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Originally published In Press as doi:10.1074/jbc.M204962200 on July 22, 2002

J. Biol. Chem., Vol. 277, Issue 39, 36329-36337, September 27, 2002
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Transcriptional Regulation during p21WAF1/CIP1-induced Apoptosis in Human Ovarian Cancer Cells*

Qun WuDagger , Paul KirschmeierDagger , Tish HockenberryDagger , Tong-Yuan Yang§, Diana L. Brassard§, Luquan Wang, Terri McClanahan||, Stuart BlackDagger , Giovanni Rizzi§, Mary Lynn Musco§, Asra MirzaDagger , and Suxing LiuDagger **

From the Dagger  Tumor Biology Department and  Human Genomic Research Department, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, § Biotechnology Development, Schering-Plough Research Institute, Union, New Jersey 07083, and || DNAX Research Institute, Palo Alto, California 94304

Received for publication, May 20, 2002, and in revised form, July 12, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study we used adenovirus vector-mediated transduction of either the p53 gene (rAd-p53) or the p21WAF1/CIP1 gene (rAd-p21) to mimic both p53-dependent and -independent up-regulation of p21WAF1/CIP1 within a human ovarian cancer cell line, 2774, and the derivative cell lines, 2774qw1 and 2774qw2. We observed that rAd-p53 can induce apoptosis in both 2774 and 2774qw1 cells but not in 2774qw2 cells. Surprisingly, overexpression of p21WAF1/CIP1 also triggered apoptosis within these two cell lines. Quantitative reverse transcription-PCR analysis revealed that the differential expression of BAX, BCL2, and caspase 3 genes, specific in rAd-p53-induced apoptotic cells, was not altered in rAd-p21-induced apoptotic cells, suggesting p21WAF1/CIP1-induced apoptosis through a pathway distinguishable from p53-induced apoptosis. Expression analysis of 2774qw1 cells infected with rAd-p21 on 60,000 cDNA microarrays identified 159 genes in response to p21WAF1/CIP1 expression in at least one time point with 2.5-fold change as a cutoff. Integration of the data with the parallel microarray experiments with rAd-p53 infection allowed us to extract 66 genes downstream of both p53 and p21WAF1/CIP1 and 93 genes in response to p21WAF1/CIP1 expression in a p53-independent pathway. The genes in the former set may play a dual role in both p53-dependent and p53-independent pathways, and the genes in the latter set gave a mechanistic molecular explanation for p53-independent p21WAF1/CIP1-induced apoptosis. Furthermore, promoter sequence analysis suggested that transcription factor E2F family is partially responsible for the differential expression of genes following p21WAF1/CIP1. This study has profound significance toward understanding the role of p21WAF1/CIP1 in p53-independent apoptosis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

p53 elicits its tumor suppressor activity by inducing cell cycle arrest and/or apoptosis of tumor cells (1, 2). p21WAF1/CIP1 is a downstream effector of p53 that mediates both G1 and G2/M phase arrest (3-7). Mechanistically, the p21WAF1/CIP1-mediated arrest of the G1 and G2/M cell cycle transition has been suggested to include a p21WAF1/CIP1-Cdk2 and p21WAF1/CIP1-PCNA protein interaction (8, 9). This interaction is thought to compete with both Cdc25c binding and also methyltransferase binding (3, 10). Although p21WAF1/CIP1 was induced during p53-mediated apoptosis, its expression did not appear to be required for p53-mediated apoptosis, and the p21WAF1/CIP1-growth arrest activity actually protects cells from p53-induced apoptosis (6, 11, 12).

There is sufficient evidence to demonstrate that up-regulation of p21WAF1/CIP1 can be independent of p53 (13-16). In addition, BRCA1 has been shown to require p21WAF1/CIP1 for its cell cycle arrest (17). The importance of p53-independent regulation of p21WAF1/CIP1 activity can be inferred by involvement in cellular differentiation, influence on gene expression and chromosomal repositioning, and correlation with preoperative chemotherapeutic efficacy (18-22). Recently, more evidence (9, 23-25) has demonstrated that p21WAF1/CIP1 could mediate apoptosis in a p53-independent manner. Although a downstream effector of p53 mediated G1 arrest, the role of p21WAF1/CIP1 in the apoptotic pathway remains controversial.

