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2018, Cilt 57, Sayı 1, Sayfa(lar) 036-045
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Influence of PD-0332991 treatment on cell cycle regulatory genes in breast cancer stem cells
Hasan Onur Çağlar1, Sunde Yılmaz Süslüer2, Cumhur Gündüz2, Ayfer Haydaroğlu3
1Ege University Health Science Institute, Department of Stem Cell, İzmir, Turkey
2Ege University Faculty of Medicine, Department of Medical Biology, İzmir, Turkey
3Ege University Faculty of Medicine, Department of Radiation Oncology, İzmir, Turkey
Keywords: PD-0332991, breast neoplasms, neoplastic stem cells, G1 cell cycle arrest, cell cycle
Abstract
Aim: Palbociclib (PD-0332991) is an inhibitor for cyclin-dependent kinase 4/6 complex and exhibits more activity in luminal ER+ breast cancer. However, sensitivity of breast cancer stem cells (BCSCs) to PD-0332991 treatment and expression patterns of cell cycle regulatory genes after PD-0332991 treatment in BCSCs are still unclear. This study aims to determine response of BCSCs to PD-0332991 treatment.

Materials and Methods: An experimental in vitro study was designed on breast cancer cell lines. MCF-7 and BCSCs cell lines were used in this study. Water soluble tetrazolium salt-1 (WST-1) test was used for the cytotoxicity assay. Cell cycle distribution pattern and apoptosis were examined with flow cytometry according to IC50 values at 48th h. Real-Time PCR was used to detect expression profiles of CDKN1A, CHEK1, CDKN2A, CDC25A, and CCND1 genes.

Results: PD-0332991 decreased cell proliferation in both cell lines. G0/G1 arrest was detected for both cell lines. There was no apoptotic effect of PD-0332991 on MCF-7 cells and BCSCs. In MCF-7 cells, expression levels of CDKN1A, CDKN2A, and CCND1 genes were increased as 3.11, 3.21, and 1.05 folds, respectively. Expression levels of CHEK1 and CDC25A genes were decreased as 4.75 and 3.73 folds, respectively. In BCSCs, expression levels of CDKN1A, CHEK1, CDKN2A, and CCND1 were decreased as 1.15, 2.01, 1.32, and 1.68 folds, respectively. No expression of CDC25A gene was found in BCSCs group.

Conclusion: In this study, it was observed that PD-0332991 leads to different expression profiles for cell cycle regulatory genes between BCSCs and breast cancer cells.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Disscussion
  • Conclusion
  • References
  • Introduction
    Breast cancer is the most common cancer type among women and it is the second leading cause of the cancer-related death after respiratory system and lung cancers 1. Approximately, estimated 231,840 new cases of breast cancer are expected to be diagnosed in the USA according to a report by Siagel et al. 1. Novel effective drugs are a requirement to provide therapies due to high incidence and mortality rates of breast cancer. Cell cycle inhibitors have been evaluated for breast cancer treatment 2,3. Palbociclib (PD0332991) is a cyclin dependent kinase-4/6 (CDK4/6) inhibitor, leading to G1 arrest in cancer cells 4. This inhibitor binds specifically to CDK-4/6 complex involving G1/S transition in cell cycle 5-7. PD-0332991 blocks specifically cell cycle progression in G1 phase due to no activity on other kinases 2. Thus, the cancer cells are allowed to be arrested specifically in G1 phase.

    Studies demonstrated that PD-0332991 is more effective for estrogen positive (ER+) breast cancer cells 4,8-11. Finn et al. showed that ER+ breast cancer cells as well as HER-2 positive cells are sensitive to PD-0332991 treatment 11 Sensitivity of breast cancer cells to PD-0332991 treatment is associated with expression patterns of cell cycle regulatory genes such as retinoblastoma (RB1), CDKN2A (p16), and cyclin D1 11. However, expression patterns of genes that determine molecular subtypes of breast cancer are not implicated in sensitivity to PD-0332991 treatment. Paternot et al. 12 identified that PD-0332991 stabilizes CDK-4/6-cyclin D3 complex devoid of p21 and p27 proteins. Depending on withdrawal of PD-0332991, stabilized cyclin D3-CDK4/6 complex via PD-0332991 contributes to entry S phase in cell cycle 12. Thus, it has been suggested that genes which regulate G1/S checkpoint in cell cycle may also assess sensitivity of breast cancer cells to PD-0332991 treatment 11,12.

