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Integrin alphav-mediated inactivation of p53 controls a MEK1-dependent melanoma cell survival pathway in three-dimensional collagen.

Bao W, Strömblad S - J. Cell Biol. (2004)

Bottom Line: We found that integrin alphav inactivated p53 and that suppression of p53 activity by dominant negative p53 or p53-small interfering RNA obviated the need for integrin alphav for melanoma cell survival in 3D-collagen and for tumor growth in vivo.Furthermore, we found that melanoma cell integrin alphav was required for MAPK kinase (MEK) 1 and extracellular signal-regulated kinase (ERK)1/2 activity in 3D-collagen, whereas inhibition of MEK1 activity induced apoptosis.Surprisingly, MEK1 and ERK1/2 activities were restored in integrin alphav-negative melanoma cells by suppression of p53, whereas concomitant block of MEK1 induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Karolinska Institutet, Stockholm, 141 86, Sweden.

ABSTRACT
Integrin alphav is required for melanoma cell survival and tumor growth in various models. To elucidate integrin alphav-mediated melanoma cell survival mechanisms, we used a three-dimensional (3D) collagen gel model mimicking the pathophysiological microenvironment of malignant melanoma in the dermis. We found that integrin alphav inactivated p53 and that suppression of p53 activity by dominant negative p53 or p53-small interfering RNA obviated the need for integrin alphav for melanoma cell survival in 3D-collagen and for tumor growth in vivo. This indicates that integrin alphav-mediated inactivation of p53 functionally controls melanoma cell survival. Furthermore, we found that melanoma cell integrin alphav was required for MAPK kinase (MEK) 1 and extracellular signal-regulated kinase (ERK)1/2 activity in 3D-collagen, whereas inhibition of MEK1 activity induced apoptosis. Surprisingly, MEK1 and ERK1/2 activities were restored in integrin alphav-negative melanoma cells by suppression of p53, whereas concomitant block of MEK1 induced apoptosis. This suggests that integrin alphav controls melanoma cell survival in 3D-collagen through a pathway involving p53 regulation of MEK1 signaling.

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Regulation of melanoma cell caspase cleavage by integrin αv in 3D-collagen. Cleaved caspase-8 and caspase-9 were detected by immunoblotting in the integrin αv-positive and αv-negative melanoma M21 (αv+) and M21L (αv−) cells (A) as well as M0 (αv−) and M0-αv (αv+) cells (B) cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. Active caspase-8 from cycloheximide-treated Jurkat cells served as a positive control for caspase-8 cleavage. Detection of actin served as loading control. The displayed blots are representative among three independent experiments.
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fig2: Regulation of melanoma cell caspase cleavage by integrin αv in 3D-collagen. Cleaved caspase-8 and caspase-9 were detected by immunoblotting in the integrin αv-positive and αv-negative melanoma M21 (αv+) and M21L (αv−) cells (A) as well as M0 (αv−) and M0-αv (αv+) cells (B) cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. Active caspase-8 from cycloheximide-treated Jurkat cells served as a positive control for caspase-8 cleavage. Detection of actin served as loading control. The displayed blots are representative among three independent experiments.

Mentions: Unligated integrin αvβ3 may induce epithelial cell apoptosis in 3D-collagen by recruitment and activation of caspase-8 (Stupack et al., 2001). To examine potential caspase activation of integrin αv-positive and αv-negative melanoma cells cultured in 3D-collagen, we analyzed the presence of cleaved (active) caspases 8 and 9 in M21 (αv+), M21L (αv−), M0 (αv−), and M0-αv (αv+) cells (Fig. 2, A and B). We found no cleaved caspase-8 in any of these four melanoma cell types when cultured in 3D-collagen, whereas caspase-8 cleavage was clearly visible in cycloheximide-treated Jurkat cells serving as positive control. Interestingly, caspase-9 were cleaved to a higher degree in integrin αv-negative M21L and M0 cells as compared with integrin αv-positive M21 and M0-αv cells, respectively, in 3D-collagen, indicating that loss of integrin αv leads to activation of caspase-9. Given that caspase-9 is active in the mitochondrial apoptotic pathway (Green and Kroemer, 1998), this suggests that the observed apoptosis in M21L (αv−) and M0 (αv−) cells might be mediated via mitochondria.


