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Long-patch base excision repair of apurinic/apyrimidinic site DNA is decreased in mouse embryonic fibroblast cell lines treated with plumbagin: involvement of cyclin-dependent kinase inhibitor p21Waf-1/Cip-1

Abstract

Molecular interactions among cell cycle and DNA repair proteins have been described, but the impact of many of these interactions on cell cycle control and DNA repair remains unclear. The cyclin-dependent kinase inhibitor, p21, is known to be involved in DNA damage-induced cell cycle arrest and blocking DNA replication and repair. Participation of p21 has been implicated in nucleotide excision repair. However, the role of p21 in the base excision repair (BER) pathway has not been thoroughly studied. In the present investigation, we treated isogenic mouse embryonic fibroblast (MEF) cell lines containing wild-type (MEF-polβ) or DNA polymerase β (polβ) gene-knockout (MEFpolβKO) with oxidative DNA-damaging agent, plumbagin, and examined its effect on p21 levels and BER activity. Plumbagin treatment caused a S-G2/M phase arrest and cell death of both MEF cell lines, induced p21 levels, and decreased p21-mediated long-patch (LP) BER by blocking DNA ligase activity in the polβ-dependent pathway and by blocking both FEN1 and DNA ligase activity in polβ-independent pathway. These findings suggest that plumbagin induced p21 levels play a regulatory role in cell cycle arrest, apoptosis, and polβ-dependent and -independent LP-BER pathways in MEF cells.

Introduction

Exogenous and endogenous mutagenic agents attack the genomes of all living cells. DNA bases damaged by these agents may be cytotoxic and/or mutagenic, and thus, they are a major source of intermediates in carcinogenesis (reviewed by Krokan et al., 1997). The base excision repair (BER) systems of both prokaryotes and eukaryotes repair modified bases generated by ionizing radiation, alkylating agents, and endogenous hydrolytic and oxidative processes (reviewed by Seeberg et al., 1995). Biochemical and genetic studies indicate that BER occurs through two sub-pathways that are differentiated by repair gap size and the enzymes involved (reviewed by Wilson, 1998). These sub-pathways are designed as ‘single-nucleotide BER’ also referred to as ‘short patch (SP) BER’, and ‘multi-nucleotide BER’ also referred to as ‘long patch (LP) BER’. In both sub-pathways, repair is initiated by excision of a damaged base by a DNA glycosylase leaving an abasic or AP site in DNA. The resulting AP sites, which are also generated spontaneously or by radiation and chemicals, are subsequently acted upon by an AP endonuclease (APE) to generate a 3′ hydroxyl group and a 5′-deoxyribosephosphate (dPR) terminus. In SP-BER, DNA polymerase β (pol β) extends the 3′ terminus by a single nucleotide and removes the dRP moiety with its dRP lyase activity. Finally, the nick is sealed by DNA ligase I or DNA ligase III/XRCC1 (Srivastava et al., 1998). When AP sites are oxidized or reduced, they become resistant to β-elimination and cannot be excised by the dRP lyase activity of pol β. In these cases, the modified AP site is repaired via the LP-BER pathway where a DNA polymerase (β, δ, or ε) incorporates 2–15 nucleotides displacing the strand containing the modified AP site. This DNA flap structure is cleaved by FEN1 and the nick is sealed by a DNA ligase (Biade et al., 1998; Dianov et al., 1999; Fortini et al., 1998; Frosina et al., 1996; Klungland and Lindahl, 1997; Prasad et al., 2000, 2001). The importance of BER in maintaining genomic integrity and reducing cancer susceptibility is evidenced by the fact that mutations and/or different levels of expression of polβ have been observed in many colon and lung tumors (Bhattacharyya et al., 1999; Canitrot et al., 2000; Srivastava et al., 1999; Wang et al., 1992).

Regulation of DNA replication and repair following DNA damage is critical to mutation avoidance. The cyclin-dependent kinase inhibitor, p21Waf1-Cip1 (hereafter p21), is one protein involved in regulating cell cycle arrest and inhibition of DNA replication following DNA damage providing time for DNA to be repaired. Interactions between p21 and proliferating cell nuclear antigen (PCNA) are believed to be responsible for blocking DNA synthesis (Waga and Stillman, 1998). PCNA stimulates DNA synthesis by acting as a processivity factor for pols δ and ε (reviewed by Jonsson and Hubscher, 1997). In vitro, binding of p21 to PCNA blocks the ability of PCNA to act as a processivity factor for pols δ and ε, modulates the primer-template recognition complex, and inhibits DNA replication. In vivo, the interaction of p21 with PCNA is critical in regulation of DNA replication and cell cycle progression. Residues in the C-terminal domain, amino acids 141–160, of p21 bind to PCNA to inhibit replication and residues in the N-terminal domain bind to cyclin-Cdk complexes to inhibit cell cycle progression (reviewed by Dotto, 2000). PCNA is required for other pathways including LP-BER and has also been shown to interact with and stimulate a number of other proteins required for DNA replication and repair including FEN1, mismatch repair proteins MSH2 and MLH1, the nucleotide excision repair endonuclease XP-G, human DNA-(cytosine-5) methyl transferase, and DNA ligase I (reviewed by Dotto, 2000; Kelman, 1997; Warbrick, 2000). Thus, the possibility exists that p21 inhibits other pathways including LP-BER by binding PCNA.

