PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro methods
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Materials and methods
MDA-MB-134, MDA-MB-415, MDA-MB-436, MDA-MB-175, UACC-893, UACC-812, and MDA-MB-157 cells were cultured in L15 medium supplemented with 10% heat-inactivated FBS, 2 mmol/l glutamine and 1% penicillin G-streptomycin-fungizone solution (PSF) (Irvine Scientific, Santa Ana, CA, USA). CAL-51, KPL-1, and Hs578t cells were grown in DMEM (Cellgro, Manassas, VA, USA) supplemented with 10% heat-inactivated FBS and PSF, as above. SUM-190 and SUM-225 cells were cultured in HAM's F12 supplemented with 5% heat-inactivated FBS, PSF, 5 mg/ml insulin and 1 mg/ml hydrocortisone. 184A1, 184B5, and MCF 10A cells were grown in a 50/50 mix of mammary epithelial basal medium (MCDB 170) (US Biological, Swampscott, MA, USA) supplemented with 1.5 ml/l bovine pituitary extract (Invitrogen, Carlsbad, CA, USA), 20 µl/l epidermal growth factor (Invitrogen), 10 ml insulin (Sigma, Saint Louis, MO, USA), 1 ng/ml c...
Proliferation assays
Cells were seeded in duplicate at 5,000 to 10,000 cells per well in 24-well plates. The day after plating, PD 0332991 was added at 1 µM and twofold dilutions over six concentrations were performed to generate a dose-response curve. Control wells without drug were also seeded. Cells were counted on day 1 when the drug was added as well as after 6 days when the experiment ended. After trypsinization, cells were placed in Isotone solution and counted immediately using a Coulter Z2 particle counter (Beckman Coulter Inc., Fullerton, CA, USA). Suspension lines were counted using a Coulter Vi-Cell counter (Beckman Coulter Inc.).
Western blot analysis
Cells in log-phase growth were treated with 100 nM PD 0332991 and were harvested at various timepoints by washing in PBS and lysis at 4°C in RIPA lysis buffer. Insoluble material was cleared by centrifugation at 10,000 × g for 10 minutes and protein was quantitated using bicinchoninic (BCA) (Pierce Biochemicals, Rockford, IL, USA). Protein content was resolved by SDS-PAGE electrophoresis, and was transferred to nitrocellulose membranes (Invitrogen). Total pRb expression was detected using a rabbit polyclonal antibody to pRb (Abcam, Cambridge, MA, USA). Rb phosphorylation was detected using rabbit polyclonal antibody to phospho-serine 780 (Cell-signaling, Danvers, MA, USA). Blots were washed and incubated with a goat-anti-rabbit IgG horseradish peroxidase conjugate (Upstate, Bellerica, MA, USA), developed using ECL Plus chemifluorescent reagent (Amersham Biosciences, Pistcata...
Cell cycle analysis and apoptosis studies
Effects of PD 0332991 on the cell cycle were assessed using Nim-DAPI staining (NPE Systems, Pembroke Pines, FL, USA). Cells were plated evenly in control and experimental wells, were allowed to grow to log phase and were then treated with 100 nM PD 0332991 for the defined times. To perform cell cycle analysis, cells were washed with PBS; then trypsin was applied to release cells, which were then centrifuged at 10,000 × g for 5 minutes. Supernatant was aspirated and cells were then resuspended in 100 µl Nim-DAPI (NPE Systems) and gently vortexed.
Cell cycle analysis and apoptosis studies
Cells were analyzed with UV using a Cell Lab Quanta SC flow cytometer (Beckman-Coulter Inc.). Apoptosis assays were performed using an Annexin V-FITC apoptosis detection kit (MBL, Woburn, MA, USA) and flow cytometry. Cells were plated and treated as for cell cycle studies and were exposed to 100 nM PD 0332991 for 5 days. After incubation, cells were processed as directed in the kit and were analyzed using a FITC signal detector and propidium iodide detector using a Cell Lab Quanta SC flow cytometer.
