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Dual regulatory roles of human AP-endonuclease (APE1/Ref-1) in CDKN1A/p21 expression.

Sengupta S, Mitra S, Bhakat KK - PLoS ONE (2013)

Bottom Line: Interestingly, APE1 and AP4 showed mutual dependence for p21 repression.Moreover, ectopic p53 in p53- cells inhibited AP4's association with APE1, their binding to the promoter and p21 repression.These results together establish APE1's role as a co-activator or co-repressor of p21 gene, dependent on p53 status.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, United States of America.

ABSTRACT
The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein involved in repair of oxidative DNA damage as well as in transcriptional regulation, is often overexpressed in tumor cells. APE1 was earlier shown to stimulate p53's DNA binding and its transactivation function in the expression of cyclin-dependent kinase inhibitor p21 (CDKN1A) gene. Here, we show APE1's stable binding to p53 cis elements which are required for p53-mediated activation of p21 in p53-expressing wild type HCT116 cells. However, surprisingly, we observed APE1-dependent repression of p21 in isogenic p53- HCT116 cells. Ectopic expression of p53 in the p53- cells abrogated this repression suggesting that APE1's negative regulatory role in p21 expression is dependent on the p53 status. We then identified APE1's another binding site in p21's proximal promoter region containing cis elements for AP4, a repressor of p21. Interestingly, APE1 and AP4 showed mutual dependence for p21 repression. Moreover, ectopic p53 in p53- cells inhibited AP4's association with APE1, their binding to the promoter and p21 repression. These results together establish APE1's role as a co-activator or co-repressor of p21 gene, dependent on p53 status. It is thus likely that APE1 overexpression and inactivation of p53, often observed in tumor cells, promote tumor cell proliferation by constitutively downregulating p21 expression.

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Repression of p21 by APE1 in p53- cells and effect of ectopic p53 in this repression.(A & B) Real Time RT-PCR analysis showing relative quantitation of p21 transcript level in (A) HCT116p53 cells with WT and NΔ42 APE1 overexpression; *: p value (n = 4) calculated from control (empty vector transfection) vs. WT or NΔ42 APE1 overexpression, and (B) control (control siRNA) vs. APE1-depleted HCT116p53 cells; *: p value <0.05 (n = 4) calculated from control vs. APE1-depleted cells. (C) Effect of ectopic p53 expression on p21 transcript level in control vs. APE1-depleted HCT116p53 cells. First, cells were transfected with control siRNA or APE1 siRNA, the next day both the cell types were again transfected with empty vector or p53 expression vector and after 48 hrs the cells were harvested; signal from empty vector transfection in both control and APE1-depleted cells were set as reference samples; *: p value <0.05 (n = 3) calculated based on the effect of ectopic p53 expression over empty vector transfection in control vs. APE1-depleted cells. (D) Effect of APE1 depletion in control (empty vector transfected) vs. ectopic p53-expressing HCT116p53 cells; the same experiment was performed as in C but analyzed differently; signal from control siRNA-transfected cells in both empty vector transfected and ectopic p53 expressing cases were set as reference samples; *: p value <0.05 (n = 3) calculated based on the effect of APE1-depletion in empty vector transfected vs. ectopic p53 expressing cells. (E) Representative Western analysis of p53, APE1, p21 and α-Tubulin levels in the same HCT116p53 cells as in B–D. (F & G) Real Time RT-PCR analysis of p21 level in Saos2 cells as in C & D. *: p value <0.05 (n = 2).
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pone-0068467-g003: Repression of p21 by APE1 in p53- cells and effect of ectopic p53 in this repression.(A & B) Real Time RT-PCR analysis showing relative quantitation of p21 transcript level in (A) HCT116p53 cells with WT and NΔ42 APE1 overexpression; *: p value (n = 4) calculated from control (empty vector transfection) vs. WT or NΔ42 APE1 overexpression, and (B) control (control siRNA) vs. APE1-depleted HCT116p53 cells; *: p value <0.05 (n = 4) calculated from control vs. APE1-depleted cells. (C) Effect of ectopic p53 expression on p21 transcript level in control vs. APE1-depleted HCT116p53 cells. First, cells were transfected with control siRNA or APE1 siRNA, the next day both the cell types were again transfected with empty vector or p53 expression vector and after 48 hrs the cells were harvested; signal from empty vector transfection in both control and APE1-depleted cells were set as reference samples; *: p value <0.05 (n = 3) calculated based on the effect of ectopic p53 expression over empty vector transfection in control vs. APE1-depleted cells. (D) Effect of APE1 depletion in control (empty vector transfected) vs. ectopic p53-expressing HCT116p53 cells; the same experiment was performed as in C but analyzed differently; signal from control siRNA-transfected cells in both empty vector transfected and ectopic p53 expressing cases were set as reference samples; *: p value <0.05 (n = 3) calculated based on the effect of APE1-depletion in empty vector transfected vs. ectopic p53 expressing cells. (E) Representative Western analysis of p53, APE1, p21 and α-Tubulin levels in the same HCT116p53 cells as in B–D. (F & G) Real Time RT-PCR analysis of p21 level in Saos2 cells as in C & D. *: p value <0.05 (n = 2).

