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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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APE/ref-1 protein levels in control and gemcitabine-treated MiaPaCa and Panc-1 monolayers. (A) Representative flow cytometry data for MiaPaCa cells treated with 60 μM gemcitabine for 48 h compared to untreated control, autofluorescence background and calibration beads. Gated on light scatter to exclude dead cells. (B) APE/ref-1 protein levels in MiaPaCa and Panc-1 controls and cells treated with 20, 40 and 60  μm gemcitabine for 48 h, expressed as mean equivalent fluorescein (MEFL) values obtained from the calibration beads. Bars represent means from three separate experiments±s.e. Stars indicate results that are statistically significant (P<0.05) with respect to the nontreated samples.
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fig1: APE/ref-1 protein levels in control and gemcitabine-treated MiaPaCa and Panc-1 monolayers. (A) Representative flow cytometry data for MiaPaCa cells treated with 60 μM gemcitabine for 48 h compared to untreated control, autofluorescence background and calibration beads. Gated on light scatter to exclude dead cells. (B) APE/ref-1 protein levels in MiaPaCa and Panc-1 controls and cells treated with 20, 40 and 60  μm gemcitabine for 48 h, expressed as mean equivalent fluorescein (MEFL) values obtained from the calibration beads. Bars represent means from three separate experiments±s.e. Stars indicate results that are statistically significant (P<0.05) with respect to the nontreated samples.

Mentions: Cells were adjusted to 1 × 106 cells ml−1 and fixed with 2% formaldehyde for 10 min at 37°C, followed by 90% ice-cold methanol for 30 min. Prior to antibody staining, cells were rinsed with 2 ml phosphate-buffered saline (PBS) containing 4% FBS. Based on a preliminary dilution curve showing that this concentration is saturating, 1 : 1000 dilution of APE/ref-1 antibody (Novus Biologicals, Littleton, CO, USA) was added to the pellet for 30 min at room temperature, then washed twice in PBS plus 4% FBS. A goat-anti-mouse FITC-labelled secondary antibody (Caltag Laboratories, Burlingame, CA, USA) was added to the pellet for 15 min at room temperature, and the sample washed twice with PBS plus 4% FBS and resuspended in 1 ml of PBS plus 4% FBS. Samples were analysed by flow cytometry (Epics Elite; Beckman-Coulter, Miami, FL, USA) using 488 nm excitation and collecting through a 525 nm bandpass filter. To standardise the flow cytometry results, calibration particles (Rainbow Beads; Spherotech Inc., Libertyville, IL, USA) were measured in each sample run. A calibration curve was generated based on the mean fluorescence values for the Rainbow Beads, as illustrated in Figure 1Figure 1


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)

APE/ref-1 protein levels in control and gemcitabine-treated MiaPaCa and Panc-1 monolayers. (A) Representative flow cytometry data for MiaPaCa cells treated with 60 μM gemcitabine for 48 h compared to untreated control, autofluorescence background and calibration beads. Gated on light scatter to exclude dead cells. (B) APE/ref-1 protein levels in MiaPaCa and Panc-1 controls and cells treated with 20, 40 and 60  μm gemcitabine for 48 h, expressed as mean equivalent fluorescein (MEFL) values obtained from the calibration beads. Bars represent means from three separate experiments±s.e. Stars indicate results that are statistically significant (P<0.05) with respect to the nontreated samples.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747714&req=5

fig1: APE/ref-1 protein levels in control and gemcitabine-treated MiaPaCa and Panc-1 monolayers. (A) Representative flow cytometry data for MiaPaCa cells treated with 60 μM gemcitabine for 48 h compared to untreated control, autofluorescence background and calibration beads. Gated on light scatter to exclude dead cells. (B) APE/ref-1 protein levels in MiaPaCa and Panc-1 controls and cells treated with 20, 40 and 60  μm gemcitabine for 48 h, expressed as mean equivalent fluorescein (MEFL) values obtained from the calibration beads. Bars represent means from three separate experiments±s.e. Stars indicate results that are statistically significant (P<0.05) with respect to the nontreated samples.
Mentions: Cells were adjusted to 1 × 106 cells ml−1 and fixed with 2% formaldehyde for 10 min at 37°C, followed by 90% ice-cold methanol for 30 min. Prior to antibody staining, cells were rinsed with 2 ml phosphate-buffered saline (PBS) containing 4% FBS. Based on a preliminary dilution curve showing that this concentration is saturating, 1 : 1000 dilution of APE/ref-1 antibody (Novus Biologicals, Littleton, CO, USA) was added to the pellet for 30 min at room temperature, then washed twice in PBS plus 4% FBS. A goat-anti-mouse FITC-labelled secondary antibody (Caltag Laboratories, Burlingame, CA, USA) was added to the pellet for 15 min at room temperature, and the sample washed twice with PBS plus 4% FBS and resuspended in 1 ml of PBS plus 4% FBS. Samples were analysed by flow cytometry (Epics Elite; Beckman-Coulter, Miami, FL, USA) using 488 nm excitation and collecting through a 525 nm bandpass filter. To standardise the flow cytometry results, calibration particles (Rainbow Beads; Spherotech Inc., Libertyville, IL, USA) were measured in each sample run. A calibration curve was generated based on the mean fluorescence values for the Rainbow Beads, as illustrated in Figure 1Figure 1

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