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All-trans retinoic acid (ATRA) is a potential therapeutic agent for the treatment of hematopoietic malignancies, because of its function as an inducer of terminal differentiation of leukemic blasts. Although the efficacy of ATRA as an anticancer drug has been demonstrated by the successful treatment of acute promyelocytic leukemia (APL), the molecular mechanisms of ATRA-induced cell cycle arrest of myeloid cells have not been fully investigated. In this study, we show that the onset of ATRA-induced G(0)/G(1) arrest of human monoblastic U-937 cells is linked to a sharp down-regulation of c-Myc and cyclin E levels and an increase in p21(WAF1/CIP1) expression. This is followed by an increase in p27(Kip1) protein expression due to enhanced protein stability. The importance of an early decrease in Myc expression for these events was demonstrated by the failure of a U-937 subline with constitutive exogenous expression of v-Myc to cell cycle arrest and regulate cyclin E and p27(Kip1) in response to ATRA. Preceding the initiation of G(1) arrest, a transient rise in retinoblastoma protein (pRb), p107, and cyclin A levels was detected. Later, a rapid fall in the levels of cyclins A and B and a coordinate dephosphorylation of pRb at Ser780, Ser795, and Ser807/811 coincided with the accumulation of cells in G(1). These results thus identify a decrease in c-Myc and cyclin E levels and a posttranscriptional up-regulation of p27(Kip1) as important early changes, and position them in the complex chain of events regulating ATRA-induced cell cycle arrest of myeloid cells.

Deregulated expression of the myc-family of oncogenes in hematopoietic and other cell types plays an important role in tumorigenesis, and results in increased proliferative potential and block of cellular differentiation. We have previously shown that IFN-gamma restores phorbol ester-induced differentiation and cell cycle arrest in v-myc transformed human U-937 monoblasts. To investigate whether other cytokine signals could also abrogate such a block, IL-1, IL-3, IL-4, IL-6, IL-7, IL-10, IL-11, LIF, oncostatin M, M-CSF, G-CSF and GM-CSF, and TGFbeta1, TNF-alpha, IFN-alpha were examined. We show that GM-CSF and IL-6, in combination with the phorbol ester 12-O-tetradecanoyl-phorbol acetate (TPA), restored differentiation and cell cycle arrest. In contrast, treatment by TGFbeta1 +/- TPA resulted in an efficient G1/G0 arrest, but did not appear to induce terminal differentiation. Restoration of differentiation and cell cycle arrest was accomplished despite maintained expression of the v-Myc protein. Our results show that the cytokine-induced signals reduced Myc-dependent transcription of an artificial target promoter/reporter gene construct, correlating in most, but not all, cases with decreased association of v- and c-Myc with its essential partner, Max. Thus, cytokine-induced signals may counteract the activity of deregulated Myc, and contribute to the normalization of differentiation, arrest in the G1/G0 phase of the cell cycle, or both.