Characterization of cyclin E expression in multiple myeloma and its functional role in seliciclib-induced apoptotic cell death

Abstract

Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cell that eventually develops resistance to chemotherapy. Drug resistance, differentiation block and increased survival of the MM tumor cells result from high genomic instability. Chromosomal translocations, the most common genomic alterations in MM, lead to dysregulation of cyclin D, a regulatory protein that governs the activation of key cell cycle regulator--cyclin dependent kinase (CDK). Genomic instability was reported to be affected by over expression of another CDK regulator--cyclin E (CCNE). This occurs early in tumorigenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore sought to investigate the role of cyclin E in MM. CCNE1 expression was found to be heterogeneous in various MM cell lines (hMMCLs). Incubation of hMMCLs with seliciclib, a selective CDK-inhibitor, results in apoptosis which is accompanied by down regulation of MCL1 and p27. Ectopic over expression of CCNE1 resulted in reduced sensitivity of the MM tumor cells in comparison to the paternal cell line, whereas CCNE1 silencing with siRNA increased the cell sensitivity to seliciclib. Adhesion to FN of hMMCLs was prevented by seliciclib, eliminating adhesion-mediated drug resistance of MM cells. Combination of seliciclib with flavopiridol effectively reduced CCNE1 and CCND1 protein levels, increased subG1 apoptotic fraction and promoted MM cell death in BMSCs co-culture conditions, therefore over-coming stroma-mediated protection. We suggest that seliciclib may be considered as essential component of modern anti MM drug combination therapy.

Figure 1. CCNE1 expression profile in hMMCLS.

(A) Various multiple myeloma cell lines (NCI H929, RPMI8226, CAG, ARP1 and U266) and plasma cell leukemia cell line ARH77 in logarithmic growth phase were extracted and subjected to immunoblotting, utilizing CCNE1 antibodies. CDK2 expression served as an internal loading control. (B) Various multiple myeloma cell lines (NCI H929, RPMI8226, CAG, ARP1 and U266) and plasma cell leukemia cell line ARH77 in logarithmic growth phase were extracted and subjected to qRT-PCR in quadruplicates. Data are represented as mean±standard deviation of the ratio of CCNE1 and GAPDH between all hMMCLs and ARP1. (A–B) Experiments were performed at least 3 times and one representative result is presented.

Figure 2. Heterogenous resistancy to seliciclib in hMMCLs.

(A) The indicated hMMCLs were incubated in the absence or presence of increasing concentrations of seliciclib for 3 days. Cell viability was determined by MTT assay. Data are represented as mean±standard deviation. Experiments were performed at least 3 times and one representative result is presented. Seliciclib resulted in a decrease in cell viability with an IC50 ranging from 25 to 90 µM. (B) Cell cycle analysis by PI staining was performed on hMMCLs incubated in the absence or presence of 50 µM seliciclib for 12 hours. Cells were collected, fixed, stained with propidium iodide (PI) and analyzed by flow cytometry. DNA distribution in the cells is presented. (C) Quantification of cell cycle stage analysis of control and seliciclib-treated (50 µM, 12 hours culture) hMMCLs. Analysis of representative lines of highly sensitive (NCI H929), a moderately-sensitive (ARP1) and a resistant (ARH77) lines are displayed. The subdyploid DNA peak (subG1) represents apoptotic cell fraction. Data are represented as mean±standard deviation of 3 different experiments. Probability values of t-test are presented **p<0.01.

Figure 3. Seliciclib induces apoptosis in sensitive hMMCLs.

hMMCLS were incubated in the absence or presence of 50 µM seliciclib. Following 5 hours or 16 hours of culture cells were collected, fixed, stained using annexin V/PI and analyzed by flow cytometry. The extent of apoptosis is expressed as a percentage of cells positively stained using annexin V. (A) Seliciclib-sensitive NCI H929 and seliciclib-resistant ARH77 were incubated in the presence or absence of seliciclib for the indicated time points. Experiments were performed at least 3 times and one representative result is presented. (B) Representative analysis of seliciclib-induced apoptosis following a 16 hour incubation time in control, DMSO and seliciclib treated cells. The percentage of live (annexin V/PI-negative) and apoptotic (annexin V-positive) cells is given. Data represents the mean and standard deviation of 3 different experiments. Probability values of t-test are presented *p<0.05 **p<0.01.

Figure 4. Seliciclib downregulates MCL1 expression in hMMCLs.

(A) The indicated multiple myeloma cell lines in logarithmic growth phase were extracted and subjected to immunoblotting, utilizing MCL1 antibodies. In all experiments CDK2 expression serves as an internal loading control. Experiments were performed at least 3 times and one representative result is presented. (B–C) MCL1 downregulation by seliciclib. Cells were extracted and subjected to immunoblotting. (B) Various hMMCLs were incubated in the presence of seliciclib 50 µM or DMSO for 6 hours and the level of MCL1 expression was analyzed. (C) RPMI8226, CAG and NCI H929 cells were incubated in the absence or presence of seliciclib 50 µM for the indicated time points, or in the presence of increasing concentrations of seliciclib for 8 hours. (D) Reduction in MCL1 phosphorylation by seliciclib. Cells were treated with 50 µM or DMSO for 8 hours and the levels of total and phosphorylated MCL1 were analyzed by immunoblotting using specific antibodies. β-actin was used to confirm equal protein loading. (E) CAG cells were incubated in the absence or presence of seliciclib or MG132 (10 µM) exclusively or combined. MCL1 level of expression was verified by immunoblotting. β-actin was used to confirm equal protein loading.