In this study, we use adenovirus vector-mediated transduction of p53 (rAd-p53)1 or p21WAF1/CIP1 gene (rAd-p21) to mimic p53-dependent and -independent up-regulation of p21WAF1/CIP1 within the human ovarian cancer cell line, 2774, and its derivative cell lines, 2774qw1 and 2774qw2. These cell lines harbor endogenous mutant p53, which allows the ectopic expression of wild-type p53. We observed that in this particular system rAd-p21 induced apoptosis in 2774 and 2774qw1 cells in a p21WAF1/CIP1-specific manner. Furthermore, a small scale and a genome-wide expression analysis were performed to investigate the downstream events regulated by p21WAF1/CIP1.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Lines and Cell Culture-- The human ovarian cancer cell line, 2774, was obtained from American Type Cell Collection (ATCC). 2774 was maintained in RPMI 1640 medium containing 10% fetal bovine serum. The derived cell lines, 2774qw1 and 2774qw2, were cultured under identical conditions to the parental cell line, 2774.

Sequence Analysis of p53 Transcript-- Sequence analyses were carried out by Geneka Biotechnology, Inc. (Montreal, Canada). Briefly, reverse transcription from random decamers was performed on RNA obtained from 2774qw1 and 2774qw2 cells by using the RNeasy kit from Qiagen (Valencia, CA). 35 cycles of PCR with Vent polymerase and p53-specific primers (forward primer, 5'-TCCGGGTCACTGCCATGGAGGAGCCG-3'; reverse primer, 5'-TGGAGAATGTCAGTCTGAGTCAGGCC-3') were used to amplify p53 cDNA. DNA sequencing was performed using multiple IR dye-labeled primers (Li-Cor, Lincoln, NB) and the SequiTherm EXCEL II DNA sequencing kit-LC (Epicentre Technologies, Madison, WI). The reactions were analyzed on a model 4200 Long ReadIR automated DNA sequencer (Li-Cor).

Infection of Adenoviruses-- Human ovarian tumor cells, 2774, and 2774qw1 and 2774qw2 cells were infected with rAd-p21, rAd-p53, or rAd-empty vector at concentrations of 1 × 1010 particles/ml for 1 h. Following the incubation, the media for all cell samples were replaced with fresh media, not containing virus, and cells were harvested at the indicated hour post-infection.

Apoptosis Assays-- NEXINTM assay (Guava, Inc.) was used for the staining of the cells with annexin V, and the TUNEL assay was performed with ApopTagTM staining kit (Intergen). Apoptosis analysis followed the manufacturer's instructions.

Cell Cycle Analysis-- 2774qw1 cells infected with rAd-p21, rAd-p53, and rAd-empty vector were harvested at 4, 8, 12, 16, 20, and 24 h post-infection. All time points were performed in duplicate. The cells were trypsinized, and washed with D-PBS. The sample was fixed by slowly dropping 2 ml of cold 95% ethanol into the tube followed by storage at 4 °C. After fixation, the cells were washed, re-pelleted, and resuspended in 0.05 mg/ml propidium iodide, 0.0005% Triton X-100, 44.5 mM EDTA, 100 units/ml RNase in PBS. The sample was incubated at room temperature for 30 min, filtered through a 35-µm nylon mesh filter, and analyzed on a FACSCalibur (BD PharMingen). Cell cycle data originally obtained with Cell Quest software (BD PharMingen) was re-analyzed using MODFIT software (Verity Software, Topsham, ME).

Quantitative RT-PCR-- RNA preparation and quantitative reverse transcription-PCR (RT-PCR) was performed essentially as described previously (26).

cDNA Microarray Hybridization-- The total RNA was purified from 2774qw1 cells infected with 1010 particles/ml of rAd-p21 or rAd-empty vector at various time points, and poly(A) mRNA was isolated using oligotex beads (Qiagen, Chatsworth, CA). For each probe pair, poly(A) mRNA was fluorescently labeled with Cy3 and Cy5 fluorescent dyes and hybridized on Incyte GeneAlbum 1-6 at Incyte (Incyte Genomics, Palo Alto, CA) using their proprietary technology as described (27).2

