Chemotherapy adds to cell death induced by Mcl-1 and A1 inhibition.

<p>(A) 1205Lu melanoma cells were treated with the indicated siRNAs together with 1 µM 5-fluorouracil (5-FU) or not (no treatment) and analyzed on day 3 after transfection. Left panel: Cell viability was determined. Viability of control siRNA-treated cells without 5-FU treatment was set to 100%. Right panel: Cell death analysis measured by FACS after staining with Annexin V and propidium iodide. (B) Analysis of cell viability (left panel) or cell death (right panel) of 1205Lu cells on day 6 treated as described in A. (C) Analysis of cell viability (left panel) or cell death (right panel) of primary human fibroblasts on day 6 treated as described in A. If not otherwise indicated, asterisks represent significant increase in cell death compared to control siRNA-treated cells. Mean +/− SD of 3 independent experiments is shown in A, B, and C. AN, Annexin V; PI, propidium iodide.</p

Expression of antiapoptotic Bcl-2 proteins in non-malignant skin cells and melanoma cell lines.

<p>(A) Expression of Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 protein in primary human fibroblasts, keratinocytes, and melanocytes was analyzed by immunoblotting. For each cell type, 2 donors are shown. (B) Expression of Bcl-2, Bcl-xL, Bcl-w and Mcl-1 protein in melanocytes isolated from 4 donors. (C) Expression of Bcl-2, Bcl-xL, Bcl-w and Mcl-1 protein in melanoma cell lines of different progression levels. Blot is representative for 2 independent experiments. (D) Levels of A1 mRNA were measured by quantitative RT-PCR. Mean +/− SD of 3 donors is shown for non-malignant cells, mean +/− SD of 3 cell passages is shown for melanoma cell lines. β-actin served as loading control in immunoblots. The same protein sample of melanocytes from donor #2 was loaded on all gels as a reference to allow the comparison of expression levels among the immunoblots shown in A, B, and C. RGP: radial-growth phase (non-invasive); VGP: vertical growth phase (invasive).</p

Inhibition of Mcl-1 or A1 leads to melanoma-specific cell death.

<p>(A) Primary human fibroblasts and 1205Lu melanoma cells were treated with the indicated siRNAs and expression of Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 was analyzed after 48 hours by immunoblotting. (B) A1 mRNA was measured by quantitative RT-PCR in cells treated with A1-specific- or control siRNA for 48 hours (left and middle panel). Non-targeted Bcl-2 proteins were analyzed at the same time point by immunoblotting (right panel). (C) Cell death of fibroblasts was determined by quantification of Annexin V (AN)- and propidium iodide (PI)-positive cells 72 hours after transfection of the indicated siRNAs. (D) Cell death analysis of 1205Lu melanoma cells treated as described in C. Asterisks indicate a significant increase in cell death versus control siRNA-treated cells. (E) Fluorescence-activated cell sorting (FACS) of apoptotic and dead fibroblasts (upper panel) or 1205Lu melanoma cells (lower panel) treated as described in C. Numbers indicate the portion of cells in each quadrant that defines Annexin V- or propidium iodide-positive or negative cells. Mean +/− SD of at least 3 independent experiments is shown in B, C, and D. α-tubulin served as loading control in immunoblots. Blots are representative for 3 independent experiments.</p

Combined inhibition of Mcl-1 and A1 results in efficient induction of apoptosis in 1205Lu melanoma cells.

<p>(A) 1205Lu melanoma cells were transfected with A1- or Mcl-1-specific siRNAs or control siRNAs as indicated. Cell death was assessed 72 hours after transfection. Mean +/− SD of 3 independent experiments is shown. Asterisks represent significant increase in cell death compared to single inhibition. (B) 1205Lu melanoma cells were transfected with different siRNA sequences targeting A1 or Mcl-1 (A1b, Mcl-1b). Analysis was carried out as described in A. Asterisks represent significant increase in cell death compared to single inhibition. Also, cell death induction by A1b, Mcl-1b, and A1b/Mcl-1b versus control was significant (not indicated in the figure). (C) Caspase activation of 1205Lu cells treated as described in A was assessed by immunoblotting with antibodies detecting the proform as well as the active subunits. Caspase 9 (upper panel) and caspase 3 (lower panel) were analyzed 48 hours after transfection. (D) 1205Lu cells treated with siRNAs as described in A with or without the pan-caspase inhibitor zVAD-FMK (100 µM). Cell death was measured 72 hours after transfection. Mean +/− SD of 3 independent experiments is shown. Asterisks represent significant decrease in cell death of zVAD-FMK-treated cells compared to cells not treated with zVAD-FMK. AN, Annexin V; PI, propidium iodide.</p

Representative light microscopy images of 1205Lu melanoma cells (A) or fibroblasts (B) 6 days after transfection of the indicated siRNAs alone (upper panel) or together with 1 µM 5-FU (lower panel).

<p>Scale bar = 0.5 mm.</p

Combined inhibition of Mcl-1 and A1 results in efficient induction of cell death in a panel of melanoma cell lines, but not in keratinocytes.

<p>(A) Primary human keratinocytes (upper left panel), non-invasive melanoma cells (WM3211), invasive (WM793), and metastatic melanoma cells (WM1232, WM239A, WM1158) were transfected with the siRNAs as indicated. Cell death was determined 72 hours after transfection. Mean +/− SD of 3 (keratinocytes, WM1232, WM239A, WM1158) or 5 (WM3211 and WM793) independent experiments is shown. (B) A1 mRNA was measured by quantitative RT-PCR in indicated cells treated with A1-specific- or control siRNA for 48 hours. Mean +/− SD of 3 independent experiments is shown. Asterisks represent significant increase in cell death compared to control siRNA-treated cells. AN, Annexin V; PI, propidium iodide.</p