DNA repair is often coupled with the arrest of cells in G1 and S phase of the cell cycle (Holmquist, 1998). However, prolonged arrest of cells in the G2/M phase of the cell cycle would be more advantageous by providing more time to repair the damaged DNA and posing less threat to genomic integrity than DNA damage present during G1 and S phase of the cell cycle (Nasmyth, 1996). In any case, if the damage is un-repairable, then cell death (apoptosis) provides a fall-back mechanism by which genomic integrity is maintained. In past years, the clinical and in vitro evidence of collateral sensitivity to multiple agents suggested that it is the signaling of DNA damage (the downstream events) that differ in cancer cells, which resulted in chemosensitivity (Bedford et al., 1998; Hill et al., 1994; Koberle et al., 1997; Sark et al., 1995). One of the highly studied DNA damage-induced downstream signaling molecules is p53, which by regulating p21 gene expression causes cell cycle arrest and apoptosis in target cells (Jaiswal and Narayan, 2002; reviewed by Boulaire et al., 2000; Prosperi, 1997; Taylor and Stark, 2001). In previous studies, although controversial (Li et al., 1994a; Mcdonald et al., 1996; Pan et al., 1995; Sheikh et al., 1997; Shivji et al., 1994), the role of p21 is reported in NER (reviewed by Prosperi, 1997). However, whether DNA damage-induced levels of p21 collaborate in cell cycle arrest through BER pathway is not well established.

In the present study, the role of p21 in cell cycle arrest, apoptosis and DNA repair was examined using plumbagin, 5-hydroxy-2-methyl-1,4-napthoquinone, a natural yellow pigment found in plants of the Plumbaginaceae, Droseraceae, Ancistrocladaceae and Dioncophyllaceae families. Plumbagin is one of a series of well-established napthoquinones found in the fleshy husk surrounding the walnuts, a valuable commercial crop in the United States. In common with other napthoquinones, it is an active redox agent. In bacterial assay systems, plumbagin-induced oxidative damage is associated with plumbagin-mediated generation of reactive oxygen species (ROS; Jamieson et al., 1994; Kato et al., 1994; Prieto-Alamo et al., 1993). ROS can react with cellular components, including proteins, lipids, and nucleic acids, to induce DNA adducts such as 8-hydroxy-2′-deoxyguanosine (8-OH-dG) (reviewed by Ames, 1991; Beckman and Ames, 1997). It is believed that base excision repair (BER) pathway predominantly repairs these oxidized bases (Bjoras et al., 1997; reviewed by Brozmanova et al., 2001; Cadet et al., 2000). The DNA-damaging property of plumbagin makes it an effective chemotherapeutic agent. Recently, plumbagin has also been shown to have a protective effect in azoxymethane-induced intestinal carcinogenesis in rats (Sugie et al., 1998). These studies suggest that plumbagin may be a potential chemopreventive agent for human intestinal neoplasia, especially, since minimal toxicity has been observed in the animal model (Sugie et al., 1998). Elucidation of the mechanism(s) by which plumbagin induces anti-tumorigenic and anti-carcinogenic activity is necessary for providing a solid foundation for its use as an agent for therapeutic strategies. In the present study, we hypothesize that ROS generated by plumbagin affects the levels of p21, which in turn regulates enzymes involved in the BER pathway. Depending upon the extent of DNA damage, increase in the p21 level, and efficiency of repair, cells will be either arrested in the cell cycle to enable DNA repair or undergo apoptosis.

Results

Growth arrest and protein levels of p21, polβ, and PCNA of MEF cells treated with plumbagin

In the present studies, to determine the role of polβ-dependent and -independent base excision repair (BER) pathways on DNA damage-induced cell cycle arrest, we used a mouse embryonic fibroblast (MEF) cell line (MEF-polβ) and the matched littermate of polβ gene-knockout cell line (MEF-polβKO). These cell lines were treated with different concentrations of plumbagin for 10 h. Cell cycle profiles were evaluated by propidium iodide staining of nuclei, and the analysis of their distribution in different phases of the cell cycle was done by FACScan flow cytometer. An increased arrest occurred in the S-G2/M phase of both MEF-polβ and MEF-polβKO cell lines after plumbagin treatment (Figure 1a). Treatment of cells with 5 μM plumbagin resulted in increased cell death (population of cells in the sub-G1 phase). A decrease in the number of cells in G2/M phase, an increase in cell death, and no change in the number of cells arrested in S phase was observed for MEF-polβ cells treated with 5 μM plumbagin. On the other hand, in the MEF-polβKO cell line, the cell death was followed directly from the S phase arrested cells treated with 5 μM plumbagin. These results suggest that plumbagin-treated growth arrest of MEF-polβ and MEF-polβKO cell lines occur at the G2/M and S phases of the cell cycle, respectively, which lead to apoptosis at higher concentrations of plumbagin treatment.

Figure 1
figure1

(a) Cell cycle profile of MEF cell lines treated with plumbagin. The ranges for G0/G1, S, G2/M and sub-G1 phase cells were established based upon the corresponding DNA content of the histograms. At least 10 000 cells per sample were considered in the gated regions for calculations. The data are representative of two independent experiments. (b) Plumbagin-induced protein levels in MEF cell lines. The MEF cell lines were treated with different concentrations of plumbagin for 10 h. Cellular extracts were prepared and processed for Western blot analysis for determining the protein levels of p53, p21, polβ and PCNA. Photographs of the autoradiograms are representatives of three independent experiments. A quantitative analysis of p21 protein levels is given in (c). The data are mean±s.e. of three experiments. * and ** are significantly different than the untreated MEF-polβ and MEF-polβKO cell lines, respectively