    Cancer stem-like cells (CSCs) are distinct and highly tumorigenic cell population in tumor tissue 13. CSCs, as a unique population in tumor tissue, express stem cell markers and they also exhibit stem cell-like features such as self-renewing 13. It is known that breast cancer stem-like cells (BCSCs) are one of the causes of resistance against radio- and chemotherapy, leading to treatment failure 13-15. Although BCSCs are found in all subtypes of breast cancer, basal-type breast cancer tumors have higher population of BCSCs than luminal type breast cancer tumors 16,17. Furthermore, the loss of p16 expression increases the percentage of BCSCs in estrogen negative breast cancer cell lines 18. Expression profiles of cell cycle regulatory genes in BCSCs may be a reason for differently observed sensitivity of PD-0332991 among breast cancer subtypes. A number of cell cycle related genes such as CCNA1, CCND1, CCND2, and CDKN2A exhibit an abnormal expression pattern in breast cancer stem cells when compared with its ordinary cancer cell line 19. Suppression of NANOG gene, maintaining stem cell features, in breast cancer cells reduces particularly expression of cyclin D1 protein 20. In addition, the down-regulation of NANOG gene increases G0/G1 arrest for breast cancer cells 20. Although cell cycle related genes, responsible for sensitivity to PD-0332991, show a different expression pattern between BCSCs and breast cancer cells, influence of expression pattern of cell cycle regulatory genes on response to PD-0332991 in BCSCs are still unclear. In this study, we therefore aimed to show expression patterns of cell cycle regulatory gene, acting together in G0/G1 arrest, in breast cancer stem cells after PD-0332991 treatment.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Disscussion
  • Conclusion
  • References
  • Methods
    An experimental in vitro study was designed on breast cancer cell lines. MCF-7 breast cancer cell line and breast cancer stem cell line (CelProgen, CA, USA, Cat No: 36102-29-T25), which is positive for such stem cell markers as CD133, CD44, SSEA3/4, and aldehyde dehydrogenase, were used in the current study.

    Cell culture and reagent
    PD-0332991-isethionate was purchased from Sigma-Aldrich (Sigma-Aldrich, USA). MCF-7 breast cancer cell line was purchased from ATCC (ATCC, HTB-22, Wesel, Germany). RPMI 1640 medium, containing 10% FBS, was used for MCF-7 cells. Human breast cancer stem-like cell line was purchased from CelProgen (San Pedro, CA, USA). Human breast cancer stem cell complete growth media, including all components to maintain cell culture, was used. Cells were maintained at 37oC under a humidified atmosphere of 5% CO2 in air. The passage numbers of BCSC and MCF-7 cell lines were five and seven, respectively.

    WST-1 cell proliferation assay
    WST-1 test (Roche Diagnostic GmbH, Mannheim, Germany) was performed to show cytotoxicity of PD-0332991 on MCF-7 and BCSC cell lines. Before the cytotoxicity assay was performed, the cell numbers per well were optimized via WST-1 test in order to eliminate a deceptive apoptotic effect due to doubling time differences between cell lines. The cells, starting with 1x106 in a well and diluted in 1/2 ratio, were seeded on 96 well-plate. After 72h incubation with the media, without agent, proliferation characteristic was determined with WST-1 test for BCSCs and MCF-7 cells. Thus, in assay, MCF-7 cell line was seeded as 1x104 cells per well in 96 well-plates. For BCSC cell line, 7x103 cells per well were seeded in 96 well-plates. PD-0332991 treatments for both cell lines were depending on time and concentrations. Cells were treated with the logarithmic dilution of PD-0332991 ranging from 100μM to 0.0001μM for 24h, 48h, and 72h. WST-1 reagent, 10μl, was added to 100μl medium containing PD-0332991. Absorbance values at 450nm were taken with the microplate reader (Thermoscientific, Multiskan FC, Finland) after incubation periods. Absorbance value at 620nm was used as the reference wavelength. The viability of the cells was calculated according to absorbance value made up of WST-1 reduction (21, 22). The absorbance of control cells was assumed as showing 100% viability for normalization.

