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Effects of gemcitabine on APE/ref-1 endonuclease activity in pancreatic cancer cells, and the therapeutic potential of antisense oligonucleotides.

Lau JP, Weatherdon KL, Skalski V, Hedley DW - Br. J. Cancer (2004)

Bottom Line: To test this hypothesis, we examined the effects of disrupting APE/ref-1 using antisense on gemcitabine toxicity.Antisense oligonucleotides decreased protein levels three-fold in MiaPaCa-2 and five-fold in Panc-1 in comparison to controls, associated with reduced endonuclease activity.While clonogenic assays showed only slight decreases in colony formation in cells treated with either antisense oligonucleotides or gemcitabine alone, the combination with APE/ref-1 antisense resulted in a 2-log enhancement of gemcitabine toxicity in Panc-1 cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Therapeutics, Ontario Cancer Institute/Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9.

ABSTRACT
Apurinic/apyrimidinic endonuclease (APE) is a key enzyme involved in DNA base excision repair (BER) that is often expressed at elevated levels in human cancers. Pancreatic cancer cells treated with the nucleoside analogue gemcitabine (2', 2'-difluoro-2'deoxycytidine) showed increases in APE/redox effector factor (ref-1) protein levels (approximately two-fold for Panc-1 and six-fold for MiaPaCa-2), with corresponding increases in endonuclease activity. These results suggested that the activation of APE/ref-1 might be an adaptive response that contributes to gemcitabine resistance by facilitating BER. To test this hypothesis, we examined the effects of disrupting APE/ref-1 using antisense on gemcitabine toxicity. Antisense oligonucleotides decreased protein levels three-fold in MiaPaCa-2 and five-fold in Panc-1 in comparison to controls, associated with reduced endonuclease activity. Combination treatments with antisense oligonucleotides and gemcitabine partially suppressed the increase in APE/ref-1 activity seen in cells exposed to gemcitabine alone. While clonogenic assays showed only slight decreases in colony formation in cells treated with either antisense oligonucleotides or gemcitabine alone, the combination with APE/ref-1 antisense resulted in a 2-log enhancement of gemcitabine toxicity in Panc-1 cells. Overall these findings suggest that APE/ref-1 plays a significant role in gemcitabine resistance in some pancreatic cancer cells, and support the further investigation of novel treatments that target this protein.

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Endonuclease activity present in gemcitabine-treated cells. (A) Film images of 26-mer substrate vs 14-mer product in MiaPaCa and Panc-1 cells after various doses of gemcitabine for 48 h. Positive controls consisted of purified recombinant APE/ref-1 protein and negative controls included the BER inhibitor methoxyamine. (B) Range of gemcitabine concentrations vs the fraction of 14-mer product formed. Stars indicate results that are statistically significant (P<0.05) with respect to the nontreated samples. The concentration of gemcitabine is expressed as μM. All values are means of three independent experiments±s.e.
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fig2: Endonuclease activity present in gemcitabine-treated cells. (A) Film images of 26-mer substrate vs 14-mer product in MiaPaCa and Panc-1 cells after various doses of gemcitabine for 48 h. Positive controls consisted of purified recombinant APE/ref-1 protein and negative controls included the BER inhibitor methoxyamine. (B) Range of gemcitabine concentrations vs the fraction of 14-mer product formed. Stars indicate results that are statistically significant (P<0.05) with respect to the nontreated samples. The concentration of gemcitabine is expressed as μM. All values are means of three independent experiments±s.e.

Mentions: To examine the effects on endonuclease activity, MiaPaCa-2 and Panc-1 cells were treated with various concentrations of gemcitabine for 48 h, then lysed and assayed for catalytic activity. More 14-mer product was formed, with a corresponding decrease in 26-mer substrate, at increasing concentrations of gemcitabine (Figure 2AFigure 2


Effects of gemcitabine on APE/ref-1 endonuclease activity in pancreatic cancer cells, and the therapeutic potential of antisense oligonucleotides.

Lau JP, Weatherdon KL, Skalski V, Hedley DW - Br. J. Cancer (2004)

Endonuclease activity present in gemcitabine-treated cells. (A) Film images of 26-mer substrate vs 14-mer product in MiaPaCa and Panc-1 cells after various doses of gemcitabine for 48 h. Positive controls consisted of purified recombinant APE/ref-1 protein and negative controls included the BER inhibitor methoxyamine. (B) Range of gemcitabine concentrations vs the fraction of 14-mer product formed. Stars indicate results that are statistically significant (P<0.05) with respect to the nontreated samples. The concentration of gemcitabine is expressed as μM. All values are means of three independent experiments±s.e.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747714&req=5

fig2: Endonuclease activity present in gemcitabine-treated cells. (A) Film images of 26-mer substrate vs 14-mer product in MiaPaCa and Panc-1 cells after various doses of gemcitabine for 48 h. Positive controls consisted of purified recombinant APE/ref-1 protein and negative controls included the BER inhibitor methoxyamine. (B) Range of gemcitabine concentrations vs the fraction of 14-mer product formed. Stars indicate results that are statistically significant (P<0.05) with respect to the nontreated samples. The concentration of gemcitabine is expressed as μM. All values are means of three independent experiments±s.e.
Mentions: To examine the effects on endonuclease activity, MiaPaCa-2 and Panc-1 cells were treated with various concentrations of gemcitabine for 48 h, then lysed and assayed for catalytic activity. More 14-mer product was formed, with a corresponding decrease in 26-mer substrate, at increasing concentrations of gemcitabine (Figure 2AFigure 2

Bottom Line: To test this hypothesis, we examined the effects of disrupting APE/ref-1 using antisense on gemcitabine toxicity.Antisense oligonucleotides decreased protein levels three-fold in MiaPaCa-2 and five-fold in Panc-1 in comparison to controls, associated with reduced endonuclease activity.While clonogenic assays showed only slight decreases in colony formation in cells treated with either antisense oligonucleotides or gemcitabine alone, the combination with APE/ref-1 antisense resulted in a 2-log enhancement of gemcitabine toxicity in Panc-1 cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Therapeutics, Ontario Cancer Institute/Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9.

ABSTRACT
Apurinic/apyrimidinic endonuclease (APE) is a key enzyme involved in DNA base excision repair (BER) that is often expressed at elevated levels in human cancers. Pancreatic cancer cells treated with the nucleoside analogue gemcitabine (2', 2'-difluoro-2'deoxycytidine) showed increases in APE/redox effector factor (ref-1) protein levels (approximately two-fold for Panc-1 and six-fold for MiaPaCa-2), with corresponding increases in endonuclease activity. These results suggested that the activation of APE/ref-1 might be an adaptive response that contributes to gemcitabine resistance by facilitating BER. To test this hypothesis, we examined the effects of disrupting APE/ref-1 using antisense on gemcitabine toxicity. Antisense oligonucleotides decreased protein levels three-fold in MiaPaCa-2 and five-fold in Panc-1 in comparison to controls, associated with reduced endonuclease activity. Combination treatments with antisense oligonucleotides and gemcitabine partially suppressed the increase in APE/ref-1 activity seen in cells exposed to gemcitabine alone. While clonogenic assays showed only slight decreases in colony formation in cells treated with either antisense oligonucleotides or gemcitabine alone, the combination with APE/ref-1 antisense resulted in a 2-log enhancement of gemcitabine toxicity in Panc-1 cells. Overall these findings suggest that APE/ref-1 plays a significant role in gemcitabine resistance in some pancreatic cancer cells, and support the further investigation of novel treatments that target this protein.

Show MeSH
Related in: MedlinePlus