Limits...
Elevated expression of MITF counteracts B-RAF-stimulated melanocyte and melanoma cell proliferation.

Wellbrock C, Marais R - J. Cell Biol. (2005)

Bottom Line: Microphthalmia-associated transcription factor (MITF) is an important melanocyte differentiation and survival factor, but its role in melanoma is unclear.MITF reexpression in B-RAF-transformed melanocytes inhibits their proliferation.These data suggest that MITF is an anti-proliferation factor that is down-regulated by B-RAF signaling and that this is a crucial event for the progression of melanomas that harbor oncogenic B-RAF.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction Team, Cancer Research UK Centre of Cell and Molecular Biology, The Institute of Cancer Research, London SW3 6JB, England, UK.

ABSTRACT
The protein kinase B-RAF is a human oncogene that is mutated in approximately 70% of human melanomas and transforms mouse melanocytes. Microphthalmia-associated transcription factor (MITF) is an important melanocyte differentiation and survival factor, but its role in melanoma is unclear. In this study, we show that MITF expression is suppressed by oncogenic B-RAF in immortalized mouse and primary human melanocytes. However, low levels of MITF persist in human melanoma cells harboring oncogenic B-RAF, suggesting that additional mechanisms regulate its expression. MITF reexpression in B-RAF-transformed melanocytes inhibits their proliferation. Furthermore, differentiation-inducing factors that elevate MITF expression in melanoma cells inhibit their proliferation, but when MITF up-regulation is prevented by RNA interference, proliferation is not inhibited. These data suggest that MITF is an anti-proliferation factor that is down-regulated by B-RAF signaling and that this is a crucial event for the progression of melanomas that harbor oncogenic B-RAF.

Show MeSH

Related in: MedlinePlus

MITF inhibits proliferation of B-RAF–transformed melanocytes. (A) Melan-a–VE clones VE11 and VE16 were transfected with a Hygromycin resistance plasmid plus empty vector or the MITF expression vector. Western blot shows MITF expression 24 h after transfection in comparison with endogenous MITF in melan-a cells. The cells were then selected for Hygromycin resistance and stained for colonies 15 d after transfection. Results are means from triplicate determinations with error bars to represent the SD. A representative stained cell image is shown to the right of the graph. (B) Western blot analysis of stably expressed HA.ER and HA.ER-MITF in melan-a–VE cells untreated or treated with 200 nM 4-hydroxy-tamoxifen (4-OHT) for 24 h. HA.ER and HA.ER-MITF were revealed with the anti-HA antibody. (C) Luciferase assay of melan-a–VE HA.ER or HA.ER-MITF cells transfected with a tyrosinase promoter luciferase reporter. 24 h after transfection, cells were untreated or treated with 200 nM 4-OHT for a further 24 h. Error bars represent SD from the mean. (D) Growth curve of melan-a–VE HA.ER- or HA.ER-MITF–expressing cells in the absence or presence of 200 nM 4-OHT. Error bars represent SD from the mean.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171350&req=5

fig3: MITF inhibits proliferation of B-RAF–transformed melanocytes. (A) Melan-a–VE clones VE11 and VE16 were transfected with a Hygromycin resistance plasmid plus empty vector or the MITF expression vector. Western blot shows MITF expression 24 h after transfection in comparison with endogenous MITF in melan-a cells. The cells were then selected for Hygromycin resistance and stained for colonies 15 d after transfection. Results are means from triplicate determinations with error bars to represent the SD. A representative stained cell image is shown to the right of the graph. (B) Western blot analysis of stably expressed HA.ER and HA.ER-MITF in melan-a–VE cells untreated or treated with 200 nM 4-hydroxy-tamoxifen (4-OHT) for 24 h. HA.ER and HA.ER-MITF were revealed with the anti-HA antibody. (C) Luciferase assay of melan-a–VE HA.ER or HA.ER-MITF cells transfected with a tyrosinase promoter luciferase reporter. 24 h after transfection, cells were untreated or treated with 200 nM 4-OHT for a further 24 h. Error bars represent SD from the mean. (D) Growth curve of melan-a–VE HA.ER- or HA.ER-MITF–expressing cells in the absence or presence of 200 nM 4-OHT. Error bars represent SD from the mean.

Mentions: To examine the biological consequences of MITF regulation by B-RAF, we reexpressed MITF in melan-a–VE cells. This caused a significant (73–84%) reduction in the number of colonies that were formed by these cells (Fig. 3 A). To clarify whether this effect was caused by inhibition of proliferation or induction of apoptosis, we developed melan-a–VE cell lines expressing an estrogen receptor (ER) version of MITF (ER-MITF; Carreira et al., 2005) that can be regulated by 4-hydroxy-tamoxifen (4-OHT) and developed a control cell line expressing only the ER fragment. Both proteins are expressed at similar levels (Fig. 3 B). ER-MITF activates the tyrosinase promoter in a 4-OHT–dependent manner, whereas the ER fragment does not (Fig. 3 C), demonstrating that ER-MITF is functional. Critically, ER-MITF activation does not induce apoptosis in melan-a–VE but significantly impairs its proliferation (38% reduction, P = 0.0116; Fig. 3 D).


