PMA-induced Cell Differentiation Research Articles

Abstract 3915: Inhibition of cdc2 is linked to PMA-induced cell differentiation

Abstract The critical role of cdc2 (CDK1) in G2-M transition of the cell cycle control in mammalian cells has been well documented. Recent studies have also demonstrated that cdc2 is capable of driving G1 progress and G1-S transition via association with multiple interphase cyclins and through the interactions with Rb proteins. However, whether this pluripotent CDK regulates cell differentiation is unclear. Thus, the effect of phorbol 12-myristate 13-acetate (PMA) on cell differentiation and the expression of cdc2 in a human myeloid leukemia cell line, TF-1a, was investigated. When TF-1a cells were treated with 10−5, 10−6, and 10−7 PMA for 48 and 72h, they showed marked (≥50%) macrophage-like changes, evidenced by significant decrease in nucleus/cytoplasm ratio and increase (1-3 folds) in the expression of IL-1β, a macrophage marker. PMA treatment also caused time-dependent inhibition of cdc2 in both cytosol and nucleus of the cells, with a maximal inhibition being observed by 48 and 72h, which paralleled with the cell differentiation course. In contrast, there was no significant cell differentiation and inhibition of cdc2 being observed in control human myeloid leukemia TF-1 cells treated with PMA. Previous studies suggest that prolonged activation of MAPK pathway is linked to cell differentiation. PMA treatment also rapidly induced phosphorylation of MAPK kinases (MEK and ERK), which persisted for 24h, after which phosphorylated MEK and ERK were returned to base level, while the expression of cdc2 was still significantly downregulated, as compared with the control cells treated with DMSO. Pretreatment of TF-1a cells with sense cdc2 oligo nucleotides partially inhibited PMA-induced differentiation and upregulation of IL-1β. Taken together, our data suggest that MAPK pathway may be responsible for initiation of cell differentiation whereas inhibition of cdc2 is required for late differentiation of TF-1a cells in response to PMA stimulation. Whether activation of the MAPK pathway inhibits expression of cdc2 is currently under investigation (Supported by Faculty Incentive Grant and NIH-NIGMS MBRS RISE: R25 GM059244-13, Barry University). Note: This abstract was not presented at the meeting. Citation Format: Daria Vasilyeva, Pairat Dolinsky, Shashana Fielder, Alice Nakasone, Gerhild Packert, Xiaotang Hu. Inhibition of cdc2 is linked to PMA-induced cell differentiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3915. doi:10.1158/1538-7445.AM2015-3915

The DDX5 protein is involved in proliferation and differentiation of human cultured cells

Abstract—The expression of the DDX5 protein (RNA helicase p68) in cells correlates with processes of proliferation and differentiation. However, involvement of the protein in these processes has not been proven yet. In this paper, we have studied the effect of DDX5 protein inactivation (with small interfering RNAs) on the proliferation of Jurkat cell line and DDX5 expression dynamics during differentiation of U-937 cells induced by phorbol 12-myristate 13-acetate (PMA). Flow cytometry showed that the content of DDX5 in Jurkat cells in G0 and G1 phases was lower than compared with the G2/M phases. After induction of U-937 cell differentiation, an expression peak was observed during the first 2–3 h, followed by further reduction of the DDX5 concentration (4 h). The number of macrophage surface markers CR3 on the cell membrane increased only 24 h after PMA-induced cell differentiation. Hence, our data suggest that (1) the DDX5 protein is essential for cell proliferation and (2) the DDX5 protein concentration in U-937 cells increases during the first hours after stimulation with PMA followed by its decrease (after 4 ho) long before the macrophage marker appears on the cell surface.

Megakaryocytic differentiation of K562 cells induced by PMA reduced the activity of respiratory chain complex IV.

Mitochondria are involved in the regulation of cell differentiation processes, but its function changes and molecular mechanisms are not yet clear. In this study, we found that mitochondrial functions changed obviously when K562 cells were induced to megakaryocytic differentiation by phorbol 12-myristate 13-acetate (PMA). During the cell differentiation, the reactive oxygen species (ROS) level was increased, mitochondrial membrane potential declined and respiratory chain complex IV activity was decreased. Treatment with specific inhibitor of mitochondrial respiratory chain complex IV led to a significant inhibition in mitochondrial membrane potential and reduction of PMA-induced cell differentiation. However, treatment with cyclosporine A, a stabilization reagent of mitochondrial membrane potential, did not improve the down-regulation of mitochondrial respiratory chain complex IV induced by PMA. Furthermore, we found that the level of the complex IV core subunit COX3 and mitochondrial transport-related proteins Tim9 and Tim10 were decreased during the differentiation of K562 cells induced by PMA, suggesting an important role of these factors in mitochondrial functional changes. Our results suggest that changes in mitochondrial functions are involved in the PMA-induced K562 cell differentiation process, and the maintenance of the steady-state of mitochondrial functions plays a critical role in the regulation of cell differentiation.

