Phorbol ester-stimulated phosphorylation of PU.1: association with leukemic cell growth inhibition

Abstract

PU.1, a member of the ets transcription factor family, has been previously shown to be necessary for tetradecanoylphorbol-13 acetate (TPA)-induced U937 leukemic cell maturation. We examined the effects of TPA on PU.1 content and PU.1 DNA binding activity in U937 cells. Unstimulated cells expressed PU.1 mRNA transcripts and TPA did not increase these levels. However, TPA treatment induced phosphorylation of PU.1. Gel-shift analysis using a labeled PU.1 oligomer showed that TPA induced a unique PU.1 binding activity. This binding activity was phosphorylation-dependent, as indicated by the ability of phosphatase treatment to abolish its detection. The PU.1 binding activity was generated at TPA-13 concentrations stimulating growth arrest and was blocked by the PKC inhibitor GF109203X, which antagonized TPA-induced growth inhibition. Bryostatin 1, another protein kinase C activator, induced only a modest degree of U937 growth inhibition and antagonized TPA-stimulated growth arrest. Bryostatin 1 was unable to induce this TPA-generated PU.1 binding activity. High bryostatin 1 concentrations inhibited generation of this TPA-induced band shift. These data suggest that TPA-induced growth inhibition is associated with phosphorylation of PU.1 and generation of a unique PU.1 binding activity.

Selective activation of NF‐κB and E2F by low concentration of arsenite in U937 human monocytic leukemia cells

“…To confirm the above observation experimentally, by detecting molecular markers indicative of macrophage differentiation, we conducted an EMSA study on DNA-binding activity of PU.1 protein, which is a key regulator of macrophage differentiation. It has been reported that TPA-induced growth inhibition of U937 cells is associated with phosphorylation of PU.1 and generation of unique PU.1-binding activity [23]. Although we could confirm the results of this previous study on the effect of TPA, we could not find any effect of arsenite on the DNA-binding activity of PU.1.…”

Divergent effects of bryostatin 1 and phorbol myristate acetate on cell cycle arrest and maturation in human myelomonocytic leukemia cells (U937)

“…In contrast, exposure to bryostatin 1 did not have an inhibitory effect on cell proliferation. Consistent with the results of previous studies [7,27], co-administration of bryostatin 1 reduced the growth-inhibitory effects of PMA (data not shown).…”

“…In human promyelocytic leukemia cells (HL-60), bryostatin 1 variably induces maturation [29,43]; moreover, it blocks PMA effects in sublines immune to its differentiation-inducing actions [27]. In the human monocytic leukemic cell line U937, bryostatin 1 has been reported to exert antiproliferative effects by some investigators [4], although others have found its activity to be marginal, and consistently less than that exerted by PMA [7,36]. Recently, the antiproliferative effects of bryostatin 1 toward U937 cells has been related to dehosphorylation (and inactivation) of cyclin-dependent kinase-2 (CDK2), although such effects were most pronounced at relatively high bryostatin 1 concentrations (e.g., 200 nM) [4].…”

Nf1 Haploinsufficiency Alters Myeloid Lineage Commitment and Function, Leading to Deranged Skeletal Homeostasis

“…(35,45) Intriguingly, however, supraphysiologic levels of Pu.1 expression do not necessarily correlate with increased myelomonocytic lineage commitment. In fact, recent studies suggest that phosphorylation of Pu.1 plays a central role in modulating its DNA binding affinity, (37,46) and is thereby critical to its ability to activate myeloid-specific gene programs that promote terminal differentiation. (39,40) For instance, although human myeloid leukemic cell lines have been shown to overexpress Pu.1, (47) the transcript levels of Pu.1-dependent myeloid genes, such as CD11b and c-fms, are relatively low by comparison.…”

“…Phosphorylation of Pu.1 is known to retard its migration in SDS-PAGE electrophoresis. (36,37) To confirm that these band shifts represent phosphorylated forms of Pu.1, nuclear lysates were treated with potato acid phosphatase prior to electrophoresis. Phosphatase treatment resulted in complete conversion of the shifted bands to the lower molecular weight, unphosphorylated Pu.1 species (Fig.…”