    Cell cycle assay by flow cytometry
    IC50 values of PD-0332991 at 48th h were used in cell cycle experiment for both cell lines. Non-treated cells were used as control. BD Cycletest Plus DNA Reagent Kit (BD Pharmigen, CA, USA) was used for cell cycle assay. Protocol was performed according to manufacturer’s instructions. For the determination of cell cycle pattern by flow cytometry, BCSCs and MCF-7 cells were seeded at a density of 5x105 cells per 25cm2 cell culture flasks. For defined concentrations, depending on IC50 values at 48th h, 8 ml media with agent was prepared and the cells were treated. Media, 8ml without agent, was used for the control group which has the same cell number as the treatment group. After 48h incubation, cells were removed from surface of cell culture flask and cells were taken to falcon tubes. The cell suspensions were centrifuged at 300xg for 5 min. After removing the supernatant, the pellet was resuspended with 1ml washing buffer. Washing step was repeated twice. Washing buffer was removed without disturbing the pellet. 250μl of solution A (trypsin buffer) was added to the pellet and mixed gently. Tubes were incubated at room temperature for 10 min. After incubation, 200μl of solution B was added to tubes without removing solution A. Incubation step was repeated. 200μl of solution C (propidium iodide stain solution with RNase enzyme) kept in 4ºC was added to tubes. Samples were read at the flow cytometry (BD Accuri C6 flow cytometry, Becton–Dickinson, USA). The argon laser light source, emitting at 488nm wavelength, was used for excitation. A fluorescence detector equipped with the 585/40 band-pass filter was used to show the PI-DNA content (FL2-A) for the analysis.

    Apoptosis assay by flow cytometry
    Both cell lines were treated with IC50 values of PD-0332991 at 48th h. Non-treated cells were used as control. BCSCs and MCF-7 cells were seeded at a density of 5x105 cells per 25cm2 cell culture flasks. The control group had the same cell number as treatment group. PE-Annexin V Apoptosis Detection Kit I (BD Pharmigen, San Diego, CA, USA) was used to detect apoptosis after PD-0332991 treatment in both cell lines. Protocol was performed according to manufacturer’s instructions. Cells were washed twice with 1X PBS. After washing step, 1X binding buffer was added to tubes. 5μl of PE-Annexin V and 7-AAD antibodies were added to tubes and samples were incubated for 15 min at room temperature in the dark. After incubation, 400μl of binding buffer was added to samples and the prepared samples were read at the flow cytometry (BD Accuri C6 Flow Cytometer, Becton–Dickinson, USA) within 1h. The argon laser light source, emitting at 488nm wavelength, was used for excitation. The 670nm band-pass filter for 7-AAD fluorescence (FL3-A) and a fluorescence detector equipped with the 585/40 band-pass filter for FL2A were used for analysis. A total of 20.000 events were acquired for analysis with using Cell Quest Software.

    Gene expression analysis
    In order to detect mRNA expression of cell cycle regulatory genes, total RNA were isolated from PD-0332991 treated and non-treated cell lines. Total RNA purification Kit (Jena Bioscience, Jena, Germany) was used to isolate total RNA from cell lines according to manufacturer’s instructions. Total RNA (1-5μg) was converted to cDNA with using SCRIPT cDNA Synthesis Kit (Jena Bioscience, Jena, Germany). For the detection of gene expression, 1.0 μL cDNA were mixed with 10 μl qPCR GreenMaster with UNG Mix (Jena Bioscience, Jena, Germany) and sterile bidistilled water was added to PCR mix. The expression studies of CDKN1A, CHEK1, CDKN2A, CDC25A, and CCND1 genes were performed with LightCyler (Roche 480 LightCycler, Mannheim, Germany) according to the GreenMaster protocol (Jena Bioscience, Jena, Germany). Relative quantification method described by Livak et al. (23) was used to analyze data. ACTB, HPRT1, 18SRNA, and GAPDH genes were chosen as housekeeping genes for normalization. Ct values were obtained for both the target genes and the housekeeping genes in all cell lines. The expression data was normalized to Ct values obtained from non-treated cell lines to calculate 2-ΔΔCt. Primers for gene expression were designed with Probe Finder Software (Roche Applied Science). Primers were given in Table-1.