Mentions: In contrast to our results about APE1’s involvement as a co-activator in p53-mediated p21 activation in HCT116WT cells, we observed the opposite role of APE1 in the isogenic p53- (HCT116p53) cells. Overexpression of WT APE1 but not NΔ42 mutant decreased endogenous p21 mRNA level (Fig. 3A). Furthermore, APE1 downregulation enhanced p21 expression (Fig. 3B). These results suggest that APE1 can regulate p21 expression both positively and negatively, depending on the status of p53 in the cell. We then tested the effect of ectopic p53 in APE1’s regulation of p21 expression in p53 cells. As expected ectopic expression of p53 increased p21 level in these cells, and interestingly we could not observe any significant effect of p53 expression on p21 activation in APE1-downregulated cells (Fig. 3C). The reference samples in both control cells and APE1-depleted cells are empty vector transfected cells and the effect of p53 overexpression in these two cell types were measured. Because APE1 had opposite effects on p21 expression in HCT116WT vs. HCT116p53 cells, we asked whether ectopic expression of p53 in HCT116p53 cells could abrogate APE1’s repressor function. In empty vector transfected cells APE1-depletion could activate p21 mRNA level and this activation was inhibited in ectopically p53-expressing cells (Fig. 3D). In both empty vector transfected and ectopically p53-expressing cells, the reference samples were with control siRNA transfection and the effect of APE1 depletion was measured. The effect of APE1 overexpression and depletion in p53- and ectopically-expressing cells on p21 regulation was also reflected to some extent at the protein level (Fig. 3E). To confirm that APE1’s repressor activity for p21 expression in the absence of p53 is a general phenomenon, we used another p53- cell of different origin, namely Saos-2 osteosarcoma line. Fig. 3F showed that ectopic expression of p53 activated p21 expression which was inhibited by APE1 depletion (as observed for HCT116p53 cells in Fig. 3C). On the other hand, Fig. 3G showed that APE1 downregulation in these p53-negative cells enhanced p21 mRNA level and this activation was prevented by ectopic p53 (as observed for HCT116p53 cells in Fig. 3D). Thus, the negative regulatory role of APE1 for p21 expression is evident only in the absence of p53. These results together indicate that APE1 can function both as a co-activator or a co-repressor for p21 expression which is dependent on p53 status.


Dual regulatory roles of human AP-endonuclease (APE1/Ref-1) in CDKN1A/p21 expression.

Sengupta S, Mitra S, Bhakat KK - PLoS ONE (2013)