Elevated expression of MITF counteracts B-RAF-stimulated melanocyte and melanoma cell proliferation.

Wellbrock C, Marais R - J. Cell Biol. (2005)

MITF inhibits proliferation of B-RAF–transformed melanocytes. (A) Melan-a–VE clones VE11 and VE16 were transfected with a Hygromycin resistance plasmid plus empty vector or the MITF expression vector. Western blot shows MITF expression 24 h after transfection in comparison with endogenous MITF in melan-a cells. The cells were then selected for Hygromycin resistance and stained for colonies 15 d after transfection. Results are means from triplicate determinations with error bars to represent the SD. A representative stained cell image is shown to the right of the graph. (B) Western blot analysis of stably expressed HA.ER and HA.ER-MITF in melan-a–VE cells untreated or treated with 200 nM 4-hydroxy-tamoxifen (4-OHT) for 24 h. HA.ER and HA.ER-MITF were revealed with the anti-HA antibody. (C) Luciferase assay of melan-a–VE HA.ER or HA.ER-MITF cells transfected with a tyrosinase promoter luciferase reporter. 24 h after transfection, cells were untreated or treated with 200 nM 4-OHT for a further 24 h. Error bars represent SD from the mean. (D) Growth curve of melan-a–VE HA.ER- or HA.ER-MITF–expressing cells in the absence or presence of 200 nM 4-OHT. Error bars represent SD from the mean.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171350&req=5

fig3: MITF inhibits proliferation of B-RAF–transformed melanocytes. (A) Melan-a–VE clones VE11 and VE16 were transfected with a Hygromycin resistance plasmid plus empty vector or the MITF expression vector. Western blot shows MITF expression 24 h after transfection in comparison with endogenous MITF in melan-a cells. The cells were then selected for Hygromycin resistance and stained for colonies 15 d after transfection. Results are means from triplicate determinations with error bars to represent the SD. A representative stained cell image is shown to the right of the graph. (B) Western blot analysis of stably expressed HA.ER and HA.ER-MITF in melan-a–VE cells untreated or treated with 200 nM 4-hydroxy-tamoxifen (4-OHT) for 24 h. HA.ER and HA.ER-MITF were revealed with the anti-HA antibody. (C) Luciferase assay of melan-a–VE HA.ER or HA.ER-MITF cells transfected with a tyrosinase promoter luciferase reporter. 24 h after transfection, cells were untreated or treated with 200 nM 4-OHT for a further 24 h. Error bars represent SD from the mean. (D) Growth curve of melan-a–VE HA.ER- or HA.ER-MITF–expressing cells in the absence or presence of 200 nM 4-OHT. Error bars represent SD from the mean.
Mentions: To examine the biological consequences of MITF regulation by B-RAF, we reexpressed MITF in melan-a–VE cells. This caused a significant (73–84%) reduction in the number of colonies that were formed by these cells (Fig. 3 A). To clarify whether this effect was caused by inhibition of proliferation or induction of apoptosis, we developed melan-a–VE cell lines expressing an estrogen receptor (ER) version of MITF (ER-MITF; Carreira et al., 2005) that can be regulated by 4-hydroxy-tamoxifen (4-OHT) and developed a control cell line expressing only the ER fragment. Both proteins are expressed at similar levels (Fig. 3 B). ER-MITF activates the tyrosinase promoter in a 4-OHT–dependent manner, whereas the ER fragment does not (Fig. 3 C), demonstrating that ER-MITF is functional. Critically, ER-MITF activation does not induce apoptosis in melan-a–VE but significantly impairs its proliferation (38% reduction, P = 0.0116; Fig. 3 D).

Bottom Line: Microphthalmia-associated transcription factor (MITF) is an important melanocyte differentiation and survival factor, but its role in melanoma is unclear.MITF reexpression in B-RAF-transformed melanocytes inhibits their proliferation.These data suggest that MITF is an anti-proliferation factor that is down-regulated by B-RAF signaling and that this is a crucial event for the progression of melanomas that harbor oncogenic B-RAF.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction Team, Cancer Research UK Centre of Cell and Molecular Biology, The Institute of Cancer Research, London SW3 6JB, England, UK.

ABSTRACT
The protein kinase B-RAF is a human oncogene that is mutated in approximately 70% of human melanomas and transforms mouse melanocytes. Microphthalmia-associated transcription factor (MITF) is an important melanocyte differentiation and survival factor, but its role in melanoma is unclear. In this study, we show that MITF expression is suppressed by oncogenic B-RAF in immortalized mouse and primary human melanocytes. However, low levels of MITF persist in human melanoma cells harboring oncogenic B-RAF, suggesting that additional mechanisms regulate its expression. MITF reexpression in B-RAF-transformed melanocytes inhibits their proliferation. Furthermore, differentiation-inducing factors that elevate MITF expression in melanoma cells inhibit their proliferation, but when MITF up-regulation is prevented by RNA interference, proliferation is not inhibited. These data suggest that MITF is an anti-proliferation factor that is down-regulated by B-RAF signaling and that this is a crucial event for the progression of melanomas that harbor oncogenic B-RAF.

Show MeSH
Related in: MedlinePlus