8-h time period saw a significant decrease in the amount of labeled pRb extracted from A549-5 (P < 0.003 by Student's t-test; used to compare densitometric band area of pRb from A549-5 at time 0 with densitometric band area of pRb from A549-5 at time 8 h, n = 2). These results clearly show that pRb in the cyclin D1 antisense transfected cells is degraded much more rapidly than pRb in wild-type A549.

Cell death due to withdrawal of serum from cyclin D1 antisense transfected cells. To determine if decreased expression of cyclin D1 compromised the ability of tumor cells to live in a growth factor-poor environment, A549, NCI-H441, empty vector transfected control pools, and two representative D1AS transfected cell lines (A549-5 and H441-HB2) were plated in duplicate wells at 30% confluence in the presence of 10% FBS and, in the case of empty vector and D1AS transfectants, 500 µg/ml G418. Later (24 h), serum was removed from one-half of the cells by extensive washing with PBS, followed by incubation in RPMI 1640 containing 0.1% FBS and, where appropriate, 500 µg/ml G418 for 72 h (day 3). At that time, adherent cells were trypsinized and counted (Fig. 4, A and B). Untransfected A549 and NCI-H441 and empty vector transfected pool cells exhibited a modest rate of growth during this time. However, the cyclin D1 antisense transfected cells showed marked evidence of cell death, with the number of viable cells present after serum starvation decreasing from the original number plated by 50-85%. For this experiment, the difference in adherent cell number (represented as a percentage of the original number plated, normalized to 100%) at day 3 between the D1AS transfected cells and the control cell lines was significant; P values of <0.005 for A549-5 and 0.05 for H441-HB2 were obtained using Student's t-test (n = 4).


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Fig. 4.   Death of cyclin D1 antisense transfected cells after withdrawal of serum. Cells were plated in 6-well plates at 2 × 105 cells/cm2 in duplicate. Later (24 h), medium was removed, and cells were washed extensively with PBS and then were incubated in medium containing 0.1% fetal bovine serum (FBS) for 72 h. At that time, cell numbers were counted. Data represent the means ± SD for counts from two separate experiments (n = 4). Viability of adherent cells was determined using trypan blue exclusion analysis and, for all counts, averaged from 5 to 10%. Results are represented as a percentage of increase or decrease in cell number in relation to the original cell count at the time serum was removed, which was normalized to 100%. A: untransfected A549, empty vector transfected A549pcDNA3 and cyclin D1 antisense transfected clone A549-5 (* P < 0.005). B: untransfected NCI-H441, empty vector transfected H441pcDNA3, and cyclin D1 antisense transfected clone H441-HB2 (* P < 0.05).

Characterization of cyclin D1 protein expression in cells after serum deprivation. The nature of the cells that died during a period of serum deprivation was inferred by immunohistochemical analysis of cultures of control and D1AS transfected cells after 48 h of serum deprivation, followed by recovery for 24 h in medium containing 10% FBS. A monoclonal antibody to cyclin D1 was used to stain fixed cells of each type. At time 0, the pcDNA3 empty vector transfected cell lines A549pcDNA3 (Fig. 5A) and NCI-H441pcDNA3 (Fig. 5D) exhibited marked nuclear and perinuclear staining, indicating high levels of cyclin D1 expression. However, at the same time point, 50-85% of the corresponding D1AS transfected cell lines A549-5 (Fig. 5B) and H441-HB2 (Fig. 5E) exhibited little or no specific staining with the anti-cyclin D1 antibody. After 48 h of serum deprivation, the empty vector transfected cells, numbering ~110-145% of the original numbers, and the D1AS transfected cells, numbering 15-50% of the original numbers, were changed to standard medium containing 10% FBS. Cells were fixed and stained 24 h later, and results showed that 100% of the cells remaining in the cyclin D1 antisense transfected cultures expressed high levels of cyclin D1 [A549-5 (Fig. 5C) and H441-HB2 (Fig. 5F)]. None of the cells that survived the 48 h of serum deprivation exhibited the lack of cyclin D1 immunostaining observed in the D1AS transfected cells at time 0. We infer from this result that those cells remaining represented the untransfected G418 resistant portion of the population, which did not contain the plasmid expressing the D1AS RNA and thus had a level of endogenous cyclin D1 equal to that of the original tumor cell line. We also infer that the unaffected expression of cyclin D1 was one of the factors that allowed these cells to survive during the period of serum deprivation.