Effects of PD 0332991 on cell cycle and apoptosis
To evaluate the effects of PD 0332991 on the cell cycle and to correlate them with the antiproliferative effects of the compound, we treated a subset of both sensitive cell lines and resistant cell lines with PD 0332991 at 100 nM for 48 hours and then performed flow cytometry using Nim-DAPI staining. Clear and pronounced G 0 /G 1 arrest was seen in cell lines that had lower IC 50 values (IC 50 < 150 nM) compared with those with higher IC 50 values (IC 50 > 1,000 nM) (Figure ). There was no evidence of apoptosis in even the most sensitive cell lines when PD 0332991 was used as a single agent (data not shown).
Effects of PD 0332991 on cell cycle and apoptosis
Effects of PD 0332991 on cell cycle. (a) Sensitive cell lines (IC 50 < 150 nM) show marked G 0 /G 1 arrest and a decrease in the S-phase fraction as compared with (b) resistant cell lines (IC 50 > 1,000 nM) after incubation with 100 nM PD 0332991 for 24 hours. Solid bars, control samples; hatched bars, treated samples. Error bars represent the standard error for two separate experiments.
Combinations of PD 0332991 plus tamoxifen and PD 0332991 plus trastuzumab in ER-positive and HER2-amplified breast ca...
Given that both the ER-positive cell lines as well as HER2-amplified cell lines within the panel demonstrated greatest sensitivity to PD 0332991, the combination of targeted therapeutics plus the CDK4/6 inhibitor was evaluated in both of these molecular subtypes. In ER-positive breast cancer, hormonal blockade with tamoxifen is an effective treatment for early and advanced disease. For the three ER-positive lines evaluated, when considering the entire dose-response curve, the combination was synergistic with mean CI < 1 across clinically relevant concentrations of both drugs (Figure ). Effects of the combination on the cell cycle are shown in Additional data file.
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Protein expression by western blot
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MDA-MB-134, MDA-MB-415, MDA-MB-436, MDA-MB-175, UACC-893, UACC-812, and MDA-MB-157 cells were cultured in L15 medium supplemented with 10% heat-inactivated FBS, 2 mmol/l glutamine and 1% penicillin G-streptomycin-fungizone solution (PSF) (Irvine Scientific, Santa Ana, CA, USA). CAL-51, KPL-1, and Hs578t cells were grown in DMEM (Cellgro, Manassas, VA, USA) supplemented with 10% heat-inactivated FBS and PSF, as above. SUM-190 and SUM-225 cells were cultured in HAM's F12 supplemented with 5% heat-inactivated FBS, PSF, 5 mg/ml insulin and 1 mg/ml hydrocortisone. 184A1, 184B5, and MCF 10A cells were grown in a 50/50 mix of mammary epithelial basal medium (MCDB 170) (US Biological, Swampscott, MA, USA) supplemented with 1.5 ml/l bovine pituitary extract (Invitrogen, Carlsbad, CA, USA), 20 µl/l epidermal growth factor (Invitrogen), 10 ml insulin (Sigma, Saint Louis, MO, USA), 1 ng/ml cholera toxin (Calbiochem, San Diego, CA, USA), 0.5 mg/l hydrocortisone (Sigma), and RPMI 1640 supplemented with 10% heat-inactivated FBS, 2 mmol/l glutamine, and 1% PSF. The remaining cell lines were cultured in RPMI 1640 (Cellgro) supplemented with 10% heat-inactivated FBS, 2 mmol/l glutamine, and...
Cells were seeded in duplicate at 5,000 to 10,000 cells per well in 24-well plates. The day after plating, PD 0332991 was added at 1 µM and twofold dilutions over six concentrations were performed to generate a dose-response curve. Control wells without drug were also seeded. Cells were counted on day 1 when the drug was added as well as after 6 days when the experiment ended. After trypsinization, cells were placed in Isotone solution and counted immediately using a Coulter Z2 particle counter (Beckman Coulter Inc., Fullerton, CA, USA). Suspension lines were counted using a Coulter Vi-Cell counter (Beckman Coulter Inc.).