Promoter Sequence Analysis-- The promoter sequences for the genes on microarray were collected as described previously (26). The human subset of transcription factor binding site consensus sequences was retrieved from TRANSFAC data base (28, 29). The "FindPatterns" program (Wisconsin Sequence Analysis Package, version 10) was used to match consensus DNA-binding site in the promoters.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of Two Cell Clones Derived from Human Ovarian Cancer Cell Line 2774-- The human ovarian cancer cell line 2774 carries an arginine to histidine mutation in codon 273 of the p53 gene and was initially selected for this study based on its infectivity (>90%) with adenovirus (30). To determine the temporal profile of cell cycle status of 2774 cells after infection with rAd-p53 or rAd-p21, FACS analysis of cellular DNA determined that the 2774 cells represent a mixed cell population (data not shown). Therefore, several single clones were isolated, and multiple distinct cell morphologies were observed. 2774qw1 and 2774qw2 are single cell clones based on FACS analysis and were representative of two of these cell types (data not shown).

The p53 status of these three cell lines was determined by sequencing of the coding sequence of the p53 gene. The results indicated that the parental 2774 cell line and the two single cell clones harbor a missense mutation at codon 273 (CGT to CAT, arginine to histidine). This is one of the common mutational hot spots common in the p53 protein (31). Interestingly, we also observed that 2774qw1 and 2774qw2 cells harbor different additional p53 mutations. The amino acid at the codon 72 of the p53 sequence from 2774qw1 cells was a proline substitution for the arginine, which was not found in the p53 sequence from the 2774qw2 cells. The amino acid at codon 309 of the p53 sequence from 2774qw2 cells was a serine substitution for the proline, which was not found in the p53 sequence from the 2774qw1 cells. This suggests heterogenicity of the parental cell line, 2774.

Adenoviruses Expressing p53 and p21WAF1/CIP1 Induce Significant Apoptosis, Respectively, in 2774qw1 Cells but Not in 2774qw2 Cells-- To determine the optimal concentration of adenoviruses used for transducing p53, multiple concentrations of rAd-p53 and adenovirus containing an empty vector (rAd-empty vector) were used to infect 2774qw1 cells for 2-24 h. The maximal rAd-p53-induced apoptosis was observed in 2774qw1 cells infected with 1 × 1010 particle/ml of rAd-p53, whereas little vector toxicity with the cells infected with the same concentration of rAd-empty vector.3 A similar concentration of rAd-p21 was used in the following experiments. The levels of expression of transgenes, p53 and p21WAF1/CIP1, were monitored in 2774qw1 cells at 2-24 h post-infection of rAd-p53 and rAd-p21, respectively. Quantitative RT-PCR analysis detects exogenous p53 mRNA as early as 2 h post-infection with rAd-p53, with the p53 protein observed 6 h post-infection (32). The activation of endogenous p21WAF1/CIP1, a known p53 target gene, by the overexpression of exogenous p53 was observed in the rAd-p53-infected cells. The level of p21WAF1/CIP1 expression induced by rAd-p21-infection was 100-fold higher than what was detected in the rAd-p53-infected cells. It should be noted that the primer/probe reagents used in these RT-PCR experiments can not discriminate between the exogenous and endogenous p21WAF1/CIP1 mRNA. The viral transduction efficiency in 2774, 2774qw1, and 2774qw2 was estimated by infection of 1 × 1010 particle/ml of the adenovirus expressing beta -galactosidase. The results of beta -galactosidase staining indicated that ~86-89% transduction efficiency occurred in 2774qw1 cells at 2-24 h post-infection, and similar results were obtained with 2774 and 2774qw2 cell lines.

We next evaluated whether rAd-p53 infection could induce apoptosis in the two 2774-derived clones, 2774qw1 and 2774qw2, using annexin V staining. The parental cell line 2774 was included in these assays as a reference. The cells at 20 h post-infection of rAd-p53 or rAd-empty vector were fixed and stained with annexin V. Apoptotic cells were quantified by counting annexin V-positive cells, as described under "Materials and Methods." Strikingly, rAd-p53 induced a significant level of apoptosis in 2774 and 2774qw1 cells (Fig. 1A). In contrast to these cell lines, rAd-p53-infected 2774qw2 cells does not elicit apoptosis (Fig. 1A). We were surprised to observe that rAd-p21 can also trigger apoptosis within the parental 2774 and 2774qw1 cell lines (Fig. 1A). Taken together, our data indicated that 2774qw1 and 2774qw2 derived from the same parental 2774 cell line exhibit distinct cellular morphologies and apoptotic effects in response to rAd-p53 and rAd-p21 infection.