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Fig. 5.   Characterization of cyclin D1 protein expression in cells after serum deprivation. Cells were plated at 2 × 105 cells/cm2 on chamber slides. Later (24 h), medium was removed, and wells were washed extensively with PBS and then were incubated in medium containing 0.1% FBS. At that time, chambers for each cell line were fixed for immunostaining. After 48 h of serum deprivation, the medium in the remaining wells was changed to standard medium containing 10% FBS. Later (24 h), chambers containing cells that were serum deprived for 48 h and then allowed to recover in normal medium were also fixed. At that time, all chambers were immunostained for cyclin D1. A-D: all cell lines at time 0 for serum deprivation. A: empty vector transfected cell line A549pcDNA3. B: empty vector transfected cell line NCI-H441pcDNA3. C: cyclin D1 antisense transfected cell line A549-5. D: cyclin D1 antisense transfected cell line NCI-H441-HB2. E and F: D1 antisense transfected cell lines at 48 h of serum deprivation plus 24 h of recovery in medium containing 10% FBS. E: A549-5. F: NCI-H441-HB2.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Cyclin D1 antisense transfection resulted in striking changes at the level of expression of those proteins that control traversal of G1 and the G1 to S transition: cyclin D1 itself, cyclin D2, cdk4, cyclin A, cdk2, and pRb. In addition, the expression of the cdk inhibitor p21, which also regulates G1 events, was altered significantly in the antisense transfected A549 cell lines. However, levels of S to G2/M phase components, cyclin B, and cdk1 were equivalent for both transfected and untransfected cells. These data (Fig. 2) appear to indicate that manipulation of cyclin D1 levels has the most influence on G1 events. In normal cells, feedback mechanisms involving these proteins appear to control their respective expression levels and, therefore, cell cycle progression. However, in tumor cells, deletions and mutations have resulted in inappropriate levels of expression of cyclin D1.

In the cyclin D1 antisense transfected cells described here, the decrease in cyclin D1 expression can be attributed directly to the presence of abundant cyclin D1 antisense RNA in the stably transfected cells, leading to inhibition of translation of endogenous cyclin D1. Decreased cyclin D1 expression may, through feedback mechanisms, control the expression of other cell cycle-related proteins. Alternatively, the changes in other proteins may be a consequence of the retarded cell growth that results from decreased cyclin D1 expression. Although the exact mechanism is difficult to establish from the current experiments, the negative effect of cyclin D1 antisense expression on tumor cell growth is clear.

A striking effect associated with the reduction in cyclin D1 levels was the altered expression of pRb (Fig. 3A). A decrease in pRb phosphorylation was expected, because cyclin D1 has been previously shown to be a critical kinase for the tumor suppressor protein. This change could be seen most clearly in the A549 cells, which grow quite rapidly, such that the majority of cells in a random population are in S phase, and a large percentage of the pRb expressed is hyperphosphorylated. In the NCI-H441 cells, which grow more slowly, more pRb is in its underphosphorylated form, but some of the hyperphosphorylated species can be observed in a random population. In contrast, the pRb expressed in the cyclin D1 antisense transfected clones, even in a random population, is almost all in the underphosphorylated form. This indicates that the amount of cyclin D1 required to shift the endogenous pRb from the hypophosphorylated to the hyperphosphorylated form is severely lacking in the transfected cells.

A lack of phosphorylation was not the only effect that decreased expression of endogenous cyclin D1 had on pRb. The level of pRb protein expressed in the transfected cells was also consistently lower than that in the untransfected cells. Pulse-chase experiments showed that the pRb expressed in the transfected cells was much less stable than pRb from the untransfected cells or from cells transfected with the G418 resistance vector alone. This may be due to a lack of necessary basal phosphorylation by cyclin D1-associated kinases, which may serve to stabilize pRb. Consistent with these observations is the fact that the expression of pRb in terminally differentiating cells is often very low (31), indicating that cells that are ready to withdraw from the cell cycle no longer need to express pRb. Thus only cells that are about to reenter the cell cycle need to express pRb at high levels so that ordered progression through the cell cycle can be achieved.

