Proliferation Arrest in B-Raf Mutant Melanoma Cell Lines upon MAPK Pathway Activation  Roland Houben, Sonja Ortmann, Astrid Drasche, Jakob Troppmair, Marco J. Herold, Jürgen C. Becker  Journal of Investigative Dermatology  Volume 129, Issue 2, Pages 406-414 (February 2009) DOI: 10.1038/jid.2008.214 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Induction of ERK phosphorylation and a senescence type proliferation arrest by 4-hydroxytamoxifen (OHT) stimulation of Skmel28-Raf-ER cells. SKmel28 Raf-ER cells expressing c-Raf-1-BXB-ER, SKmel28 Raf-ERK375R expressing a kinase dead RAF-ER protein, and Skmel28 EYZ cells stably transfected with the empty vector were incubated in the presence of 200nM OHT. As control, the adequate amount of the solvent (EtOH) was added to the medium. (a) Twenty-four hours following OHT stimulation, total cell lysates were subjected to western blot analysis and probed with the indicated antibodies. For western blot analysis, 2 × 105 cells per well were seeded in 6-well plates. (b) At the indicated time points following OHT stimulation, detached and adherent cells were pooled, pelleted, and resuspended in trypan blue solution. The number of cells excluding trypan was plotted against time. (c) The percentage of cells, not excluding trypan blue, was plotted against time. For assessment of proliferation and viability 4 × 103 cells per well were seeded in duplicates in 48-well plates. (d) Skmel28 Raf-ER cells were seeded with 0.5 × 104 cells per well in triplicates in 96-well plates. Following 20hours in the absence or presence of OHT, cells were incubated for 3hours with BrdU. BrdU incorporation was than measured by an immunoassay. (e) After 6 days in the presence or absence of OHT, Skmel28 Raf-ER cells were trypsinized and subjected to an X-Gal assay to visualize expression of β-galactosidase. (f) Following 2 days in the absence or presence of OHT Skmel28 Raf-ER cells were analyzed by flow cytometry for the expression of ki-67. Journal of Investigative Dermatology 2009 129, 406-414DOI: (10.1038/jid.2008.214) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Correlation of ERK phosphorylation and proliferation upon titration of 4-hydroxytamoxifen (OHT) to Skmel28-Raf-ER cells. Skmel28-Raf-ER cells were incubated with the indicated concentration of OHT and 24hours following OHT stimulation total cell lysates were subjected to western blot analysis and probed with the indicated antibodies. Proliferation of the cells was assessed using the MTS assay after 5 days of incubation with OHT. For the MTS assay, the cells were seeded in triplicates with 1 × 103 cells per well in 96-well plates. Journal of Investigative Dermatology 2009 129, 406-414DOI: (10.1038/jid.2008.214) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Induction of ERK phosphorylation and proliferation arrest by 4-hydroxytamoxifen (OHT) stimulation of Mel2a-Raf-ER, FM88-Raf-ER, and NIH3T3-Raf-ER cells. The melanoma cell-line derivates, Mel2a-Raf-ER and FM88-Raf-ER as well as NIH3T3-Raf-ER, expressing c-Raf-1-BXB-ER were incubated in the presence of 200nM OHT. To unstimulated cells adequate amount of the solvent (EtOH) was added to the medium. (a) At the indicated time points following OHT stimulation total cell lysates were subjected to western blot analysis and probed with the indicated antibodies. For western blot analysis 2 × 105 cells per well were seeded in six-well plates. (b–d) At the indicated time points following OHT stimulation, detached and adherent cells were pooled, pelleted, and resuspended in trypan blue solution. The number of cells excluding trypan was plotted against time. For assessment of proliferation and viability 1 × 104 cells per well were seeded in duplicates in 24-well plates. Journal of Investigative Dermatology 2009 129, 406-414DOI: (10.1038/jid.2008.214) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 MEK dependency of the 4-hydroxytamoxifen (OHT)-induced proliferation arrest and mitogen-activated protein kinase (MAPK) pathway-dependent signaling crosstalk. SKmel28-Raf-ER cells were cultured in the presence or absence of 200nM OHT and the MEK inhibitor U0126 was added with varying concentrations (as indicated). (a) After 4 days of incubation, detached and adherent cells were pooled, pelleted, and resuspended in trypan blue solution. The numbers of cells excluding trypan blue are depicted. For proliferation analysis, cells were seeded with 2 × 103 cells per well in 96-well plates. (b) Twenty-four hours following addition of 200nM OHT and/or U0126 at the indicated concentration cell lysates were subjected to western blot analysis and probed with the indicated antibodies. For western blot analysis, 2 × 105 cells per well were seeded in 6-well plates. Journal of Investigative Dermatology 2009 129, 406-414DOI: (10.1038/jid.2008.214) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 The mitogen-activated protein kinase (MAPK) pathway activation induced proliferation arrest shifts from a reversible to an irreversible state. Cells were pretreated with 200nM 4-hydroxytamoxifen (OHT) for 0, 2, and 5 days, respectively. Thereafter cells were seeded with 1 × 103 cells per well in 96-well plates. Cells pretreated with OHT were subsequently cultivated in the presence of 200nM OHT whereas the cells not pretreated were cultured either with or without OHT. As indicated, varying concentrations of the MEK inhibitor U0126 were added. Proliferation of the cells during a 5-day incubation period was assessed using the MTS assay. The extinction values measured in the absence of U0126 were set as 100% and relative extinctions are plotted. Journal of Investigative Dermatology 2009 129, 406-414DOI: (10.1038/jid.2008.214) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Induction of p21 and decreased phosphorylation of the RB protein upon mitogen-activated protein kinase (MAPK) pathway activation. Following incubation in the presence or absence of 200nM OHT, total cell lysates of the indicated Raf-ER expressing cell lines were subjected to western blot analysis and probed with the indicated antibodies. The phospho-RB signals in the murine NIH3T3 Raf-ER cells were only detectable upon prolonged exposition of the film. Journal of Investigative Dermatology 2009 129, 406-414DOI: (10.1038/jid.2008.214) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Cell-cycle distribution following activation or inactivation of the mitogen-activated protein kinase (MAPK) pathway. Cells were seeded with 2 × 105 cells per well in 6-well plates. Untreated cells as well as 4-hydroxytamoxifen (OHT; 200nM) or U0126 (20μM) treated cells were harvested at the indicated time points. Untreated cells were harvested 24hours following seeding. Journal of Investigative Dermatology 2009 129, 406-414DOI: (10.1038/jid.2008.214) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions