Figure 1.
Expression of CDK2 and MITF in a panel of melanoma cell lines.
A) CDK2 mRNA and protein expression levels in a panel of melanoma cell lines. mRNA levels in the melanoma panel were measured via microarray analysis. Data shows melanoma cell lines harboring the BRAF V600E mutation (blue) and the NRAS mutations (red). Protein expression was measured by Western blotting. B) MITF mRNA and protein expression levels in a panel of melanoma cell lines. mRNA levels in the melanoma cell lines were measured via microarray analysis. Data shows melanoma cell lines harboring the BRAF V600E mutation (blue) and the NRAS mutations (red). MITF protein expression was measured by Western blotting. Blots were stripped once and probed for actin to show equal protein loading. Relative CDK2 expression is denoted as “CDK2 LOW” and “CDK2 HIGH”.
Figure 2.
Dinaciclib inhibits the growth and survival of melanoma cell lines grown under both 2D adherent culture and 3D organotypic cell culture conditions.
A) Growth inhibition for melanoma cells treated with dinaciclib measured by the MTT assay. Cells were treated with increasing concentrations of dinaciclib (1 nM–10 µM) for 72 hours before treatment with the MTT reagent. Absorbances were read at 570 nm and expressed as a percentage of control. B) Dinaciclib reduces the viability and invasion of melanoma cells grown as three-dimensional collagen-implanted spheroids. Preformed melanoma spheroids that harbored either the BRAF V600E mutation (1205Lu) or NRAS mutation (WM1366) were embedded into collagen and overlaid with medium. Cells were then treated with either 10 nM or 30 nM dinaciclib for 72 hours before being stained with calcein-AM and ethidium bromide. Spheroids were visualized and photographed using an inverted fluoresce microscope. Green, viable cells; red, dead cells.
Figure 3.
Dinaciclib causes a G2/M arrest and apoptosis in a panel of melanoma cell lines.
A) Dinaciclib causes a G2/M arrest within 12 hours of treatment. Cell cycle analysis of a BRAF V600E (1205Lu) mutated or NRAS mutated (WM1366) cell line. Cells were treated with 30 nM of dinaciclib for either 12 hours or 24 hours; then cells were fixed for 24 hrs, and then stained for DNA content with propidium iodide. B) Dinaciclib causes apoptosis in a panel of melanoma cell lines independent of BRAF V600E (blue), NRAS (red) mutational status and CDK2 levels. Cells were treated with 30 nM dinaciclib for 48 hrs and apoptosis was assessed via Annexin-V staining. Data represent % of cells that were Annexin V +. Data shows mean of three independent experiments +/− S.E.
Figure 4.
Dinaciclib induces regression of WM1366 melanoma xenografts.
A) Photographs of representative vehicle and dinaciclib-treated WM1366 tumors taken after day 14 of treatment. After tumor establishment, mice were dosed twice daily with either vehicle (20% HPBCD) or dinaciclib (5 mg/kg in 20% HPBCD) intraperitoneally for 14 days. B) Growth curves of vehicle or dinaciclib-treated mice. Tumor volumes were measured twice a week and were normalized to the start volumes. Dinaciclib led to tumor regression after 14 days of treatment (p = .0046). C) Dinaciclib treatment led to the downregulation of pRBser807/811, upregulation of p53, and inhibition of Bcl-2 Tumor samples were extracted from SCID mice after 14 days of treatment with dinaciclib or vehicle and were analyzed via western blot. Blots were stripped once and equal protein loading was confirmed by probing the blots for actin expression.
Figure 5.
Dinaciclib treatment induces the up-regulation of p53 expression and caspase-3 cleavage and a downregulation of the pro-survival molecules Bcl-2, XIAP, and Mcl-1 in vitro.
Western Blot data of dinaciclib treated 1205Lu cells treated with 30 nM of dinaciclib for increasing periods of time (0–48 hrs). Along with a decrease in pRBser807/811, dinaciclib induced a marked upregulation of p53, increase in cleaved caspase-3, and down regulation of the pro-survival molecules Bcl-2, XIAP, and Mcl-1. The membrane was probed for actin expression to ensure equal protein loading.
Figure 6.
shRNA knockdown of p53 expression reverses dinaciclib-induced apoptosis.
A) Knockdown of p53 levels using a lentiviral shRNA construct. WM35 melanoma cells were infected with a lentivirus encoding for shRNA against p53 and were selected by flow cytometric sorting for GFP (designated WM35 shp53). Western blot shows knockdown of p53 protein. Actin shows equal protein loading. B) WM35 cells expressing p53 shRNA show diminished cell accumulation in the sub-G1 phase of the cell cycle following dinaciclib treatment. Cells were treated with 30 nM of dinaciclib for either 12 hours or 24 hours, before being fixed and stained for DNA content with propidium iodide. C) Apoptosis in a wild-type p53 melanoma cell line is dependent upon p53. WM35 and WM35 shp53 cells were treated with 30 nM dinaciclib for 48 hrs and were assessed for apoptosis via Annexin-V staining. WM35 shp53 cells exhibited a dramatic reduction of apoptosis when compared to WM35. D) The knockdown of p53 led to decreased pRB (ser807/811) and a decrease in cleaved PARP in cells treated with dinaciclib. WM35 and WM35 shp53 cells were treated with 30 nM of dinaciclib for 8, 16, 24, and 48 hrs, after which protein were assessed for the levels of p53, pRBser807/811, PARP, and Mcl-1. Actin was used as loading control.