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  		Fig. 1.   Induction of apoptosis by rAd-p53 and rAd-p21. A, apoptotic response of three cell lines harvested at 20 h post-infection of rAd-p53, rAd-p21, and rAd-empty vector. The percentage of apoptotic cells was determined after staining cells with annexin V. Values are the means of triplicate ± S.D. in one of three independent experiments. B and C, fluorescence microscopy of 2774 (B) and 2774qw1 (C) cells 20 h post-infection of rAd-p53, rAd-p21, rAd-empty vector, or no virus. Apoptosis-positive cells are shown in green in the center of the propidium iodide-stained cells (red).

Microscopic analysis of TUNEL staining was used to further evaluate the apoptotic effect of 2774 and 2774qw1 cells infected with rAd-p53 and rAd-p21 observed in Guava Nexin assay. The nuclei of control cells showed no staining without adenovirus infection (MOCK), indicating that cells were healthy and their nuclei were intact. Furthermore, 2774qw1 cells did not exhibit TUNEL staining at 20 h post-infection of rAd-empty vector. In contrast, a fraction of the 2774qw1 cells exhibited the typical strong yellow-green TUNEL staining at 20 h post-infection with either rAd-p53 or rAd-p21 (Fig. 1B). The observations of these TUNEL-positive cells demonstrated typical characteristics of apoptosis, such as condensed and shrunken nuclei, and were clearly distinguishable from normal nuclei. These data confirmed that the observed DNA fragmentation induced by both rAd-p53 and rAd-p21 infection of 2774qw1 cells correlated with morphological apoptotic features. Similar observations were made in parental 2774 cell line (Fig. 1C), whereas no apoptotic response was detected in 2774qw2 cells (data not shown).

Effect of rAd-p53 or rAd-p21 on Cell Cycle Progression of 2774qw1 Cells-- To assess the effect of rAd-p53 and rAd-p21 on cell cycle progression, population distribution throughout the cell cycle in 2774qw1 cells was determined using flow cytometry. A comparison of the cell cycle profiles of rAd-p21-, rAd-p53-, and rAd-empty vector-infected 2774qw1 cells confirms that p21WAF1/CIP1 and p53 transgene-specific effects can be detected. By using the percent of cells in G0/G1 phase as an analytical end point, infection with rAd-p53 or rAd-p21 shifted cells to a G1 arrest in a time-dependent manner (Fig. 2A). Notably, 20 and 24 h after rAd-p21 infection the 2774qw1 cells demonstrate a significant G1 arrest (p < 0.05) in comparison to uninfected cells (Fig. 2A). Interestingly, infection with rAd-p53 resulted in a significant reduction in S phase cell number that started 20 h post-infection. Infection with rAd-p21 also decreases the S-phase population but to a lesser extent compared with rAd-p53 (Fig. 2B). The differences seen when comparing the G0/G1 or S phase populations of rAd-p21- or rAd-p53-infected cells to rAd-empty vector-infected cells show significant difference as determined by a Student's t test (p < 0.1 and p < 0.05, respectively; Fig. 2B). Taken together, these results demonstrate a 90-95% confidence in the data and signify an impact on cell cycle progression with p21WAF1/CIP1 and p53 transgene expression. These results agree with the apoptosis time course presented in Fig. 1B which shows a break in the trend of data at 16 h post-infection and significant differences detected at 20 and 24 h post-infection. Analysis of the percent of cells in G2/M did not show a statistically significant difference between cells infected with rAd-p21, rAd-p53, or rAd-empty vector (Fig. 2C).


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  		Fig. 2.   Effects of rAd-p53, rAd-p21, and rAd-empty vector on cell cycle progression of 2774qw1 cells. A, percent of cell population at G1/G0 phase. B, percent of cells in S phase. C, percent of cells in G2/M phase. Results presented are the mean of two independent experiments. p values represent the probability of the rAd-empty vector samples being significantly different from rAd-p21 or rAd-p53 using a Student's t test. * indicates p < 0.05, and ** indicates p < 0.1. rAd-Empty indicates rAd-empty vector.

