Phorbol ester induces c-sis gene transcription in stem cell line K-562.

Abstract

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced megakaryoblastic differentiation and c-sis expression in the human hematopoietic stem cell line K-562. This induction occurred at the transcriptional level, as determined by a nuclear runoff transcriptional assay, and was not a generalized effect of TPA, since the treatment of other hematopoietic cell lines and normal peripheral blood lymphocytes with TPA did not result in the appearance of c-sis mRNA.The study of cellular oncogenes (c-onc) has become an important tool in exploring the mechanisms of neoplasia (2). Many proto-oncogenes are also expressed in normal cells during growth and differentiation (7,13,21). Simian sarcoma virus bears in its structure the viral oncogene sis (v-sis) which has its homologous cellular counterpart, c-sis (7). Expression of this oncogene has been reported in glioblastoma and sarcoma cell lines (10,22,28), but it is also expressed in normal endothelial and placental cells (1,13,15). c-sis codes for the B chain of platelet-derived growth factor (4, 9, 16, 23, 27), a potent mitogen for connective tissue cells which is released from platelet alpha granules during clot formation (17). Recently, the sequence of a cDNA clone (from normal endothelial cells) coding for c-sis was reported (6). However, the expression of this oncogene has not been studied in megakaryocytes, the main source of platelet-derived growth factor in the organism, because it is extremely difficult to obtain enough megakaryocytes from normal bone marrow. We report here the expression of c-sis in the stem cell line K-562 during phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA])-induced megakaryocytic differentiation of these cells (12,24,25). The c-sis transcript can be detected as early as 4 h after adding TPA to cultures, even though megakaryocytic differentiation could only be detected after day 2 of treatment. We demonstrate that c-sis expression may occur concomitantly with the megakaryocytic differentiation of K-562 cells rather than as result of nonspecific effects of TPA, since other cell lines and normal peripheral blood lymphocytes treated with TPA do not show c-sis-related transcripts. c-sis induction occurs at the transcriptional level as determined by a nuclear runoff assay.The K-562 cell line was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillinstreptomycin. Log-phase K-562 cells were treated with TPA (Sigma Chemical Co., St. Louis, Mo.) (final concentration, 10-9 M) and kept in culture for variable lengths of time ranging from 1 h to 6 days. Megakaryoblastic differentiation was assessed by flow cytometric analysis with the monoclonal antibody lOES (5; kindly provided by B. Coller, State University of New York at Stony Brook) which recognizes the GpIIb-IIIa complex, a specific megakaryocytic marker expressed early in the differentiation of this lineage (19,26). Cells positive for the GpIIb-IIIa complex were detected after '* Corresponding author.2 days of TPA treatment, and by 4 days, the majori...

PMA-induced phenotypic changes in K562 cells: MAPK-dependent and -independent events

“…11,[17][18][19] We detected PMA-induced expression of Pyk2 kinase, a protein expressed in megakaryocytes, 45 that was not dependent on the activity of the MEK/MAPK pathway. Since PMA-stimulated differentiation of K562 cells mimics the pathway of natural megakaryocyte precursors, 6 it remains to be detemined whether Pyk2 expression is regulated in a similar fashion in vivo.…”

“…16 PMA treatment also leads to the induction of transcription factors such as jun/fos 17 and egr-1 18 and of protooncogenes such as c-ski 19 and c-sis. 11 Treatment of K562 cells with PMA also leads to the p53-independent expression of the cell cycle inhibitor p21waf1/cip. 20 In addition to the effect on cell growth, the changes associated with the adhesive properties of the K562 cells are also evident upon PMA treatment and are likely due, in part, to changes in the cytoskeleton 14 and in integrin expression.…”

“…10 This differentiation process is characterized by changes in cell morphology and adhesive properties, cell growth arrest, endomitosis and expression of markers associated with megakaryocytes. [10][11][12][13][14][15] The signaling cascade that leads to PMA-induced cell cycle arrest, polyploidy and differentiation of K562 cells has been partially described and the role of PKC in the megakaryocyte differentiation process has been established. 16 Studies carried out with K562 cells revealed that PMA induces the translocation of specific isoforms of protein kinase C (PKC) to particulate fractions (plasma and nuclear membranes) with subsequent reduction in the levels of the ␤11 PKC isoform and an increase in the ␣ PKC isoform.…”

26S proteasome exhibits endoribonuclease activity controlled by extra-cellular stimuli

“…[6][7][8][9][10][11][12] One of the well-known mechanisms of mRNA destruction is via mRNA decay, whereby 3'-untranslated regions (UTR) of mRNA that contain AU-rich elements (ARE) are degraded by exo-and endoribonucleases. Recently, it has been shown that ARE sequences often associate with Alu-repeats.…”

Regulation of the Rb Gene by Normal and Mutated Ras, Tpa and Egf

“…Relevant to this hypothesis is the finding that TPA induces transcription of cellular protooncogenes, e.g. c-fos (16,17), c-myc (16,18) and c-sis (19). In an attempt to identify the target of transcription regulation by ras, we examined its effect on the promoter of the Rb gene, in expectation of activation of Rb transcription by ras.…”