To determine whether plumbagin treatment affected protein levels of p21 and BER proteins, polβ and PCNA, a Western blot analysis was performed on extracts prepared from MEF cells treated with plumbagin for 10 h. The level of p21 significantly increased in both MEF-polβ and MEF-polβKO cell lines (Figure 1b,c), whereas the levels of PCNA and polβ were unchanged at these concentrations of plumbagin (Figure 1b). In previous studies, we (Jaiswal and Narayan, 2002) and others (reviewed by Boulaire et al., 2000; Prosperi, 1997; Taylor and Stark, 2001) have established that p21 expression in cells is induced in a p53-dependent manner after treatment with DNA-damaging agents. However, the induction of p21 after plumbagin treatment in the MEF cell lines is p53-independent. The p53 protein was not detectable in these MEF cell lines because they expressed the SV40 T-antigen, which is involved in the degradation of p53 (Sobol et al., 2000). In previous studies, it has been clearly indicated that polβ participates in SP- and L-BER pathways, and that both pathways occur simultaneously when examined either with MEF-polβ and MEF-polβKO cell extracts (Fortini et al., 1998) or with purified BER proteins (Klungland and Lindahl, 1997). We used an in vitro BER assay system based on the extracts from MEF-polβ and MEF-polβKO cell lines to determine the effect of plumbagin treatment on SP- or LP-BER in these cells. In these experiments, cells were either treated with 5 μM plumbagin for 10 h or untreated.

Pol β-dependent and -independent BER activity in extracts from plumbagin-treated MEF cell lines

In previous studies, oxidative stress-induced expression of p21 at the mRNA and protein levels has been shown to be p53-independent but MAP kinase-dependent (Esposito et al., 1998). Recently, using 7-ketocholesterol, an oxidizing agent, Agrawal et al. (2002) have shown the role of Stat1 in p21-mediated apoptosis of MEF cells lacking p53. Currently, it is unknown whether similar mechanisms are involved in the plumbagin-induced p21 up-regulation in MEF-polβ and MEF-polβKO cell lines. Two separate double-stranded linear oligonucleotides containing either U residue (U-DNA) or tetrahydrofuran (F) (F-DNA) at the AP site were used as substrates in this assay. The substrate containing U can be repaired by SP-BER, the predominant cellular pathway, whereas the substrate containing F can only be repaired by LP-BER. In our assay system, the DNA repair activities associated with different BER proteins is measured by determining levels of 23-mer incision product formed by APE, 1 nt gap-filling product formed by polβ, strand-displacement products formed by polδ, ε or β, and 63-mer completely repaired product. The SP-BER activity as measured by repair of U-DNA in both MEF-polβ or MEF-polβKO cell extracts was slightly reduced by treatment with plumbagin, as there was a minor change in the 63-mer repaired products (Figure 2a, compare lanes 1 with 2 and 3 with 4).

Figure 2
figure2

Characterization of polβ-dependent and -independent repair of U-DNA and F-DNA in MEF cell extracts. The cell extracts from plumbagin untreated and treated MEF-polβ and MEF-polβKO cell lines were prepared for BER assays as described in Materials and methods. Photographs of the autoradiograms in (a) are representative of three independent experiments. Data shown in (b) are mean±s.e. of three experiments. The comparative P-value of the student t-test is given in the graph. U-DNA, 63-mer double-stranded linear oligonucleotide containing U residue at position 24; F-DNA, 63-mer double-stranded linear oligonucleotide containing tetrahydrofuran, an abasic analog of the natural AP site DNA at position 24. Fully ligated 63-mer products, 1 nt gap-filling product, and 23-mer-incision product are shown with arrows. The strand-displacement products are shown within a bracket

Since plumbagin treatment showed a minor effect on SP-BER, we focused our studies on LP-BER. The LP-BER activity determined with the F-DNA substrate slightly decreased in extracts from plumbagin-treated in comparison with untreated MEF-polβ cell lines (Figure 2a, compare lane 5 with 6). On the other hand, the LP-BER activity significantly decreased in extracts from plumbagin-treated MEF-polβKO cell lines (Figure 2a, compare lane 7 with 8). Our results also show that the polβ-dependent LP-BER activity is higher than polβ-independent activity in MEF-polβ cell line (Figure 2a, compare lane 5 with 7). The low efficiency of polβ-independent LP-BER may be explained partly by the low efficiency of PCNA-dependent BER on the linear DNA (Biade et al., 1998), because ring-shaped PCNA trimer rapidly diffuses off ends of linear DNA.

Interestingly, even though the overall level of SP-BER was unaffected by plumbagin treatment, the distribution of products did change. The polβ-dependent (MEF-polβ cell line) 1 nt gap-filling product increased with plumbagin treatment on both U- and F-DNA substrates (Figure 2a, compare lane 1 with 2 and 5 with 6). This accumulation of 1 nt gap-filling product in the MEF-polβ cell extracts may be due to impaired dRP lyase or DNA ligase activity for the U-DNA substrate, and it may be due to the impaired activities of either FEN 1 or DNA ligase for the F-DNA substrate. The APE-mediated incision of the F-DNA substrate in MEF-polβKO cell extracts was not significantly different in untreated and plumbagin-treated systems (Figure 2a, compare lane 3 with 4 and 7 with 8 for 23-mer incision products). These results indicate that the decreased LP-BER in MEF-polβKO cells after plumbagin treatment affects steps following APE incision.

p21 mimics polβ-dependent and -independent LP-BER activity in plumbagin-treated MEF cell lines