2774qw1 cells, but Not 2774qw2 Cells, Showed Similar Expression Profile to Its Parental 2774 Cell Line-- In the first attempt to look at the expression patterns of two clones, 2774qw1 and 2774qw2, and their parental cell line, 2774, in response to p53 or p21WAF1/CIP1 expression, quantitative RT-PCR was used to study any changes of downstream genes in mRNA expression. RNA was isolated from the corresponding cell lines 16 h post-infection with rAd-p53 or rAd-empty vector. p53 has been shown to regulate the expression of p21WAF1/CIP1, cyclin B1, BAX, and BCL2 (33-36). Caspase 3 is known to play a key role in initiation of cellular events during the apoptotic process (37). Quantitative RT-PCR analysis of these 6 genes was performed to study the effects of rAd-p53 or rAd-empty vector on 2774qw1 or 2774qw2 cells (Table I). Interestingly, p21WAF1/CIP1 and MDM2 mRNA levels show a similar change in the 2774qw1 cells and its parental cell line2774. In contrast, the 2774qw2 cells exhibited a much higher induction of p21WAF1/CIP1 and MDM2 gene expression. Overall, the 2774qw1 cells showed similar expression profile to its parental 2774 cell line, whereas the 2774qw2 cells did not.

                              
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Table I
Differential expression (fold change) of 6 genes in 2774qw1 and 2774qw2 clones and their parental 2774 cells 16 h post-infection of rAd-p53 as compared with rAd-empty

p21WAF1/CIP1-induced Apoptosis Is Independent of Caspase 3, BAX, and BCL-2 Gene Activation-- To gain insight into the apoptotic role of p21WAF1/CIP1 in a p53-independent pathway, we compared the expression profiles of 2774qw1 cells with rAd-p53 infection and rAd-p21 infection. 5 apoptosis- and cell cycle-related genes were analyzed using quantitative RT-PCR analysis. Both p21WAF1/CIP1 and caspase 3 mRNAs were activated by p53 in all time points tested. After rAd-p53-induced apoptosis takes place, all 5 genes showed differential expression (Fig. 1 and Table II). In contrast, rAd-p21-infected cells showed substantial induction of p21WAF1/CIP1, which resulted from transgene expression as expected, and repression of cyclin B1. Interestingly, we observed no detectable activation of caspase 3, BAX, and BCL2 mRNAs when p21WAF1/CIP1-induced apoptosis occurred in 2774qw1 cells (Table II). This suggests that p21WAF1/CIP1 alone induces apoptosis in a pathway distinguishable from p53-induced apoptosis.

                              
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Table II
Temporal differential expression (fold change) of 5 genes in 2774qw1 cells infected with rAd-p53 or rAd-p21 as compared with rAd-empty, respectively

Genome-wide Expression Profiling of 2774qw1 Cells Infected with rAd-p21, rAd-p53, or rAd-empty Vector-- High density microarray technology allows a genome-wide identification of genes downstream of p21WAF1/CIP1. To do so, mRNA isolated from 2774qw1 cells 4, 8, 12, 16, 20, and 24 h post-infection of 1010 particles/ml rAd-p21 or rAd-empty vector were hybridized to 6 microarrays, containing ~60,000 cDNAs. A total of 159 genes were altered in a time-dependent response to rAd-p21 infection using 2.5-fold change as a cutoff. Performance of the hybridization was assessed by analyses of 3 genes that have multiple cDNA clones represented on the microarrays (Table III). Overall, there was good consistency between the signals observed between the different cDNA clones on the microarrays. It was observed that the expression of p21WAF1/CIP1 was the most induced in the microarray experiments. The p21WAF1/CIP1 mRNA level in 2774qw1 cells infected with rAd-p21 was up-regulated 4 h post-infection (4.7-fold) and reached maximal up-regulation at 20 and 24 h post-infection (10.7- and 10.4-fold, respectively) when compared with cells infected with rAd-empty vector. The results confirmed the quantitative RT-PCR data presented in Table II. Nevertheless, we noticed that the microarray data generally underestimated the differential expression as compared with Taqman analysis. Similar observations were made in several independent microarray experiments in other laboratories (21, 38). We noticed that the majority of genes that showed differential expression was repressed. Therefore, a reverse fluorescence hybridization experiment was performed. The results indicated that the mRNA repression observed is not an artifact of dye labeling and could represent a bona fide response.3

                              
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Table III
Fold change of 3 genes (as compared with control) represented by multiple cDNA clones on the microarrays
Fold changes are compared with control.