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    Table 1: Primer Sequences Used for RT-PCR Study.

    Statistical Analysis
    GraphPad Prism 6 (GraphPad Software, Inc., CA, USA) was used for statistical analysis. The two-way ANOVA test was performed with Dunnett's multiple comparisons test to compare the mean differences between treatment concentrations and time in the cytotoxicity assay. The same test was also used to compare the mean differences for two independent variables, cell cycle phase and treatment, in the data analysis of cell cycle assay. A p value under 0.05 was accepted as significant.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Disscussion
  • Conclusion
  • References
  • Results
    PD-0332991 decreases proliferation of BCSC and MCF-7 cells

    PD-0332991 decreased cell proliferation in both MCF-7 and BCSCs groups. IC50 values at 24th h, 48th h, and 72nd h of treatments for MCF-7 cells were 34.37μM, 25.21 μM, and 26.9 μM, respectively (Figure-1a). IC50 values of PD-0332991 in BCSC cell line were 37.37 μM, 13.16 μM, and 4.12 μM at 24th h, 48th h and 72nd h, respectively (Figure-1b). Cytotoxicity at 48th h and 72nd h of treatment was higher in BCSCs compared to MCF-7 cells at 10 μM concentration. BCSCs were found to be more sensitive to treatment.


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    Figure 1: Results of WST-1 proliferation assay after PD-0332991 treatment. IC50 values at 24th h, 48th h, and 72nd h of treatment in MCF-7 cells were 34.37μM, 25.21 μM, and 26.9 μM, respectively (a). IC50 values for BCSCs were 37.37 μM, 13.16 μM, and 4.12 μM at 24th h, 48th h and 72nd h, respectively (b). **** and *** marks describe respectively p values under 0.0001 and 0.001 with respect to two way ANOVA analysis.

    G0/G1 arrest in MCF-7 cells and BCSC cells after PD-0332991 treatment
    IC50 values of PD-0332991 at 48th h were used in cell cycle analysis of MCF-7 and BCSC cell lines. For both cell lines, G0/G1 arrest was detected after treatment. In MCF-7 control group, the percentage of cells at G0/G1, S, G2/M were determined as 48.9%, 23.5%, and 29.6%, respectively. The percentages of cells in G0/G1, S, G2/M phases for PD-0332991 treated cells were 73.9%, 13.1%, and 12.2%, respectively (Figure-2a). In BCSC control group, the percentage of cells at G0/G1, S, G2/M were determined as 57.0%, 17.0%, and 27.6%, respectively. After treatment, in BCSC cell line, the percentages of cells in phases of G0/G1, S, G2/M were 71.3%, 11.8%, and 17.8%, respectively (Figure-2b). PD-0332991 treatment induced G1 arrest and significant increase in the percentage of cells in G0/G1 phase was detected for both cell lines.


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    Figure 2: Cell cycle patterns of MCF-7 cells (a) and BCSCs (b) after treatment of IC50 values at 48th h. With PD-0332991 treatment, G0/G1 arrest was detected for both cell lines. ***, ** and * marks describe respectively p values under 0.001, 0.01, and 0.05 with respect to two way ANOVA analysis.

    Apoptotic effect of PD-0332911 on BCSC and MCF-7 cell lines
    Cells were treated with concentrations (IC50 values at 48th h) of PD-0332991. Non-treated cells were used as control. In MCF-7 control group, the percentage of living cells was 91.0%. The percentage of apoptotic cells in MCF-7 control group was 2.4%. After treatment, the percentages of living and apoptotic cells were 86.3% and 9.3%, respectively (Figure-3a). In BCSC control group, the percentages of living and apoptotic cells were detected as 96.1% and 2.6%, respectively. After treatment for BCSCs, distributions of the percentages of living and apoptotic cells were 96.8% and 2.1%, respectively (Figure-3b). There was no apoptotic effect of PD-0332991 on MCF-7 cells and BCSCs.


