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Increased expression of cyclin E is associated with an increased resistance to doxorubicin in rat fibroblasts.

Sgambato A, Camerini A, Pani G, Cangiano R, Faraglia B, Bianchino G, De Bari B, Galeotti T, Cittadini A - Br. J. Cancer (2003)

Bottom Line: Cyclin E-overexpressing fibroblasts also displayed a reduction in ROS levels and a significantly lower increase following doxorubicin treatment compared with vector control cells.The expression of manganese superoxide dismutase (MnSOD) and its activity were increased (about 1.3-fold) in cyclin E-overexpressing derivatives compared with control cells.These results suggest that cyclin E overexpression might reduce tumour cells sensitivity to doxorubicin by affecting the expression of MnSOD and that determination of cyclin E expression levels might help to select patients to be treated with an anthracycline-based antineoplastic therapy.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Patologia Generale, Centro di Ricerche Oncologiche 'Giovanni XXIII', Catholic University, Largo Francesco Vito 1, 00168 Rome, Italy. asgambato@rm.unicatt.it

ABSTRACT
Cell cycle progression in eukaryotic cells is regulated by a family of cyclin-dependent kinases (CDKs). Cyclin E is a regulatory subunit of CDK2 and drives cells from G1 to S phase. Increased expression of cyclin E is a frequent event in human malignancies and has been associated with poor prognosis in various cancers. In this study, we evaluated the effects of cyclin E-overexpression on the sensitivity of rat fibroblasts to anticancer drugs. Cyclin E-overexpressing cells were less sensitive to doxorubicin-induced inhibition of cell growth but not to other antineoplastic drugs, such as paclitaxel, vincristine, etoposide and methotrexate. Cyclin E-overexpressing fibroblasts also displayed a reduction in ROS levels and a significantly lower increase following doxorubicin treatment compared with vector control cells. The expression of manganese superoxide dismutase (MnSOD) and its activity were increased (about 1.3-fold) in cyclin E-overexpressing derivatives compared with control cells. These results suggest that cyclin E overexpression might reduce tumour cells sensitivity to doxorubicin by affecting the expression of MnSOD and that determination of cyclin E expression levels might help to select patients to be treated with an anthracycline-based antineoplastic therapy.

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Reduced concentration of intracellular ROS in cyclin E-overexpressing cells. (A) dichlorodihydrofluorescein diacetate fluorescence was measured after 45 min incubation with DCF (10 μM). The values represent the means±s.d. of four different experiments performed in triplicate and are shown as per cent of Rat-1 control cells. Similar values were obtained taking into account the readings after 15 and 30 min incubation with DCF (data not shown). *Significant at P=0.001 for Rat-E vs Rat-V. (B) Intracellular ROS concentration was assessed by flow cytometry on cells loaded with DCF. The dye (10 μg ml−1) was added to cell culture 30 min before analysis. Cells were then detached from the substrate and green fluorescence was analysed using a Coulter Epics flow cytometer equipped with a 480 nm emission laser. Numbers beside the histograms indicate mean cell fluorescence and are the averages of three independent experiments.
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fig3: Reduced concentration of intracellular ROS in cyclin E-overexpressing cells. (A) dichlorodihydrofluorescein diacetate fluorescence was measured after 45 min incubation with DCF (10 μM). The values represent the means±s.d. of four different experiments performed in triplicate and are shown as per cent of Rat-1 control cells. Similar values were obtained taking into account the readings after 15 and 30 min incubation with DCF (data not shown). *Significant at P=0.001 for Rat-E vs Rat-V. (B) Intracellular ROS concentration was assessed by flow cytometry on cells loaded with DCF. The dye (10 μg ml−1) was added to cell culture 30 min before analysis. Cells were then detached from the substrate and green fluorescence was analysed using a Coulter Epics flow cytometer equipped with a 480 nm emission laser. Numbers beside the histograms indicate mean cell fluorescence and are the averages of three independent experiments.

Mentions: To evaluate whether cyclin E overexpression was associated with changes in the redox state of rat fibroblasts, we analysed the oxidative state of both cyclin E-overexpressing and control cells using the fluorescent probe DCF, which is a sensitive fluorimetric probe of the production of oxidative stress in living cells (Zhu et al, 1994). We found that the basal level of endogenous ROS was significantly lower (about 16%) (P=0.001) in cyclin E-overexpressing cells compared with vector control and parental cells (Figure 3AFigure 3


Increased expression of cyclin E is associated with an increased resistance to doxorubicin in rat fibroblasts.