A complex interaction of many different proteins controls the progression of normal cells through the G1 phase of the cell cycle. In tumor cells, this process is accelerated due to overexpression of growth-promoting genes and deletion or mutation of growth-suppressing genes. Under controlled culture conditions, tumor cells replicate in a predictable and reproducible manner and often show reduced requirements for growth factors. Therefore, the most striking effects after reduction of cyclin D1 expression in this study were the severely retarded growth rates of the stably transfected clones and the inability of those clones to survive in a growth factor-poor environment.

Because overexpression of cyclin D1 has been shown to shorten G1 (17, 20, 21), decreased expression might be expected to lengthen this phase of the cell cycle. Indeed, Zhou et al. (32) have shown by fluorescence-activated cell sorter (FACS) analysis that the length of G1 was somewhat longer in cyclin D1 antisense transfected esophageal carcinoma cells than in the original cell line. Although not measured directly in this study, a lengthened G1 phase would account for the significant (2- to 3-fold) increase in doubling time observed in the cyclin D1 antisense transfected cells.

The altered response to exogenous growth and senescence signals by the antisense transfected cells was also significant. Parental and empty vector transfected A549 and NCI-H441 cells, when deprived of serum, cannot only exist in essentially serum-free culture for at least 3 days but also appear to continue dividing, as evidenced by a slight increase in cell number during this period. Indeed, the parental lines remained viable in serum-free medium for periods as long as 10 days, with very little cell death observed (data not shown). However, the cyclin D1 antisense transfected cells showed a marked requirement for exogenous growth factors; when serum was withdrawn from these cells, 50-85% died within 3 days. This result could not be attributed to G418 toxicity, as the empty vector transfected cells showed no significant cell death when cultured under exactly the same conditions in the presence of 500 µg/ml G418. Upon restoration of medium containing 10% FBS to those 15-50% of cells remaining after 72 h of serum deprivation, immunostaining revealed cyclin D1 levels equivalent to those observed in the parental and empty vector transfected cell lines. We inferred from these data that those cells that survived 72 h of serum deprivation did so because they had developed G418 resistance (a relatively common phenomenon observed during the establishment of stably transfected, drug-resistant cell lines) but did not carry the D1AS vector. In contrast, those cells that died during this period were inferred to be those that had been successfully transfected. The percentage of cell death should therefore reflect transfection efficiency, and, in fact, the number of cells remaining after serum deprivation was lower for the A549-5 clone (by immunostaining ~85% successfully transfected) than for the NCI-HB2 clone (~50% successfully transfected by the same assay).

The result of this experiment is currently being analyzed more thoroughly because the mode of cell death, whether apoptosis or necrosis, is unknown. Previously, it was noted that transfection of antisense cyclin D1 into an osteosarcoma cell line resulted in an increased cytocidal effect due to apoptosis, even in the presence of serum (25). Taken together, these results indicate that cyclin D1 expression may be part of a critical pathway that mediates autocrine growth factor expression and inhibits lethality due to serum deprivation.

The striking effect of cyclin D1 antisense expression on lung tumor cell proliferation described here suggests a potential approach for therapies directed against rapidly growing cells in an accessible region of the body, such as the pulmonary epithelium. These studies have shown, for the first time, that a real cell cycle inhibitory effect, triggering a pathway that may lead to actual withdrawal from the cell cycle, is a consequence of administration of cyclin D1 antisense RNA in lung epithelial cancer cells.

    ACKNOWLEDGEMENTS

We thank Dr. Yuen-Kai Fung for the gift of antibody BC-1.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-44060 and HL-44977 (to D. Warburton).

Address for reprint requests: D. Warburton, Dept. of Surgery, Childrens Hospital Los Angeles Research Institute, Smith Research Tower, MS 35, 4650 Sunset Blvd., Los Angeles, CA 90027.

Received 11 February 1997; accepted in final form 23 July 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 273(5):L941-L949
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