The results from the Western blot analysis (Figure 1) show that p21 levels, not the polβ or PCNA levels, in MEF-polβ and MEF-polβKO cell lines significantly increased, suggesting that this increase is responsible for the decreased LP-BER activity (Figure 2). Furthermore, based on the known interaction of p21 with PCNA (reviewed by Dotto, 2000), we hypothesized that the increased level of p21 is responsible for the decreased LP-BER through PCNA inhibition in both polβ-dependent and polβ-independent pathways. If this is the case, then the addition of purified p21 protein to extracts from untreated cells should mimic the decreased LP-BER activity seen in plumbagin-treated cells. To test this hypothesis, LP-BER assays were supplemented with GST-fusion proteins containing full-length p21 (p21F, amino acids 1–164) the N-terminal end of p21 (p21N, amino acids 1–82), and the C-terminal end of p21 (p21C, amino acids 76–164). Addition of either p21F or p21C proteins caused a decrease in LP-BER of the F-DNA in a dose-dependent manner in both MEF-polβ and MEF-polβKO cell lines (Figure 3). Because the C-terminal domain of p21 is known to bind PCNA (reviewed by Dotto, 2000), our results suggest that p21 blocks LP-BER through interaction with PCNA. A concentration of 500 ng of p21F or p21C proteins mimicked the effects of plumbagin on LP-BER in both MEF-polβ and MEF-polβKO cell lines (Figure 3, compare lane 2 with 6 and 7, and lane 13 with 16 and 19, respectively). In these experiments, precise levels of endogenous plumbagin-induced p21 are difficulte to accurately quantitate using the bacterially expressed p21 as a reference due to the fact that the bacterially expressed protein is not always 100% biologically active. Therefore, in our experiments, the effect of bacterially expressed p21 proteins on BER activity was determined by the addition of different concentrations in the reaction mixture. Based upon a semi-quantitative estimate, it was expected that approximately 2.5-fold higher concentrations of the bacterially expressed p21 proteins would be required to mimic the plumbagin-induced p21 protein levels on the BER activity in the MEF cells (data not shown). The addition of p21F protein resulted in a decreased level of 63-mer ligation and an increased level of 23-mer incision products. In contrast, the p21N protein had no effect on LP-BER in MEF-polβ cells, but significantly decreased LP-BER in MEF-polβKO cells (Figure 3, compare lane 1 with 9–11, and lane 12 with 20–22, respectively). Given that the N-terminal domain of p21 is not known to interact with PCNA, its effect on LP-BER could be due to interaction with other BER proteins in MEF-polβKO cells. Currently, this effect is still not clear. Strand-displacement synthesis products accumulated in extracts from MEF-polβ cells supplemented with p21F or p21C proteins. On the other hand, the level of strand-displacement synthesis products was decreased in MEF-polβKO cells supplemented with p21F or p21C proteins (Figure 3, compare lane 12 with 14–16 and with 17–19, respectively). There was no effect of the p21N protein on the strand-displacement synthesis with the extracts of the MEF-polβKO cells (Figure 3, compare lane 12 with 20–22). Taken together, these results suggest that p21/PCNA may block DNA ligase activity in polβ-dependent pathway causing accumulation of the strand-displacement synthesis products. On the other hand, strand-displacement synthesis, FEN1 or DNA ligase activity may be blocked in polβ-independent pathway.

Figure 3
figure3

Determining the role of p21 on LP-BER using F-DNA as a substrate. The LP-BER reaction mixture with F-DNA was assembled with extracts of untreated and plumbagin-treated MEF-polβ and MEF-polβKO cell lines. To examine the role of p21 in plumbagin-induced system, the purified p21F, p21C, or p21N GST-fusion proteins were supplemented to the extracts from untreated cells. The LP-BER activity was determined with the repair of 32P-labeled F-DNA. (a) Shows a photograph of an autoradiogram that is the representative of three independent experiments. Lanes 3–5, 6–8, and 9–11 were supplemented with increasing amounts (100, 250 and 500 ng) of purified p21F, p21C, or p21N proteins, respectively, in the extracts of untreated MEF-polβ cells. A similar addition of the purified p21 protein was made to the extracts from untreated MEF-polβKO cells as shown in lanes 14–16, 17–19, and 20–22, respectively. Fully ligated 63-mer products, 1 nt gap-filling products, and 23-mer-incision products are shown with arrows. The strand-displacement products are shown within a bracket. A quantitative analysis of the level of the 63-mer ligated products from (a) is given in (b). The data are mean±s.e. of three experiments. * and § is significantly different than the extracts from untreated MEF-polβ and MEF-polβKO cell lines, respectively. ¶ is significantly different than plumbagin-treated versus untreated MEF-polβKO cell extracts

Since previous studies have shown that LP-BER is more efficient on covalently closed circular DNA (cccDNA) than on the linear DNA substrates (Biade et al., 1998; Li et al., 1995), we further validated our results of LP-BER of linear DNA with LP-BER of cccDNA. The pUC19 cccDNA containing random C residues was deaminated by sodium bisulfite (Shortle and Botstein, 1983). The resulting U-cccDNA was treated with uracil-DNA glycosylase (UDG) and then reduced with sodium borohydride to generate reduced AP sites on the cccDNA (R-cccDNA). This substrate is suitable for LP-BER. To examine, whether p21 can inhibit LP-BER on R-cccDNA as on F-DNA, the LP-BER assays were performed with R-cccDNA and extracts from MEF-polβ and MEF-polβKO cell lines supplemented with purified GST-p21 fusion proteins. In this assay system, the level of repaired R-cccDNA decreased in extracts from both MEF-polβ and MEF-polβKO cell lines when treated with plumbagin (Figure 4, compare lane 1 with 2 and lane 6 with 7). Addition of p21F and p21C proteins into the extract from untreated cells blocked LP-BER on R-cccDNA in both MEF-polβ and MEF-polβKO cell lines (Figure 4, compare lane 1 with 3 and 4 and lane 6 with 8 and 9, respectively). Addition of the p21N protein in the extract showed no significant effect on blockage of LP-BER on R-cccDNA in MEF-polβ cells but blocked it in MEF-polβKO cells (Figure 4, compare lane 1 with 5 and lane 6 with 10). Thus, in our assay system, the effect of p21 on LP-BER on F-DNA and R-cccDNA are similar. These results suggest that both F-DNA and R-cccDNA are suitable substrates for determining the role of p21 on LP-BER.