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    Figure 3: Apoptosis assay for MCF-7 (a) and BCSCs (b) cell lines after treatment. Non-treated cells were used as control. PD-0332991 did not induce apoptosis in BCSCs and MCF-7 cell lines.

    Gene expression profiles of G1 regulatory genes after PD-0332991 treatment
    After 48h PD-0332991 treatment in MCF-7 cells, expression levels of CDKN1A, CDKN2A, and CCND1 genes were increased 3.11, 3.21, and 1.05 folds, respectively (Figure-4a). Expression levels of CHEK1 and CDC25A genes were decreased as 4.75 and 3.73 folds, respectively. In BCSCs, expression levels of CDKN1A, CHEK1, CDKN2A, and CCND1 genes were decreased as 1.15, 2.01, 1.32, and 1.68 folds, respectively (Figure-4b). No expression of CDC25A gene was found in BCSCs group.


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    Figure 4: Expression profiles of CDKN1A, CHEK1, CDKN2A, CDC25A, and CCND1 genes in MCF-7 (a) and BCSCs cell lines (b). mRNA expression of CDC25A gene was not observed in BCSCs group.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Disscussion
  • Conclusion
  • References
  • Discussion
    Effect of PD-0332991 treatment on BCSCs was identified in this study. BCSCs were more sensitive to PD-0332991 treatment with respect to MCF-7 cells. In addition, expression patterns of cell cycle regulatory genes between BCSCs and MCF-7 cell lines were dissimilar in response to treatment.

    It was found that PD-0332991 decreased proliferation of MCF-7 cells which is reported by other studies 6,11. Finn et al. found that IC50 values of PD-0332991 for MCF-7 are at nanomolar concentration levels 11. However, in this study, IC50 values for MCF-7 cells were not at nanomolar levels. Different methods utilized for determining of cell proliferation and treatment period may cause differently observed IC50 values for MCF-7 in the literature 11,24. Robinson et al. demonstrated that IC50 value is not observed at 10μM concentration of PD-0332991 for 72h treatment in MCF-7 cells when MTT assay is evaluated for measuring cell proliferation 24. In this study, it was also found that PD-0332991 decreased cell proliferation of BCSCs. In addition, IC50 concentrations of treatment at 48th h and 72nd h were lower in BCSCs compared to MCF-7 cells. BCSCs were more sensitive to treatment than MCF-7 cells.

    Studies indicate that MCF-7 cell line is more sensitive to PD-0332991 treatment compared to MDA-MB-231 cell line which is non-luminal and HER-2 negative cell line 11. Interestingly, it is known that MDA-MB-231 cell line has higher CD44+/CD24- cell population than luminal type breast cancer cell lines such as MCF-7 16,17. The different activity of PD-0332991 among breast cancer subtypes may be associated with variable percentage of BCSCs population in tumor tissue. In the current study, cell cycle pattern and apoptosis were examined after treatment according to IC50 values at 48h. As expected for MCF-7 cells, G0/G1 arrest was detected after treatment 6,11. In addition, PD-0332991 did not induce apoptosis in MCF-7 which is in line with other studies 4,9,11. G0/G1 arrest and no apoptotic effect were also observed for BCSCs after treatment.