Repression of p21 by APE1 in p53- cells and effect of ectopic p53 in this repression.(A & B) Real Time RT-PCR analysis showing relative quantitation of p21 transcript level in (A) HCT116p53 cells with WT and NΔ42 APE1 overexpression; *: p value (n = 4) calculated from control (empty vector transfection) vs. WT or NΔ42 APE1 overexpression, and (B) control (control siRNA) vs. APE1-depleted HCT116p53 cells; *: p value <0.05 (n = 4) calculated from control vs. APE1-depleted cells. (C) Effect of ectopic p53 expression on p21 transcript level in control vs. APE1-depleted HCT116p53 cells. First, cells were transfected with control siRNA or APE1 siRNA, the next day both the cell types were again transfected with empty vector or p53 expression vector and after 48 hrs the cells were harvested; signal from empty vector transfection in both control and APE1-depleted cells were set as reference samples; *: p value <0.05 (n = 3) calculated based on the effect of ectopic p53 expression over empty vector transfection in control vs. APE1-depleted cells. (D) Effect of APE1 depletion in control (empty vector transfected) vs. ectopic p53-expressing HCT116p53 cells; the same experiment was performed as in C but analyzed differently; signal from control siRNA-transfected cells in both empty vector transfected and ectopic p53 expressing cases were set as reference samples; *: p value <0.05 (n = 3) calculated based on the effect of APE1-depletion in empty vector transfected vs. ectopic p53 expressing cells. (E) Representative Western analysis of p53, APE1, p21 and α-Tubulin levels in the same HCT116p53 cells as in B–D. (F & G) Real Time RT-PCR analysis of p21 level in Saos2 cells as in C & D. *: p value <0.05 (n = 2).
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pone-0068467-g003: Repression of p21 by APE1 in p53- cells and effect of ectopic p53 in this repression.(A & B) Real Time RT-PCR analysis showing relative quantitation of p21 transcript level in (A) HCT116p53 cells with WT and NΔ42 APE1 overexpression; *: p value (n = 4) calculated from control (empty vector transfection) vs. WT or NΔ42 APE1 overexpression, and (B) control (control siRNA) vs. APE1-depleted HCT116p53 cells; *: p value <0.05 (n = 4) calculated from control vs. APE1-depleted cells. (C) Effect of ectopic p53 expression on p21 transcript level in control vs. APE1-depleted HCT116p53 cells. First, cells were transfected with control siRNA or APE1 siRNA, the next day both the cell types were again transfected with empty vector or p53 expression vector and after 48 hrs the cells were harvested; signal from empty vector transfection in both control and APE1-depleted cells were set as reference samples; *: p value <0.05 (n = 3) calculated based on the effect of ectopic p53 expression over empty vector transfection in control vs. APE1-depleted cells. (D) Effect of APE1 depletion in control (empty vector transfected) vs. ectopic p53-expressing HCT116p53 cells; the same experiment was performed as in C but analyzed differently; signal from control siRNA-transfected cells in both empty vector transfected and ectopic p53 expressing cases were set as reference samples; *: p value <0.05 (n = 3) calculated based on the effect of APE1-depletion in empty vector transfected vs. ectopic p53 expressing cells. (E) Representative Western analysis of p53, APE1, p21 and α-Tubulin levels in the same HCT116p53 cells as in B–D. (F & G) Real Time RT-PCR analysis of p21 level in Saos2 cells as in C & D. *: p value <0.05 (n = 2).
Mentions: In contrast to our results about APE1’s involvement as a co-activator in p53-mediated p21 activation in HCT116WT cells, we observed the opposite role of APE1 in the isogenic p53- (HCT116p53) cells. Overexpression of WT APE1 but not NΔ42 mutant decreased endogenous p21 mRNA level (Fig. 3A). Furthermore, APE1 downregulation enhanced p21 expression (Fig. 3B). These results suggest that APE1 can regulate p21 expression both positively and negatively, depending on the status of p53 in the cell. We then tested the effect of ectopic p53 in APE1’s regulation of p21 expression in p53 cells. As expected ectopic expression of p53 increased p21 level in these cells, and interestingly we could not observe any significant effect of p53 expression on p21 activation in APE1-downregulated cells (Fig. 3C). The reference samples in both control cells and APE1-depleted cells are empty vector transfected cells and the effect of p53 overexpression in these two cell types were measured. Because APE1 had opposite effects on p21 expression in HCT116WT vs. HCT116p53 cells, we asked whether ectopic expression of p53 in HCT116p53 cells could abrogate APE1’s repressor function. In empty vector transfected cells APE1-depletion could activate p21 mRNA level and this activation was inhibited in ectopically p53-expressing cells (Fig. 3D). In both empty vector transfected and ectopically p53-expressing cells, the reference samples were with control siRNA transfection and the effect of APE1 depletion was measured. The effect of APE1 overexpression and depletion in p53- and ectopically-expressing cells on p21 regulation was also reflected to some extent at the protein level (Fig. 3E). To confirm that APE1’s repressor activity for p21 expression in the absence of p53 is a general phenomenon, we used another p53- cell of different origin, namely Saos-2 osteosarcoma line. Fig. 3F showed that ectopic expression of p53 activated p21 expression which was inhibited by APE1 depletion (as observed for HCT116p53 cells in Fig. 3C). On the other hand, Fig. 3G showed that APE1 downregulation in these p53-negative cells enhanced p21 mRNA level and this activation was prevented by ectopic p53 (as observed for HCT116p53 cells in Fig. 3D). Thus, the negative regulatory role of APE1 for p21 expression is evident only in the absence of p53. These results together indicate that APE1 can function both as a co-activator or a co-repressor for p21 expression which is dependent on p53 status.

Bottom Line: Interestingly, APE1 and AP4 showed mutual dependence for p21 repression.Moreover, ectopic p53 in p53- cells inhibited AP4's association with APE1, their binding to the promoter and p21 repression.These results together establish APE1's role as a co-activator or co-repressor of p21 gene, dependent on p53 status.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, United States of America.

ABSTRACT
The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein involved in repair of oxidative DNA damage as well as in transcriptional regulation, is often overexpressed in tumor cells. APE1 was earlier shown to stimulate p53's DNA binding and its transactivation function in the expression of cyclin-dependent kinase inhibitor p21 (CDKN1A) gene. Here, we show APE1's stable binding to p53 cis elements which are required for p53-mediated activation of p21 in p53-expressing wild type HCT116 cells. However, surprisingly, we observed APE1-dependent repression of p21 in isogenic p53- HCT116 cells. Ectopic expression of p53 in the p53- cells abrogated this repression suggesting that APE1's negative regulatory role in p21 expression is dependent on the p53 status. We then identified APE1's another binding site in p21's proximal promoter region containing cis elements for AP4, a repressor of p21. Interestingly, APE1 and AP4 showed mutual dependence for p21 repression. Moreover, ectopic p53 in p53- cells inhibited AP4's association with APE1, their binding to the promoter and p21 repression. These results together establish APE1's role as a co-activator or co-repressor of p21 gene, dependent on p53 status. It is thus likely that APE1 overexpression and inactivation of p53, often observed in tumor cells, promote tumor cell proliferation by constitutively downregulating p21 expression.

Show MeSH
Related in: MedlinePlus