Integrin alphav-mediated inactivation of p53 controls a MEK1-dependent melanoma cell survival pathway in three-dimensional collagen.

Bao W, Strömblad S - J. Cell Biol. (2004)

Regulation of melanoma cell caspase cleavage by integrin αv in 3D-collagen. Cleaved caspase-8 and caspase-9 were detected by immunoblotting in the integrin αv-positive and αv-negative melanoma M21 (αv+) and M21L (αv−) cells (A) as well as M0 (αv−) and M0-αv (αv+) cells (B) cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. Active caspase-8 from cycloheximide-treated Jurkat cells served as a positive control for caspase-8 cleavage. Detection of actin served as loading control. The displayed blots are representative among three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172581&req=5

fig2: Regulation of melanoma cell caspase cleavage by integrin αv in 3D-collagen. Cleaved caspase-8 and caspase-9 were detected by immunoblotting in the integrin αv-positive and αv-negative melanoma M21 (αv+) and M21L (αv−) cells (A) as well as M0 (αv−) and M0-αv (αv+) cells (B) cultured under 2D conditions (d 0) and within 3D-collagen for the indicated times. Active caspase-8 from cycloheximide-treated Jurkat cells served as a positive control for caspase-8 cleavage. Detection of actin served as loading control. The displayed blots are representative among three independent experiments.
Mentions: Unligated integrin αvβ3 may induce epithelial cell apoptosis in 3D-collagen by recruitment and activation of caspase-8 (Stupack et al., 2001). To examine potential caspase activation of integrin αv-positive and αv-negative melanoma cells cultured in 3D-collagen, we analyzed the presence of cleaved (active) caspases 8 and 9 in M21 (αv+), M21L (αv−), M0 (αv−), and M0-αv (αv+) cells (Fig. 2, A and B). We found no cleaved caspase-8 in any of these four melanoma cell types when cultured in 3D-collagen, whereas caspase-8 cleavage was clearly visible in cycloheximide-treated Jurkat cells serving as positive control. Interestingly, caspase-9 were cleaved to a higher degree in integrin αv-negative M21L and M0 cells as compared with integrin αv-positive M21 and M0-αv cells, respectively, in 3D-collagen, indicating that loss of integrin αv leads to activation of caspase-9. Given that caspase-9 is active in the mitochondrial apoptotic pathway (Green and Kroemer, 1998), this suggests that the observed apoptosis in M21L (αv−) and M0 (αv−) cells might be mediated via mitochondria.

Bottom Line: We found that integrin alphav inactivated p53 and that suppression of p53 activity by dominant negative p53 or p53-small interfering RNA obviated the need for integrin alphav for melanoma cell survival in 3D-collagen and for tumor growth in vivo.Furthermore, we found that melanoma cell integrin alphav was required for MAPK kinase (MEK) 1 and extracellular signal-regulated kinase (ERK)1/2 activity in 3D-collagen, whereas inhibition of MEK1 activity induced apoptosis.Surprisingly, MEK1 and ERK1/2 activities were restored in integrin alphav-negative melanoma cells by suppression of p53, whereas concomitant block of MEK1 induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Karolinska Institutet, Stockholm, 141 86, Sweden.

ABSTRACT
Integrin alphav is required for melanoma cell survival and tumor growth in various models. To elucidate integrin alphav-mediated melanoma cell survival mechanisms, we used a three-dimensional (3D) collagen gel model mimicking the pathophysiological microenvironment of malignant melanoma in the dermis. We found that integrin alphav inactivated p53 and that suppression of p53 activity by dominant negative p53 or p53-small interfering RNA obviated the need for integrin alphav for melanoma cell survival in 3D-collagen and for tumor growth in vivo. This indicates that integrin alphav-mediated inactivation of p53 functionally controls melanoma cell survival. Furthermore, we found that melanoma cell integrin alphav was required for MAPK kinase (MEK) 1 and extracellular signal-regulated kinase (ERK)1/2 activity in 3D-collagen, whereas inhibition of MEK1 activity induced apoptosis. Surprisingly, MEK1 and ERK1/2 activities were restored in integrin alphav-negative melanoma cells by suppression of p53, whereas concomitant block of MEK1 induced apoptosis. This suggests that integrin alphav controls melanoma cell survival in 3D-collagen through a pathway involving p53 regulation of MEK1 signaling.

Show MeSH
Related in: MedlinePlus