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
Aim. Evidence-backed execution summary for 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 from 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.
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human
Subject model for the experiment.
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- confirm full cohort details in the source paper
Materials and methods
reagent used in the protocol.
- Use
- The cell lines used in the analysis include MDA-MB-415, MDA-MB-134, HCC-1500, ZR-75-30, HCC-202, HCC-1419, HCC-38, HCC-70, HCC-1187, HCC-1806, HCC-1937, HCC-1954, MDA-MB-436, HCC-1569, Hs578t, HCC-1143, MDA-MB-175, BT-474, SK-BR-3, MDA-MB-361, UACC-893, UACC-812, UACC-732, T-47D, MDA-MB-453, MDA-MB-468, CAMA-1, MDA-...
Materials and methods
reagent used in the protocol.
- Use
- 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 g...
Transcript microarray analyses
reagent used in the protocol.
- Use
- Briefly, cells were grown to log phase and then RNA was extracted using the RNeasy Kit (Qiagen, Valencia, CA, USA). The purified RNA was eluted in 30 to 60 µl diethylpyrocarbonate (DEPC) water and the quantity of RNA measured by spectral analysis using the Nanodrop Spectrophotometer (NanoDrop Products, Wilmingt...
Proliferation assays
reagent used in the protocol.
- Use
- 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 th...
Multiple drug effects analysis
reagent used in the protocol.
- Use
- Similar to above, the ER-positive cell lines MCF-7, T47-D, and EFM-19 were plated and treated with PD 0332991 alone, with 4-hydroxytamoxifen (Sigma) alone, or with the combination, in duplicate, over six twofold dilutions at a fixed molar ratio. For combination studies with trastuzumab, BT-474, EFM-192A, and MDA-361...
Multiple drug effects analysis
reagent used in the protocol.
- Use
- For each assay, the log of the fraction growth inhibition was plotted against the log of drug concentration, and the linear regression curve fit correlation coefficient ( r value) was calculated. Multiple drug effect analysis was performed using computer software as previously described [ ].
Multiple drug effects analysis
reagent used in the protocol.
- Use
- Combination index (CI) values were derived from parameters of the median effects plots, and statistical tests were applied (unpaired, two-tail Student t test) to determine whether the mean CI values at multiple concentrations were significantly different from CI = 1. In this analysis, synergy is defined as CI values...
Western blot analysis
reagent used in the protocol.
- Use
- 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 Biochem...
Statistical methods
Growth response to PD 0332991 and molecular subtype classification data were entered into the Statistica data analysis system version 8.0 (StatSoft Inc., Tulsa, OK, USA). The Pearson chi-square test was used to assess the relationship between response and subtype.
- Use
- Growth response to PD 0332991 and molecular subtype classification data were entered into the Statistica data analysis system version 8.0 (StatSoft Inc., Tulsa, OK, USA). The Pearson chi-square test was used to assess the relationship between response and subtype.
Statistical methods
Breast cell lines were profiled on the Agilent Human 1A V1 platform that contains 17,086 probes including known genes and ESTs. The Resolver system analysis of variance (ANOVA) and hierarchical cluster analysis of the breast cell line expression profiles were used to compare the most sensitive cell lines (n = 21, IC...
- Use
- Breast cell lines were profiled on the Agilent Human 1A V1 platform that contains 17,086 probes including known genes and ESTs. The Resolver system analysis of variance (ANOVA) and hierarchical cluster analysis of the breast cell line expression profiles were used to compare the most sensitive cell lines (n = 21, IC...
Transcript microarray analyses
Briefly, cells were grown to log phase and then RNA was extracted using the RNeasy Kit (Qiagen, Valencia, CA, USA). The purified RNA was eluted in 30 to 60 µl diethylpyrocarbonate (DEPC) water and the quantity of RNA measured by spectral analysis using the Nanodrop Spectrophotometer (NanoDrop Products, Wilmingt...
- Use
- Briefly, cells were grown to log phase and then RNA was extracted using the RNeasy Kit (Qiagen, Valencia, CA, USA). The purified RNA was eluted in 30 to 60 µl diethylpyrocarbonate (DEPC) water and the quantity of RNA measured by spectral analysis using the Nanodrop Spectrophotometer (NanoDrop Products, Wilmingt...
Transcript microarray analyses
Characterization of individual breast cancer cell line transcripts was performed by comparison with a breast cell line mixed reference pool of RNA and was conducted on a single slide in which the cell line mixture RNA was labeled with cyanine-3 and RNA from the individual cell line was labeled with cyanine-5. The mi...
- Use
- Characterization of individual breast cancer cell line transcripts was performed by comparison with a breast cell line mixed reference pool of RNA and was conducted on a single slide in which the cell line mixture RNA was labeled with cyanine-3 and RNA from the individual cell line was labeled with cyanine-5. The mi...
Transcript microarray analyses
Microarray slides were read using an Agilent Scanner, and Agilent Feature Extraction software version 7.5 was used to calculate gene expression values. The feature extracted files were imported into the Rosetta Resolver ® system version 7.1 for gene expression data analysis (Rosetta Biosoftware, Seattle, WA, US...