Parallel microarray experiments were carried out using mRNA isolated from 2774qw1 cells 4, 8, 12, 16, 20, and 24 h post-infection of 1010 particles/ml rAd-p53 or rAd-empty vector. 1,501 genes were identified in response to rAd-p53 infection in at least one time point with 2.5-fold change as a cutoff.3 The combination of these two sets of microarray data enables us to identify genes downstream of p21WAF1/CIP1 in a p53-independent pathway. Overall, the number of genes in response to p53 expression exceeded that observed in response to p21WAF1/CIP1 expression. Obviously, this seems likely because p21WAF1/CIP1 itself is not a transcription factor.

Because p21WAF1/CIP1 is transcriptionally activated by wild-type p53, some genes downstream of p21WAF1/CIP1 may also be in the p53-dependent pathway as well. Our experiments identified that 76 cDNAs, representing 66 genes (61 known genes and 5 unannotated ESTs), were downstream of both p53 and p21WAF1/CIP1. Meanwhile, 96 cDNAs regulated by p21WAF1/CIP1, representing 93 genes (55 known genes and 38 unannotated ESTs), did not overlap with a p53 response. All annotated genes are marked based on the earliest time point they started showing differential expression, designated as early, intermediate, and late responsive genes are listed in Table IV. The genes that respond to both p53 and p21WAF1/CIP1 may play a dual role in p53-dependent and -independent pathways. As expected, up-regulation of p21WAF1/CIP1 mRNA was observed in both sets of microarray data. The rest of the genes could possibly provide a molecular mechanistic explanation of p53-independent p21WAF1/CIP1-induced apoptosis.

                              
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Table IV
Genes in response to rAd-p21 infection in 2774qw1 cells

E2F Transcription Factors May be Responsible for Differential Expression of the Genes Downstream of p21WAF1/CIP1-- Because p21WAF1/CIP1 is not a transcription factor, it is likely that other transcription factors may be responsible for the observed differential expression of the genes following the p21WAF1/CIP1 expression. To address this question, the DNA-binding sites of the transcription factors in the promoter of genes downstream of p21WAF1/CIP1 were searched. The promoter sequences of the genes were collected as described previously (26). Of 116 annotated genes downstream of p21WAF1/CIP1, 41 genes have promoter sequences available in GenBankTM. The search of the DNA-binding sites of the transcription factors were performed, as described under "Materials and Methods," with the consensus DNA-binding sequences of 759 human transcription factors from TRANSFAC database (28, 29). By assuming a particular transcription factor mediates the signal form p21WAF1/CIP1, the differential expression data derived from rAd-p21 infection should enrich the genes that contain consensus DNA-binding sequence for this transcription factor because its target genes should be part of the affected genes. Interestingly, only the transcription factor E2F family (of 759 human transcription factors analyzed) was found to have the consensus DNA-binding sequence in the promoter of several genes. For example, 8 of 41 genes contain DNA-binding sequence for transcription factor E2F1 and E2F2 and 3 genes for transcription factor E2F4 (Table V).

                              
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Table V
Genes downstream of p21WAF1/CIP1 that contain transcription factor DNA-binding sequence in the promoter


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The p53 protein is a key regulator for cell cycle arrest and apoptosis induced by DNA damage. These activities are partly mediated by p21WAF1/CIP1 induced by p53. Nevertheless, there is sufficient evidence to demonstrate that up-regulation of p21WAF1/CIP1 can be independent of p53 (13-16). More importantly, p53-independent up-regulation of p21WAF1/CIP1 has been shown to be involved in cellular differentiation (18, 19) and apoptosis (39). However, the molecular mechanism of p21WAF1/CIP1 during differentiation and apoptosis is poorly understood.