    Studies indicated that response of breast cancer cells to PD-0332991 is associated with expression pattern of CDK4/6–pRb–E2F pathway mediated genes such as Rb1, CDKN2A, and cyclin D1 25-27. In cell cycle, mitogenic signals induce cyclin D1 gene (CCND1) expression to initiate G1–S transition. Cyclin D1 is assembled spontaneously with CDK4 or CDK6 proteins to form active kinase complexes. Active CDK4 and CDK6 complexes phosphorylate Rb1 protein which contributes to release of the E2F transcription factors. Mediators such as CCNB1, CDK2 and CDK1 which promote transition to further steps in cell cycle are expressed by released E2F transcription factors (25-27). CDC25A activates CDK2–cyclin E and CDK2–cyclin A complexes during G1-S transition to entry S phase 28-30. In addition, Cdc25A activates also Cdk4-cyclin D complex in G1 phase 31. Chk1 kinase (CHEK1) is a negative regulatory factor for CDC25A action 32. The phosphorylation of regulatory region of CDC25A protein due to active Chk1 leads to ubiquitin mediated proteolysis 32,33. Chk1 can also be a target of the CDK4/6–pRb–E2F pathway due to the fact that Chk1 has E2F-binding sites 34.

    In this study, expression profiles of CDKN1A, CHEK1, CDKN2A, CDC25A, and CCND1 genes were examined in both cell lines after PD-0332991 treatment. Defined genes were chosen due to be direct target of CDK4/6–pRb–E2F pathway which is responsible for activity of PD-0332991. CCND1 expression was increased in MCF-7 cell line after treatment 11. In addition, elevated expressions of CDKN1A and CDKN2A were detected. For MCF-7 cells, decreased expression levels of CHEK1 and CDC25A were also observed. On the contrary, expression levels of CDKN1A, CHEK1, CDKN2A, and CCND1 were decreased in BCSCs after treatment. Expression of CDC25A was not found in BCSCs group. CDC25A is expressed in early G1 phase of cell cycle and its activity is highly observed from the G0/G1 checkpoint to the M phase 29,29. Absence of CDC25A may be associated with why BCSCs was more sensitive to PD-0332991 treatment. It was found that expression patterns of cell cycle regulatory genes involved in early and late G1 phase were different between MCF-7 and BCSCs cell lines after treatment. This difference may determine biological response of breast cancer cells to PD-0332991.

    Breast cancer cells, overexpressing CDKN2A and CDKN1A, exhibit senescence mediated features such as hypertrophic appearance and high β-galactosidase activity 36. Furthermore, induced expression of CDKN2A and CDKN1A triggers senescence in G1 arrested cells 36,37. After treatment, MCF-7 cells may be drifted to senescence. Contrary to MCF-7 cells, expression levels of CCND1, CDKN1A, and CDKN2A were decreased in BCSCs after treatment. Decreased expression of CDK inhibitors (CKIs) and CCND1 in BCSCs may be an adaptation to treatment for maintaining stem cell-like features. Inhibition of CCND1 expression by siRNA increases migration features and ALDH expression of estrogen negative breast cancer cells 38. Suppressed CCND1 expression in ER- cell lines induces also mammosphere formation. Similar results for induced cancer stem cell-like features are observed when CDK4/6 is inhibited by siRNA or PD-0332991 38. Expression pattern of CKIs may support for maintaining stem cell-like features. Depletion of p16 by RNA interference increases the percentage of BCSCs in estrogen negative breast cancer cell lines 18. In addition, suppression of CDKN2A or CDKN1A increases efficiency of generating induced pluripotent stem cells (iPS) in order not to be observed p21- and p16-mediated senescence 39-41.

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Discussion
  • Conclusion
  • References
  • Conclusion
    In this study, the effect of PD-0332991 on expression changes of cell cycle regulatory genes in breast cancer stem cells was shown for the first time. In the current study, it was observed that PD-0332991 leads to different expression profiles for cell cycle regulatory genes between BCSCs and breast cancer cells. It was suggested that expression profile of CDK4/6–pRb–E2F pathway related genes after PD-0332991 treatment may determine divergent response due to maintenance of cancer stem cell features.

    Conflict of interest
    Authors declare that there is no conflict of interest.

    Acknowledgements
    This study is financially supported by Ege University Science and Technology Center-Technology Transfer Office (EBILTEM-TTO) (Grant numbers: 15-KSUAM-001 and 15-KSUAM-002).

  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Discussion
  • Conclusion
  • References
  • References

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  • Top
  • Abstract
  • Introduction
  • Methods
  • Results
  • Discussion
  • Conclusion
  • References
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