- Use
- Microarray slides were read using an Agilent Scanner, and Agilent Feature Extraction software version 7.5 was used to calculate gene expression values. The feature extracted files were imported into the Rosetta Resolver ® system version 7.1 for gene expression data analysis (Rosetta Biosoftware, Seattle, WA, US...
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 th...
- Use
- 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 th...
Multiple drug effects analysis
For each assay, the log of the fraction growth inhibition was plotted against the log of drug concentration, and the linear regression curve fit correlation coefficient ( r value) was calculated. Multiple drug effect analysis was performed using computer software as previously described [ ].
- Use
- For each assay, the log of the fraction growth inhibition was plotted against the log of drug concentration, and the linear regression curve fit correlation coefficient ( r value) was calculated. Multiple drug effect analysis was performed using computer software as previously described [ ].
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...
- Use
- 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...
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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.
Measurement outputs
What raw and processed outputs should exist?
Growth response to PD 0332991 and molecular subtype classification data were entered into the Statistica data analysis system version 8.0 (StatSoft Inc., Tulsa, OK, USA). The Pe...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Forty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC 50 values. T...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Breast cell lines were profiled on the Agilent Human 1A V1 platform that contains 17,086 probes including known genes and ESTs. The Resolver system analysis of variance (ANOVA)...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The cell lines used in the analysis include MDA-MB-415, MDA-MB-134, HCC-1500, ZR-75-30, HCC-202, HCC-1419, HCC-38, HCC-70, HCC-1187, HCC-1806, HCC-1937, HCC-1954, MDA-MB-436, HC...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
Growth response to PD 0332991 and molecular subtype classification data were entered into the Statistica data analysis system version 8.0 (StatSoft Inc., Tulsa, OK, USA).
from paperScoring or quantification
Quantify the primary readouts for this experiment: Growth response to PD 0332991 and molecular subtype classification data were entered into the Statistica data analysis system version 8.0 (StatSoft Inc., Tulsa, OK, USA). The Pe...; Forty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC 50 values. T...; Breast cell lines were profiled on the Agilent Human 1A V1 platform that contains 17,086 probes including known genes and ESTs. The Resolver system analysis of variance (ANOVA)...; The cell lines used in the analysis include MDA-MB-415, MDA-MB-134, HCC-1500, ZR-75-30, HCC-202, HCC-1419, HCC-38, HCC-70, HCC-1187, HCC-1806, HCC-1937, HCC-1954, MDA-MB-436, HC....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Growth response to PD 0332991 and molecular subtype classification data were entered into the Statistica data analysis system version 8.0 (StatSoft Inc., Tulsa, OK, USA). The Pe...; Breast cell lines were profiled on the Agilent Human 1A V1 platform that contains 17,086 probes including known genes and ESTs. The Resolver system analysis of variance (ANOVA)...; Combination index (CI) values were derived from parameters of the median effects plots, and statistical tests were applied (unpaired, two-tail Student t test) to determine wheth...; Differentially expressed genes between sensitive and resistant cell lines. Results of analysis of variance (ANOVA) identifying 450 differentially expressed genes between sensiti...
from paperReporting output
Report representative outputs alongside summary comparisons for Growth response to PD 0332991 and molecular subtype classification data were entered into the Statistica data analysis system version 8.0 (StatSoft Inc., Tulsa, OK, USA). The Pe..., Forty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC 50 values. T..., Breast cell lines were profiled on the Agilent Human 1A V1 platform that contains 17,086 probes including known genes and ESTs. The Resolver system analysis of variance (ANOVA)..., The cell lines used in the analysis include MDA-MB-415, MDA-MB-134, HCC-1500, ZR-75-30, HCC-202, HCC-1419, HCC-38, HCC-70, HCC-1187, HCC-1806, HCC-1937, HCC-1954, MDA-MB-436, HC....
inferred from protocolStructured statistical methods
Growth response to PD 0332991 and molecular subtype classification data were entered into the Statistica data analysis system version 8.0 (StatSoft Inc., Tulsa, OK, USA). The Pe...; Breast cell lines were profiled on the Agilent Human 1A V1 platform that contains 17,086 probes including known genes and ESTs. The Resolver system analysis of variance (ANOVA)...; Combination index (CI) values were derived from parameters of the median effects plots, and statistical tests were applied (unpaired, two-tail Student t test) to determine wheth...; Differentially expressed genes between sensitive and resistant cell lines. Results of analysis of variance (ANOVA) identifying 450 differentially expressed genes between sensiti...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
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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"@type": "HowTo",
"name": "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",
"description": "Evidence-backed execution summary for 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 from 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.",
"totalTime": "PT9615M",
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{
"@type": "HowToStep",
"position": 1,
"name": "Materials and methods",
"text": "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..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Proliferation assays",
"text": "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.)."
},
{
"@type": "HowToStep",
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"name": "Western blot analysis",
"text": "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..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Cell cycle analysis and apoptosis studies",
"text": "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."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Cell cycle analysis and apoptosis studies",
"text": "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."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Effects of PD 0332991 on cell cycle and apoptosis",
"text": "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)."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Effects of PD 0332991 on cell cycle and apoptosis",
"text": "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."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Combinations of PD 0332991 plus tamoxifen and PD 0332991 plus trastuzumab in ER-positive and HER2-amplified breast ca...",
"text": "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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