In this study, we elaborate on the biological function(s) of p21WAF1/CIP1 by mimicking p53-dependent and -independent up-regulation of p21WAF1/CIP1 within a human ovarian cancer cell line, 2774, and its derivative cell lines, 2774qw1 and 2774qw2, using adenoviral mediated transduction of the p53 or p21WAF1/CIP1 genes. Sequence analysis of the endogenous p53 gene in these two clones, as well as the parental 2774 cell line, indicated that they all carried a mutant endogenous p53. Adenovirus expression of p53 can induce apoptosis in the parental 2774 cells and its derivative cell line, 2774qw1. Notably, the 2774qw2 cells showed no p53-induced apoptotic response. Interestingly, the 2774qw1 and 2774qw2 cells not only show distinct apoptotic effects and different cellular morphologies but also contain a different p53 mutation status.

We were surprised to observe that, in addition to inducing a G1 arrest, the adenoviruses expressing p21WAF1/CIP1 can also trigger apoptosis within 2774 and 2774qw1 cell lines. The p21WAF1/CIP1-induced apoptosis detected by annexin V staining was confirmed by TUNEL assay (Fig. 1). Although p21WAF1/CIP1 is induced during p53-mediated apoptosis, its expression does not appear to be required for p53-mediated apoptosis in mouse thymocyctes (6). However, p53-independent up-regulation of p21WAF1/CIP1 has been shown to be involved in cellular differentiation (18, 19) and apoptosis (39). There are literature references that agree with our observations. p21WAF1/CIP1 directly induced apoptosis of cervical cancer cells and papillary serous endometrial cancer cells when using the p21WAF1/CIP1 gene delivered by recombinant adenoviral vector (23, 24). p21WAF1/CIP1, along with STAT1, is also required for oxysterol-induced apoptosis (9). Similarly, it was observed that activation of p21WAF1/CIP1 and Cdc2 are involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells (25). Meanwhile, apoptosis in primary hepatocytes transfected with the human papilloma virus (HPV16) E7 protein decreases in p21WAF1/CIP1 null mutant in primary hepatocytes compared with wild-type hepatocytes (40). Furthermore, overexpression of a truncated p21WAF1/CIP1 protein lacking the PCNA interaction domain can directly induce apoptosis in human papillomavirus 16 E6-expressing cancer cell, suggesting that interaction with PCNA may inhibit apoptotic function mediated directly by p21WAF1/CIP1 (41). Nevertheless, it should be noted that the opposite results have been reported, showing an inhibition of apoptosis by p21WAF1/CIP1 (42). Indeed, our data show that the 2774qw2 cells have no apoptotic response to rAd-p21 infection. Thus, it is likely that p21WAF1/CIP1-induced apoptosis occurs in a cell type-specific manner.

One of the most intriguing aspects of microarray gene expression analysis is the correlation of expression profiles with observed cellular phenotypes. The goal of these experiments is to predict and hypothesize the mechanism of action, which can be tested in follow-up experiments. For instance, we observed that a number of genes downstream of both p53 and p21WAF1/CIP1 are related to cell proliferation. For example, transcobalamin II, alpha -tubulin, CDC2, cyclin-selective ubiquitin carrier, MKI67a, M1 subunit of nucleotide reductase, P1CDC47, P1-CDC21, and serine/threonine kinase (BTAK) were repressed following both p53 and p21WAF1/CIP1 expression (Table IV). Interestingly, these genes are up-regulated in a majority of the 8 human breast cancer cell lines but not in the RNA obtained from 3 normal counterpart donors.4 Based on these microarray experiments, we hypothesize that the opposing expression levels of these genes could be ascribed to the cancer cells that have undergone malignant transformation. In this situation, the genes controlling cell proliferation are constitutively "on" when they should be down-regulated. When cancer cells experience cell cycle arrest and/or apoptosis in response to p53, the induced p21WAF1/CIP1 mRNA and the cell proliferation-related genes are down-regulated.