Figure 4
figure4

Determining the role of p21 on LP-BER using R-cccDNA as a substrate. The LP-BER reaction conditions were same as described in Figure 3. A 500 ng of purified GST-fusion proteins were added in the extracts from untreated and plumbagin-treated MEF-polβ and MEF-polβKO cell lines. The LP-BER activity was determined with the repair of R-cccDNA (300 ng) by measuring the incorporation of [α-32P]-dCTP. Photographs of the autoradiogram is representative of two independent experiments

p21 inhibits LP-BER after APE step completion

In the above experiments, accumulation of the 23-mer incision product was consistently observed in LP-BER assays performed with extracts from either the plumbagin-treated or untreated MEF cells supplemented with p21F or p21C proteins. To determine whether the accumulation of 23-mer incision products was the result of a change in the APE incision step or a later step in BER, the 63-mer F-DNA substrate was incised by purified APE. LP-BER assays were then performed on this incised F-DNA substrate using untreated or plumbagin-treated MEF cell extracts with or without supplementation of p21F, p21C, or p21N proteins. Results showed a decreased level of 63-mer ligated products with p21F and p21C proteins in both MEF-polβ and MEF-polβKO cell extracts. The decreased level of 63-mer ligated product correlated with the increased level of 23-mer incised F-DNA substrate in both MEF-polβ and MEF-polβKO cell lines (Figure 5a, compare lane 1 with 2 and 3, and lane 5 with 6 and 7, respectively). Strand-displacement products accumulated in extracts from MEF-polβ cells supplemented with the p21F or the p21C proteins. Because pre-incision of F-DNA with purified APE did not restore LP-BER activity in cell extracts supplemented with p21F or p21C proteins, some step(s) following incision such as strand-displacement synthesis, flap cleavage (FEN1 activity), or DNA ligation must be affected.

Figure 5
figure5

(a) APE does not affect p21-mediated LP-BER in MEF-polβ and MEF-polβKO cell lines. The LP-BER assay was assembled with cell extracts and 32P-labeled F-DNA substrate pre-treated with APE for 60 min at 37°C. The entire APE reaction mixture with DNA was incubated in the LP-BER assay. A 500 ng of the purified p21F, p21C, or p21N protein was added in the extracts from untreated MEF-polβ and MEF-polβKO cell lines before the addition of the APE-treated F-DNA. Photographs of the autoradiograms are representative of three independent experiments. Lanes 9 and 10 show the APE-treated and untreated 32P-labeled F-DNA substrates, respectively. (b) Describes the role of DNA ligation on the p21-mediated LP-BER activity in MEF-polβ and MEF-polβKO cell lines. The LP-BER assay was assembled with extracts from untreated and plumbagin-treated MEF cell lines supplemented with T4 DNA ligase (10, 25 and 50 units, respectively) and 32P-labeled F-DNA substrate. Photographs of the autoradiograms are representative of three independent experiments

DNA ligation step is impaired in p21-mediated LP-BER in MEF cell lines treated with plumbagin

To examine whether DNA ligase activity is inhibited by p21 in MEF-polβ and MEF-polβKO cell lines, LP-BER assays were performed with F-DNA substrate and extracts from untreated or plumbagin-treated cell lines. Before the addition of the F-DNA into the reaction mixture, different concentrations of T4 DNA ligase were added to cell extracts. The BER reaction was carried out for 30 min as before. The data demonstrate that supplementing plumbagin-treated cell extracts with purified T4 DNA ligase restored LP-BER activity (Figure 5b). There was no accumulation of strand-displacement, 1 nt gap-filling or 23-mer incision products that was seen in earlier experiments with purified GST-p21 fusion proteins. Collectively, results from these experiments suggest that plumbagin treatment induces p21 protein level that affects polβ-dependent and -independent LP-BER at the level of DNA ligation.

Discussion

In the present study, we have examined the mechanism of p21 in plumbagin-induced cell cycle arrest and base excision repair of AP site DNA in MEF cell lines with wild-type (MEF-polβ) or polβ gene-knockout (MEF-polβKO) clones. Exposure of cells to plumbagin causes oxidative damage to DNA (Jamieson et al., 1994; Kato et al., 1994; Prieto-Alamo et al., 1993) that can be repaired by LP-BER pathway (reviewed by Ames, 1991; Beckman and Ames, 1997). Our results show an induced level of p21, a S-G2/M phase arrest, and cell death in both MEF-polβ and MEF-polβKO cell lines exposed to plumbagin. In earlier studies, the role of p21 is implicated in DNA damage-induced G1 and G2/M phase arrest of cell cycle (Cayrol et al., 1998; Dulic et al., 1999; Li et al., 1994b). Recently, Ando et al. (2001) reported that p21 participates in G2/M phase arrest in DLD1 human colon carcinoma cell line by interacting with Cdc2/cyclin B1 complex and inhibiting the Cdc2 kinase activity. These authors further report that p21 plays a regulatory role in the maintenance of DNA damage-induced G2/M phase arrest by blocking the interaction of Cdc25C, a dual phosphatase, with PCNA. Cdc25 is critical in the G2/M checkpoint of cells by controlling the phosphorylation status and subsequently, the kinase activity of Cdc2 (reviewed by Shivji et al., 1998). The inhibition of DNA synthesis and cell cycle arrest at G1/S phase transition or in S phase has been suggested due to inhibition of PCNA function and Cdk/cyclin kinase activity, respectively, by p21 (Rousseau et al., 1999). These possibilities will be tested in our future studies.