Figure 5. Effect of seliciclib on cell cycle regulators expression in hMMCLs.

(A) The indicated multiple myeloma cell lines in logarithmic growth phase were extracted and subjected to immunoblotting, utilizing CCND2, CCNE1 and p27 antibodies. β-actin expression serves as an internal loading control. (B) Seliciclib effect on CCNE1, phosphor-CCNE1, CCND2, CDK2 and p27 expression: the indicated hMMCLs were incubated in the presence or absence of 50 µM seliciclib for 4 (I) or 16 (II) hours. Control cells were incubated in the presence of DMSO. Cells were lyzed and extracts were subjected to immunoblotting, utilizing specific antibodies. (C) CAG and NCI H929 cells were incubated in the absence or presence of increasing concentration of seliciclib for over-night incubation or in 50 µM selicicilb for the indicated time points. Cells were lyzed and extracts were subjected to immunoblotting, utilizing CCND2 and CDK2 antibodies. Control cells were incubated in the presence of DMSO. Experiments were performed 3 times and one representative result is presented. (D) CAG cells were incubated in the absence or presence of seliciclib or MG132 (10 µM) exclusively or combined. p27 level of expression was verified by immunoblotting. β-actin was used to confirm equal protein loading.

Figure 6. Effect of seliciclib anf flavopiridol combined treatment on hMMCLs viability, cell cycle distribution and cyclin expression.

(A) The indicated myeloma cell lines were incubated in the absence or presence of 20 µM seliciclib, 100 ng/ml flavopiridol or combination of both agents for 2 days. Cell viability was determined by MTT assay. Data are represented as mean±standard deviation. Probability values of t-test are presented **p<0.01. (B–C) Cell cycle analysis by PI staining was performed on ARH77, RPMI8226, NCI H929 and CAG cells cultured in the absence or presence of 10 µM seliciclib, 100 ng/ml flavopiridol or combination of both agents for 2 days. (B) Representative cell cycle distribution in NCI H929 cells, treated with seliciclib, flavopiridol or their combination. (C) Combined treatment with seliciclib and flavopiridol increases subG1 and decreases G2/M fractions in hMMCLs. Data are represented as mean±standard deviation. Probability values of t-test are presented **p<0.01. (D) Effect of seliciclib and flavopiridol on CCND1 and CCNE1 expression. Cells were incubated in the presence of 25 µM seliciclib, 100 ng/ml flavopiridol or combination of both for 8 hours. Cells were lyzed and extracts were subjected to immunoblotting, utilizing CCND1 and CCNE1 antibodies. β-actin was used to confirm equal protein loading.

Figure 7. CCNE1 over-expression.

RPMI8226 a low-CCNE1-expressing cell line was stably transfected with pRCCMV2hCyclin E1. (A) Stable clones have been selected by addition of G418, and cell extracts of selected cloned were subjected to immunoblotting, utilizing CCNE1 antibodies. CDK2 expression serves as an internal loading control. (B) Stable clones have been selected by addition of G418, and cell extracts of selected cloned were subjected to qRT-PCR. Presented is the relative amount of CCNE1 as compared to the levels of GAPDH. Data are represented as mean±standard deviation. (C) CCNE1-overexpressing stable cell lines as well as paternal cell line were incubated in the absence or presence of increasing concentrations of seliciclib for 3 days. Cell viability was determined by MTT assay. Data are represented as mean±standard deviation. Experiments were performed twice and one representative result is presented.

Figure 8. CCNE1 silencing.

ARH77 and RPMI8226 cells were transfected with control or CCNE1 siRNA (500 nM) using electroporation. (A) 48 hours following the transfection the cells were lyzed and subjected to immunoblotting, utilizing CCNE1 antibody. β-actin was used to confirm equal protein loading. (B) Quantification of CCNE1 protein levels 48 hours post transfection using TINA software. Data represents the mean and standard deviation of two different experiments. Probability values of t-test are presented **p<0.01. (C) At 48 hours following the transfection the cells were treated with seliciclib (10–40 µM) for additional 48 hours. Viability was determined using MTT assay. Data are represented as mean±standard deviation (**p<0.01). Experiments were performed twice and one representative result is presented.

Figure 9. Seliciclib effect on adhesion and cytotoxicty.

(A) The indicated cells were pre-incubated in the absence or presence of increasing concentrations of seliciclib for one hour, following which the cells were plated onto 40 µg/ml fibronectin coated plates. Following one hour incubation, suspended cells were removed by repeated washes. Quantification of the adherent cells was performed by crystal violet staining. Data are represented as mean±standard deviation. (B) The indicated cell lines were allowed to adhere to 40 µg/ml FN-coated plates for one hour, following which they were exposed to increasing concentrations of seliciclib for three days. Cell viability was determined by MTT assay. Data are represented as mean±standard deviation. (A–B) Experiments were performed at least 3 times and one representative result is presented. (C) ARH77 and NCI H929 were incubated in the absence or presence of BMSCs, treated with seliciclib (10 µM for NCI H929 and 20 µM for ARH77), doxorubicin (10 ng/ml), flavopiridol (100 ng/ml) or combinations during 48 hours. Cell viability was determined by MTT assay. Signal produced by BMSCs alone was subtracted as background.