Furthermore, the genes identified in this study could provide a molecular explanation as to the p53-independent p21WAF1/CIP1-induced apoptosis. Notably, we observed that B-MYB and alpha B-crystallin were repressed by p21WAF1/CIP1 in a p53-independent manner (Table IV). Overexpression of B-MYB was reported to protect CTLL-2 cells from apoptosis by inducing the expression of BCL2 via a DNA binding-dependent manner (43). It is likely that overexpression of p21WAF1/CIP1 in 2774qw1 cells represses BCL-2 expression, through B-MYB, resulting in apoptosis (Tables II and IV). The small heat shock protein alpha B-crystallin is known as a negative regulator of apoptosis by disrupting caspase 3 maturation (44). Although caspase 3 mRNA was not induced following p21WAF1/CIP1 expression (Table II), it is possible that repression of alpha B-crystallin by p21WAF1/CIP1 promotes caspase 3 maturation. Although the roles of other differentially expressed gene products regulated by p21WAF1/CIP1 in apoptosis remain unclear, certainly some of them are involved in cell cycle regulation, the primary function of p21WAF1/CIP1.

Because p21WAF1/CIP1 is not a transcription factor, it is conceivable that the observed differential expression of the genes in response to p21WAF1/CIP1 results from indirect effects though some transcription factors downstream of p21WAF1/CIP1. The extensive computational search was performed to look for the matched consensus DNA-binding sequences of 759 human transcription factors in the promoter sequences of 41 genes. Interestingly, one transcription factor family (E2F-1, E2F-2, and E2F-4) was found to have the most genes (8 genes) that contain their consensus sequence in the promoter (Table IV). This suggests that genes downstream of p21WAF1/CIP1 may be partially transcriptionally regulated through E2Fs. Induction of p21WAF1/CIP1 could lead to inhibition of cyclins/CDKs activity, resulting in dephosphorylation of retinoblastoma protein (pRB) and inhibition of E2F-mediated transcription (45). Indeed, the adenovirus expressing a C-terminal deletion mutant of p21WAF1/CIP1 enhances E2F-1-mediated apoptosis, independent of p53, in human colon cancer cells in vitro and in vivo (46, 47). Nevertheless, other transcription factors are likely to be involved in transcription regulation as well, especially some apoptosis-associated transcription factors (Table V). For example, the Max protein, together with Myc/Mad network, affects different aspects of cell behavior, including apoptosis, proliferation, and differentiation (48). Retinoic acid receptor-alpha is known to mediate retinoic acid-induced apoptosis (49). Obviously, the molecular understanding of p21WAF1/CIP1-induced apoptosis is further complicated by at least two factors. One is that p21WAF1/CIP1 can trigger apoptosis also by non-transcriptional pathways, and the other is that the p21WAF1/CIP1-induced apoptotic event occurs simultaneously with p21WAF1/CIP1-induced cell arrest event (Figs. 1 and 2).

In summary, our data show that adenovirus expressing p21WAF1/CIP1 can induce p53-independent apoptosis in 2774 and 2774qw1 cells. Interestingly, this process occurs without altered gene expression of BAX and BCL2, whereas B-MYB and alpha B-crystallin may play important roles. Unique to our studies is the ability to integrate two microarray data sets, consisting of genes downstream of p53 and/or p21WAF1/CIP1. Our experiments have extracted genes downstream of p21WAF1/CIP1 that are p53-independent. The finding in this report can serve as a good approach to elucidate further the function(s) of p21WAF1/CIP1, other than its cell cycle arrest property, and discover a novel pathway(s) involving apoptosis and gene product(s) with novel function(s). These data should aid our understanding of p21WAF1/CIP1 cellular activity and augment the role of p21WAF1/CIP1 not only in both cell cycle regulation but also in apoptotic decisions.

    FOOTNOTES

* 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. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Tumor Biology Department, Schering-Plough Research Institute, 2015 Galloping Hill Rd., Kenilworth, NJ 07033. Fax: 908-740-3918; E-mail: suxing.liu@spcorp.com.

Published, JBC Papers in Press, July 22, 2002, DOI 10.1074/jbc.M204962200

2 Incyte Pharmaceuticals, unpublished data.

3 A. Mirza et al., submitted for publication.

4 S. Liu, unpublished data.

    ABBREVIATIONS

The abbreviations used are: rAd-p53, adenovirus expressing p53; rAd-p21, adenovirus expressing p21WAF1/CIP1; rAd-empty vector, adenovirus containing an empty vector; PCNA, proliferating cell nuclear antigen; RT, reverse transcription; FACS, fluorescence-activated cell sorter; TUNEL, terminal dUTP nick-end labeling.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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