Next, we examined how plumbagin-induced S-G2/M checkpoint and how cell death is associated with the BER of the AP site DNA. Our target molecules were p21 and PCNA, since both are involved in cell cycle arrest (Cayrol et al., 1998; Dulic et al., 1999; Li et al., 1994b) and DNA repair (reviewed by Dotto, 2000; Jonsson and Hubscher, 1997; Kelman, 1997; Warbrick, 2000; Wilson, 1998). The role of p21 in NER is reported to be through interaction with PCNA; however, it is still unclear whether p21 inhibits (Li et al., 1994a; Shivji et al., 1994, 1998) or creates resistance to NER (Cooper et al., 1999; Pan et al., 1995). In a recent study, it is suggested that p21 is required for NER at a step located downstream of the recruitment of PCNA to DNA repair sites (Stivala et al., 2001). While p21's role in NER is unsettled, its role in other DNA repair pathways including BER is also unclear. DNA re-synthesis during BER is mainly accomplished by polβ and to a lesser extent by pols δ and ε (reviewed by Wilson, 1998). The repair of a U-DNA is completed by the SP-BER and of a reduced or oxidized AP site DNA (resistant to β-elimination) by the LP-BER. The PCNA is involved in LP-BER through its interaction with FEN1 (Kim et al., 1998) and DNA ligase I (Levin et al., 2000). Thus, it is likely that p21 binds with PCNA, and in turn, impairs LP-BER either at the FEN1 or DNA ligase I step. We tested this possibility in plumbagin-induced LP-BER using an AP site analog containing the tetrahydrofuran (F)-modified linear DNA (F-DNA) or cccDNA containing reduced AP site (R-cccDNA) as substrates.

Plumbagin treatment increased p21 protein levels but had no effect on polβ and PCNA levels in both MEF-polβ (polβ-dependent) and MEF-polβKO (polβ-independent) cell lines. LP-BER, not SP-BER, decreased in both MEF-polβ and MEF-polβKO cell lines treated with plumbagin. However, the extent of inhibition of LP-BER activity was more pronounced in MEF-polβKO cell line. Nonetheless, the decreased LP-BER correlated with the increased level of p21. Thus, we postulate that p21 is involved in inhibition of LP-BER in both polβ-dependent and -independent pathways. To demonstrate that decreased LP-BER could result from plumbagin-induced levels of p21 in MEF cell lines, our in vitro BER assay system was supplemented with the purified p21 protein. We found that the supplementation of the p21F or the p21C proteins into the extracts of untreated MEF-polβ cells inhibited the LP-BER of F-DNA or R-cccDNA substrates to similar level as observed with the extracts of plumbagin-treated MEF-polβ cell extracts. The purified p21 protein-mediated decrease in the level of the 63-mer ligated product formation on F-DNA correlates with the accumulation of the strand-displacement and incision products. These results suggest that in polβ-dependent studies, the purified p21 (or plumbagin-induced levels of endogenous p21) can impact multiple steps of LP-BER pathway, i.e., APE, FEN1, or DNA ligase. In the polβ-independent LP-BER, the decreased levels of 63-mer ligated products on F-DNA in response to purified p21 supplementation or plumbagin-induced levels of endogenous p21 were less effective at the strand-displacement synthesis. Instead, the effect was more pronounced at the accumulation of the 23-mer-incision product. These results suggest that in polβ-independent LP-BER pathway, the p21 can have its effect on the APE or the FEN1 activity. It can also have its effect on the pols δ and ε activity since there are plenty of 23-mer-incision products but less strand-displacement products. Our findings ruled out the possible effect of p21/PCNA on the APE activity. In our experimental system, pre-treatment of F-DNA substrate with APE produced similar results on the LP-BER as observed without APE treatment in both MEF-polβ and MEF-polβKO cell lines. This left three possible steps of the LP-BER, i.e., strand-displacement synthesis, FEN1 flap cleavage activity, and DNA ligation at which p21/PCNA may have delivered its effect on the LP-BER after plumbagin treatment.

In earlier studies, the efficient excision of the F-DNA in polβ-dependent LP-BER pathway is reported to be PCNA-dependent, but requires DNA synthesis (Kim et al., 1998). Contrary to this, other reports suggested a role of PCNA-stimulated FEN1 activity in polβ-dependent LP-BER of the reduced AP site (generated with sodium borohydride) or the oxidized AP site (generated by ionizing radiation) (Klungland and Lindahl, 1997). Our results support the first view proposed by Kim et al. (1998). In the polβ-dependent pathway of this proposal, the PCNA is involved at the DNA ligation step and p21 can block PCNA-dependent DNA ligase activity to impair the overall LP-BER of the F-DNA. These results were further confirmed by supplementation of the T4 DNA ligase in the extracts of plumbagin-treated MEF-polβ cell extracts. Our results show that the supplementation of T4 DNA ligase recovered the level of 63-mer ligated product formation to the same level of untreated cell extracts and caused no accumulation of strand-displacement and incision products. Thus, p21/PCNA impairs polβ-dependent LP-BER by blocking DNA ligase activity. These findings support the earlier report in which PCNA is shown to stimulate DNA ligase activity and play a key role in LP-BER (Levin et al., 2000). Another explanation of p21/PCNA-mediated decrease in LP-BER activity in MEF-polβ cell extracts can be given by the interaction of polβ with DNA ligase I (Dimitriadis et al., 1998). Under these circumstances, p21 binding to PCNA interferes with DNA ligase I binding to PCNA. Since DNA ligase I also interacts with polβ, p21 can interfere with polβ-DNA ligase I interaction; thus, the completion of BER. In a recent study, a similar view concerning the role of p21 in polβ-dependent BER has been associated with DNA repair patch size of the natural AP site-mediated BER (Bennett et al., 2001). While this manuscript was in preparation, another report using double-stranded linear DNA substrate demonstrated that p21 inhibits LP-BER in vitro by disrupting PCNA-directed stimulation of FEN1, DNA ligase I, and DNA polymerase δ (Tom et al., 2001).

