Decrease in protein tyrosine phosphorylation is associated with F-actin reorganization by retinoic acid in human endometrial adenocarcinoma (RL95-2) cells

Abstract

Transformed cells often express elevated levels of tyrosine-phosphorylated proteins. Inhibition of protein tyrosine kinases causes reversion of malignant cells to the normal phenotype. In the present study, we evaluated the possibility that the reversion of human endometrial adenocarcinoma RL95-2 cells to a stationary phenotype induced by retinoic acid was associated with inhibition of tyrosine phosphorylation of cellular proteins. We found that retinoic acid decreased the levels of tyrosine-phosphorylated proteins, as assessed by immunostaining and immunoprecipitations using specific anti-phosphotyrosine antibodies. In addition, the inhibitors of tyrosine kinases herbimycin A and tyrphostin mimicked retinoic acid, inducing F-actin reorganization and increasing the size of RL95-2 cells, as determined by measurement of cell perimeters. Because focal adhesions that connect actin filaments with the plasma membrane are major sites of tyrosine phosphorylation, we further investigated whether selected focal adhesion proteins were affected by retinoic acid. We found that retinoic acid altered the localization of focal adhesion kinase. All-trans retinoic acid was effective in reducing the levels of focal adhesion kinase and paxillin protein. Thirteen-cis retinoic acid increased the levels of vinculin protein in the cytosolic fraction of cells. These changes are consistent with actin reorganization and reversion toward a stationary phenotype induced by retinoic acid in endometrial adenocarcinoma RL95-2 cells. Our results indicate that the differentiating effects of retinoids on endometrial cells are associated with decreases in tyrosine phosphorylation and changes in the levels and distribution of focal adhesion proteins. These findings suggest that signaling pathways that involve tyrosine kinases are potential targets for drug design against endometrial cancer.

Glucocorticoids Induce Osteocyte Apoptosis by Blocking Focal Adhesion Kinase-mediated Survival

“…For actin filament visualization, cells were fixed in neutral buffered formalin for 10 min, followed by permeabilization with 0.1% Triton X-100 for 5 min, and incubation with 1% bovine serum albumin in phosphatebuffered saline for 30 min to block unspecific staining. Actin filaments were stained with 5 units/ml phalloidin-Alexa Fluor 555 in phosphate-buffered saline containing 0.1% bovine serum albumin for 20 min, as previously described (34). Cells were visualized by confocal microscopy.…”

Sodium butyrate/retinoic acid costimulation induces apoptosis-independent growth arrest and cell differentiation in normal and ras-transformed seminal vesicle epithelial cells unresponsive to retinoic acid

“…Further experimental details are in Materials and Methods. rearrangement of the cytoskeleton could have played an important role in the molecular mechanism of this differentiation process (Higgins & Ryan 1989, Staiano-Coico et al 1990, Gentile et al 1992, Carter & Bellido 1999. A recent work, demonstrating that cytoplasmic actin is an abundant glutaminyl substrate for tTGase (Nemes et al 1997), points also to the possibility that the highly active tTGase induced in SVC1 and Ki-SVC1 cells by NaB/RA treatment could have contributed significantly to such hypothesized cytoskeleton rearrangement.…”