Cells in log-phase growth were treated with 100 nM PD 0332991 and were harvested at various timepoints by washing in PBS and lysis at 4°C in RIPA lysis buffer. Insoluble material was cleared by centrifugation at 10,000 × g for 10 minutes and protein was quantitated using bicinchoninic (BCA) (Pierce Biochemicals, Rockford, IL, USA). Protein content was resolved by SDS-PAGE electrophoresis, and was transferred to nitrocellulose membranes (Invitrogen). Total pRb expression was detected using a rabbit polyclonal antibody to pRb (Abcam, Cambridge, MA, USA). Rb phosphorylation was detected using rabbit polyclonal antibody to phospho-serine 780 (Cell-signaling, Danvers, MA, USA). Blots were washed and incubated with a goat-anti-rabbit IgG horseradish peroxidase conjugate (Upstate, Bellerica, MA, USA), developed using ECL Plus chemifluorescent reagent (Amersham Biosciences, Pistcataway, NJ), and imaged using chemifluorescence.
Effects of PD 0332991 on the cell cycle were assessed using Nim-DAPI staining (NPE Systems, Pembroke Pines, FL, USA). Cells were plated evenly in control and experimental wells, were allowed to grow to log phase and were then treated with 100 nM PD 0332991 for the defined times. To perform cell cycle analysis, cells were washed with PBS; then trypsin was applied to release cells, which were then centrifuged at 10,000 × g for 5 minutes. Supernatant was aspirated and cells were then resuspended in 100 µl Nim-DAPI (NPE Systems) and gently vortexed.
Cells were analyzed with UV using a Cell Lab Quanta SC flow cytometer (Beckman-Coulter Inc.). Apoptosis assays were performed using an Annexin V-FITC apoptosis detection kit (MBL, Woburn, MA, USA) and flow cytometry. Cells were plated and treated as for cell cycle studies and were exposed to 100 nM PD 0332991 for 5 days. After incubation, cells were processed as directed in the kit and were analyzed using a FITC signal detector and propidium iodide detector using a Cell Lab Quanta SC flow cytometer.
To evaluate the effects of PD 0332991 on the cell cycle and to correlate them with the antiproliferative effects of the compound, we treated a subset of both sensitive cell lines and resistant cell lines with PD 0332991 at 100 nM for 48 hours and then performed flow cytometry using Nim-DAPI staining. Clear and pronounced G 0 /G 1 arrest was seen in cell lines that had lower IC 50 values (IC 50 < 150 nM) compared with those with higher IC 50 values (IC 50 > 1,000 nM) (Figure ). There was no evidence of apoptosis in even the most sensitive cell lines when PD 0332991 was used as a single agent (data not shown).
Effects of PD 0332991 on cell cycle. (a) Sensitive cell lines (IC 50 < 150 nM) show marked G 0 /G 1 arrest and a decrease in the S-phase fraction as compared with (b) resistant cell lines (IC 50 > 1,000 nM) after incubation with 100 nM PD 0332991 for 24 hours. Solid bars, control samples; hatched bars, treated samples. Error bars represent the standard error for two separate experiments.
Given that both the ER-positive cell lines as well as HER2-amplified cell lines within the panel demonstrated greatest sensitivity to PD 0332991, the combination of targeted therapeutics plus the CDK4/6 inhibitor was evaluated in both of these molecular subtypes. In ER-positive breast cancer, hormonal blockade with tamoxifen is an effective treatment for early and advanced disease. For the three ER-positive lines evaluated, when considering the entire dose-response curve, the combination was synergistic with mean CI < 1 across clinically relevant concentrations of both drugs (Figure ). Effects of the combination on the cell cycle are shown in Additional data file.
Machine-readable layer
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