The plumbagin-induced activity of p21/PCNA on polβ-independent LP-BER is more favorable with the view reported by Klungland and Lindahl (1997). Since the F-DNA is efficiently repaired in extracts from MEF-polβ, as well as, in MEF-polβKO cell lines, a polβ-independent pathway in MEF-polβKO cell line is more likely. This pathway is pols δ and ε-dependent and requires PCNA for its activity (Klungland and Lindahl, 1997). Based on our results, it appears that p21/PCNA interacts at the FEN1 and DNA ligase steps in the polβ-independent LP-BER. No accumulation of the strand-displacement synthesis product and the increased level of 23-mer incision products in the APE pre-treated BER system suggests that p21/PCNA impairs FEN1 activity in plumbagin-treated MEF-polβKO cells. On the other hand, no accumulation of the strand-displacement synthesis, the 23-mer incision product and the recovery of the 63-mer ligated product after supplementation with T4 DNA ligase in extracts of both untreated and plumbagin-treated MEF-polβKO cells indicate that DNA ligase step is impaired in the MEF-polβKO cells. Thus, we conclude that both FEN1 and DNA ligase activity are affected by p21/PCNA in response to plumbagin treatment in MEF-polβKO cells.

In summary, our results provide an evidence that plumbagin-induced levels of p21 arrests S-G2/M phase of cell cycle followed by cell death, which is correlated with decreased activity of LP-BER. Our results suggest that depending upon the polβ level, p21/PCNA interaction with polβ-dependent or -independent LP-BER complexes is favored in the cells in response to DNA damage. Furthermore, the sensitivity of cells to DNA-damaging agents depends upon the type of damage and efficiency of repair. For example, MEF-polβ cells are less sensitive than MEF-polβKO cells in response to treatment with methylmethane sulphonate (MMS) and methylnitosourea (MNU), which induce Sn2-DNA alkylation that is repaired by polβ-dependent LP-BER (Horton et al., 2000; Sobol et al., 1996). In our experimental system, treatment of these cell lines with plumbagin show a similar sensitivity to cell cycle arrest and cell death in MEF-polβ than in MEF-polβKO cell lines. Since repair of oxidative DNA damage-induced by plumbagin is impaired in both cell lines, an accumulation of non-repaired or incompletely repaired DNA damage is acting as a trigger of apoptosis in MEF-polβ and MEF-polβKO cell lines. Thus, the DNA damage-induced p21 levels and DNA repair capacity can be an important consideration for plumbagin, a chemotherapeutic agent-induced cell cycle regulation and apoptosis of cancer cells.

Materials and methods

Cell lines and treatment

The MEF-polβ and MEF-polβKO cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 80 μg/ml hygromycin B at 37°C under a humidified atmosphere of 5% CO2 (Sobol et al., 2000). After cells reached 70% confluence, cells were treated with different concentrations of plumbagin (Sigma Chem. Co., St Louis, MO, USA) for 10 h.

FACS analysis

A detergent and proteolytic enzyme-based technique was used for nuclear isolation and DNA content analysis of cells in different phases of the cell cycle. After treatment with plumbagin, cells were harvested and processed for propidium iodide staining of the nuclei as described earlier (Vindelov and Christensen, 1990). The cellular DNA content was analysed by Becton-Dickinson FACScan flow cytometer. The ranges for G0/G1, S, G2/M, and sub-G1 phase cells were established based upon their corresponding DNA content of histograms (Narayan et al., 2001). At least 10 000 cells per sample were considered in the gated regions used for calculations.

Synthesis of linear double-stranded DNA substrates

The synthetic oligonucleotides were made on an ABI-392 DNA synthesizer using standard β-cyanoethylphosphoramide chemistry and reagents from Glen Research Corp. (Sterling, VA, USA). The nucleotide sequence of the 63-mer long oligonucleotide has a U residue at position 24 (5′-AGATGCCTGCAGCTGATGCGCUGTACGGATCCACGTGTACGGTACCGAGGGCGGGTCGACA-3′), which is referred as U-DNA. To examine the long-patch (LP) BER, we synthesized a 63-mer AP site analog, 3-hydroxy-2-hydroxymethyltetrahydrofuran (F-DNA, 5′-TAGATGCCTGCAGCTGATGCGCFGTACGGATCCACGTGTACGGTACCGAGGGCGGGTCGACA-3′), which is non-susceptible to β-elimination. The gel-purified oligonucleotides were annealed to complimentary oligonucleotides with G residue opposite to the U residue. These oligonucleotides were labeled with γ-32P-ATP at the 5′ end and then annealed with the complementary oligonucleotides with G residue opposite to the AP site. Gel purified double-stranded oligonucleotides were used in BER assays.