1P257 Role of NADPH oxidase in vitamin D_3 and PMA-induced cell differentiation(19. Radiobiology & Active oxygen,Poster)

1P257 Role of NADPH oxidase in vitamin D_3 and PMA-induced cell differentiation(19. Radiobiology & Active oxygen,Poster)

The activation of PI 3-K and PKC ζ in PMA-induced differentiation of HL-60 cells

The human myelocytic leukemia cell line HL-60 is a useful model for the study of cellular differentiation. Phorbol 12-myristate 13-acetate (PMA) induces the monocyte/macrophage-like differentiation of HL-60 cells and results in growth arrest, increasing adherence. In PMA-induced differentiation of HL-60 cells, phosphoinositide 3-kinase (PI 3-K) activity was measured as phosphatidylinositol(3)P recovery from phosphatidylinositol by in vitro kinase assay. PI 3-K activity was increased in HL-60 cells that were stimulated by 20 nM PMA and the activity was inhibited by pretreatment with 20 μM LY294002, a specific inhibitor of PI 3-K. Members of the protein kinase C (PKC) family have been suggested to be one of the downstream targets of PI 3-K. PKC ζ is one of the atypical PKCs, non-diacylglycerol-responsive PKCs, and the activity was measured by the ability of phosphorylation onto myelin basic protein. PMA also induced the activation of PKC ζ during monocytic differentiation of HL-60 cells, and LY294002-pretreated cells failed to induce PKC ζ activation. The activity of PI 3-K is essential for PKC ζ activation, and LY294002 blocks both monocytic differentiation of HL-60 cells and activation of PKC ζ during PMA-induced cell differentiation. This implies that activated PI 3-K subsequently stimulates the PKC ζ in the process of PMA-induced monocytic differentiation.

Specific activation by concanavalin A of the superoxide anion generation capacity during U937 differentiation

Phorbol myristate acetate (PMA) induces changes in the human monocyte-macrophage-like cell line U937 which reflect cellular differentiation. PMA prompted the expression of the superoxide anion (O2-) generating capacity in U937 upon appropriate stimulation. A highly specific stimulation by Concanavalin A (Con A) of O2- release was observed in PMA-differentiated U937 cells, which exceeded in 10-20 times that obtained with Con A-stimulated monocytes and neutrophils. These results indicate that a highly specific machinery required for Con A stimulation, practically absent in mature monocytes and neutrophils, is synthesized during PMA-induced U937 differentiation. A novel cytochrome b putatively involved in O2- generation was detected in U937 cells. This cytochrome b content was increased during PMA-induced cell differentiation, although no linear correlation was found between capability to produce O2- by macrophage-like U937 cells and their content of cytochrome b.

Protein-pattern changes and morphological effects due to methionine starvation or treatment with 5-azacytidine of the phorbol-ester-sensitive cell lines HL-60, CCL-119, and U-937.

Methionine starvation causes changes in the protein pattern of HL-60 promyelocytic leukemia cells as observed by two-dimensional electrophoresis. One group of proteins is apparently modified, appearing in new positions. A further series of proteins, including several principal nuclear polypeptides, is substantially diminished. The morphology of a fraction of the cells in the culture changes concomitantly, with condensation and fragmentation of the nucleus and eventual remolding of the cell to a "grape-cluster" appearance. Similar effects are produced by a DNA methylation inhibitor, 5-azacytidine, but not by various other toxic agents tested. A defect in DNA methylation, either by depletion of S-adenosyl-L-methionine (the methyl donor) or by inactivation of the relevant enzyme, may be responsible. The T-lymphoblastoid line CCL-119 and the histiocytic lymphoma line U-937 also show these effects, but most fibroblast, epithelial, and lymphoblastoid lines do not. These changes can be largely prevented in each of the three susceptible lines by prior treatment with the tumor promoter, phorbol myristate acetate (PMA), an agent known to cause differentiation in at least two of the lines. The results thus suggest interesting relationships between methionine metabolism, protein and structural changes in the cell nucleus, and PMA-induced cell differentiation.