Sgambato A, Camerini A, Pani G, Cangiano R, Faraglia B, Bianchino G, De Bari B, Galeotti T, Cittadini A - Br. J. Cancer (2003)

Reduced concentration of intracellular ROS in cyclin E-overexpressing cells. (A) dichlorodihydrofluorescein diacetate fluorescence was measured after 45 min incubation with DCF (10 μM). The values represent the means±s.d. of four different experiments performed in triplicate and are shown as per cent of Rat-1 control cells. Similar values were obtained taking into account the readings after 15 and 30 min incubation with DCF (data not shown). *Significant at P=0.001 for Rat-E vs Rat-V. (B) Intracellular ROS concentration was assessed by flow cytometry on cells loaded with DCF. The dye (10 μg ml−1) was added to cell culture 30 min before analysis. Cells were then detached from the substrate and green fluorescence was analysed using a Coulter Epics flow cytometer equipped with a 480 nm emission laser. Numbers beside the histograms indicate mean cell fluorescence and are the averages of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2741105&req=5

fig3: Reduced concentration of intracellular ROS in cyclin E-overexpressing cells. (A) dichlorodihydrofluorescein diacetate fluorescence was measured after 45 min incubation with DCF (10 μM). The values represent the means±s.d. of four different experiments performed in triplicate and are shown as per cent of Rat-1 control cells. Similar values were obtained taking into account the readings after 15 and 30 min incubation with DCF (data not shown). *Significant at P=0.001 for Rat-E vs Rat-V. (B) Intracellular ROS concentration was assessed by flow cytometry on cells loaded with DCF. The dye (10 μg ml−1) was added to cell culture 30 min before analysis. Cells were then detached from the substrate and green fluorescence was analysed using a Coulter Epics flow cytometer equipped with a 480 nm emission laser. Numbers beside the histograms indicate mean cell fluorescence and are the averages of three independent experiments.
Mentions: To evaluate whether cyclin E overexpression was associated with changes in the redox state of rat fibroblasts, we analysed the oxidative state of both cyclin E-overexpressing and control cells using the fluorescent probe DCF, which is a sensitive fluorimetric probe of the production of oxidative stress in living cells (Zhu et al, 1994). We found that the basal level of endogenous ROS was significantly lower (about 16%) (P=0.001) in cyclin E-overexpressing cells compared with vector control and parental cells (Figure 3AFigure 3

Bottom Line: Cyclin E-overexpressing fibroblasts also displayed a reduction in ROS levels and a significantly lower increase following doxorubicin treatment compared with vector control cells.The expression of manganese superoxide dismutase (MnSOD) and its activity were increased (about 1.3-fold) in cyclin E-overexpressing derivatives compared with control cells.These results suggest that cyclin E overexpression might reduce tumour cells sensitivity to doxorubicin by affecting the expression of MnSOD and that determination of cyclin E expression levels might help to select patients to be treated with an anthracycline-based antineoplastic therapy.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Patologia Generale, Centro di Ricerche Oncologiche 'Giovanni XXIII', Catholic University, Largo Francesco Vito 1, 00168 Rome, Italy. asgambato@rm.unicatt.it

ABSTRACT
Cell cycle progression in eukaryotic cells is regulated by a family of cyclin-dependent kinases (CDKs). Cyclin E is a regulatory subunit of CDK2 and drives cells from G1 to S phase. Increased expression of cyclin E is a frequent event in human malignancies and has been associated with poor prognosis in various cancers. In this study, we evaluated the effects of cyclin E-overexpression on the sensitivity of rat fibroblasts to anticancer drugs. Cyclin E-overexpressing cells were less sensitive to doxorubicin-induced inhibition of cell growth but not to other antineoplastic drugs, such as paclitaxel, vincristine, etoposide and methotrexate. Cyclin E-overexpressing fibroblasts also displayed a reduction in ROS levels and a significantly lower increase following doxorubicin treatment compared with vector control cells. The expression of manganese superoxide dismutase (MnSOD) and its activity were increased (about 1.3-fold) in cyclin E-overexpressing derivatives compared with control cells. These results suggest that cyclin E overexpression might reduce tumour cells sensitivity to doxorubicin by affecting the expression of MnSOD and that determination of cyclin E expression levels might help to select patients to be treated with an anthracycline-based antineoplastic therapy.

Show MeSH
Related in: MedlinePlus