Preparation of R-cccDNA substrate

To generate cccDNA with multiple U residues, we used mutagenesis of pUC19 DNA with sodium bisulfite (Shortle and Botstein, 1983). Gel-purified U-cccDNA will be treated with UDG. Then the AP site containing DNAs were treated with 0.1 M sodium borohydride for 30 min on ice. The R-cccDNA was gel-purified and used in LP-BER.

BER assay

To determine the ability of these substrates to undergo polβ-dependent or -independent BER, the BER reaction was assembled at 25°C with MEF-polβ and MEF-polβKO cell line extracts with U-DNA or F-DNA substrates. The BER proficient cell extract was prepared by the procedure of Biade et al. (1998). The reaction mixture in a 20 μl volume contained: 50 mM HEPES, pH 7.9, 2 mM dithiothreitol (DTT), 10 mM MgCl2, 0.5 mM NAD, 5 mM diTris-phosphocreatine, 6 U of phosphocreatinekinase, 20 μM of ATP, and 20 μM each of dNTPs. The BER was initiated by the addition of 5–10 ng of 32P-labeled U-DNA or F-DNA substrates (Biade et al., 1998; Prasad et al., 2001). When R-cccDNA was used as a substrate then the reaction was initiated by the addition of [α-32P]dCTP. The BER reaction was stopped by adding 0.4% (w/v) SDS (final concentration), 2 μg of proteinase K and 10 μg of carrier tRNA. They were incubated again for 30 min at 37°C. The DNA was recovered by chloroform/phenol extraction and ethanol precipitation and analysed on a 7 M urea-containing 12% (w/v) acrylamide gel. After electrophoresis, the gel was dried and the amount of radioactivity in the repaired DNA was quantified with InstantImager (Packard Instrument Co., Meriden, CT, USA).

Western blot analysis

To determine a change in the protein levels, a Western blot analysis was performed with proteins from extracts of untreated or plumbagin-treated cells as previously described (Narayan and Jaiswal, 1997). The following antibodies from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) were used in this study: p53, p21 and PCNA. Sam Wilson provided the anti-polβ antibody (Sobol et al., 2000).

Purification of GST-p21

The p21F, p21C, and p21N GST-fusion proteins were purified as described by Fotedar et al. (1996). Briefly, the amplified bacterial cultures with over-expressed GST-p21 proteins with isopropyl-1-thio-β-D-galactopyranoside (IPTG) were pelleted and suspended in the ice-cold buffer containing: 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM KCl, 0.5% NP-40, 1 mM DTT, 1 mM PMSF, 1.5% sarkosyl, 2 mg/ml of benzamidine, 2 μg/ml of leupeptin and pepstatin A. Cells were sonicated for 30 s bursts, chilled on ice, and freeze–thawed for three times. Into the clarified lysates, slurry of glutathione-Sepharose beads was added and incubated for 1 h at 4°C. After extensive washing of the beads, the GST-p21 proteins were eluted in a buffer containing: 50 mM Tris-HCl, pH 9.5, 20 mM reduced-glutathione and 120 mM NaCl. Samples were dialyzed for 6 h with one change of the buffer containing: 20 mM Tris-HCl, pH 8, 1 mM EDTA, 10 mM NaCl, 1 mM DTT and 10% glycerol. Purified proteins were quantified and stored at −80°C in small aliquots for future studies.

Abbreviations

AP:

apurinic/apyrimidinic

APE:

AP endonuclease

bp:

base pair

BER:

base excision repair

cccDNA:

covalently closed circular DNA

dRP:

2′-deoxyribose 5′-phosphate

dRPase:

2′-deoxyribose 5′-phosphatase

FEN1:

flap endonuclease 1

LP:

long-patch

MEF:

mouse embryonic fibroblast cell line

PAGE:

polyacrylamide gel electrophoresis

polβ:

DNA polymerase β

polβKO:

polβ-gene knocked out

PCNA:

proliferating cell nuclear antigen

SP:

short-patch

UDG:

uracil-DNA glycosylase

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Acknowledgements

We would like to thank Drs Rajendra Prasad and Samuel H Wilson from NIEHS, Research Triangle Park, NC, for providing the neutralizing anti-polβ antibody and the mouse embryonic fibroblast (MEF) cell lines with wild-type and polβ-gene-knockout clones; Dr Arun Fotedar from the Cellular and Molecular Biology Program, Sidney Kimmel Cancer Center (San Diego, CA, USA), for providing us the wild-type and mutant GST-p21 protein overexpression vectors. We thank Jessica A Salinas for technical assistance and Nirupama Gupta for editorial comments. The FACS analysis was performed in the FCC Laboratory of the ICBR of the University of Florida. These studies were supported in part by the grants awarded to S Narayan from the National Cancer Institute, NIH (CA77721); the 2001-Research Opportunity Fund by the University of Florida (Gainesville, FL, USA); and the 2001-Ralph E Powe Junior Faculty Enhancement Award by the Oak Ridge Associated Universities (Oak Ridge, TN, USA). The work of LB Bloom was supported by NSF grant MCB-9722356.

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Correspondence to Satya Narayan.

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Jaiswal, A., Bloom, L. & Narayan, S. Long-patch base excision repair of apurinic/apyrimidinic site DNA is decreased in mouse embryonic fibroblast cell lines treated with plumbagin: involvement of cyclin-dependent kinase inhibitor p21Waf-1/Cip-1. Oncogene 21, 5912–5922 (2002). https://doi.org/10.1038/sj.onc.1205789

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Keywords

  • DNA damage
  • cell cycle arrest
  • p21
  • long-patch BER

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