Max and inhibitory c-Myc mutants induce erythroid differentiation and resistance to apoptosis in human myeloid leukemia cells

Abstract

We have used the human leukemia cell line K562 as a model to study the role of c-myc in di erentiation and apoptosis. We have generated stable transfectants of K562 constitutively expressing two c-Myc inhibitory mutants: D106-143, that carries a deletion in the transactivation domain of the protein, and In373, that carries an insertion in the DNA-interacting region. We show here that In373 is able to compete with c-Myc for Max binding and to inhibit the transformation activity of c-Myc. K562 cells can di erentiate towards erythroid or myelomonocytic lineages. K562 transfected with c-myc mutants showed a higher expression of erythroid di erentiation markers, without any detectable e ects in the myelomonocytic di erentiation. We also transfected K562 cells with a zinc-inducible max gene. Ectopic Max overexpression resulted in an increased erythroid di erentiation, thus reproducing the e ects of c-myc inhibitory mutants. We also studied the role of c-myc mutants and max in apoptosis of K562 induced by okadaic acid, a protein phosphatases inhibitor. The expression of D106-143 and In373 c-myc mutants and the overexpression of max reduced the apoptosis mediated by okadaic acid. The common biochemical activity of D106-143 and In373 is to bind Max and hence to titrate out c-Myc to form non-functional Myc/ Max dimers. Similarly, Max overexpression would decrease the relative levels of c-Myc/Max with respect to Max/Max. The results support a model where a threshold of functional c-Myc/Max is required to maintain K562 cells in an undi erentiated state and to undergo drug-mediated apoptosis.

c-Myc antagonizes the effect of p53 on apoptosis and p21WAF1 transactivation in K562 leukemia cells

“…This result seems paradoxical as c-Myc induce apoptosis in di erent cell types under conditions of growth factor deprivation. However, c-Myc overexpression induces little apoptosis of K562 cells in the absence of serum (CanÄ elles et al, 1997). This is consistent with the lack of expression of p53 (Law et al, 1993), p19 ARF (Delgado et al, 2000) and CD95/ FAS (McGahon et al, 1995) in K562, as the three proteins are required for c-Myc-mediated apoptosis in ®broblasts (Hermeking and Eick, 1994;Hueber et al, 1997;Wagner et al, 1994;Zindy et al, 1998).…”

“…Cell growth and viability were assayed with hemocytometer and the trypan blue exclusion test and by colorimetry based on the reduction of the tetrazolium salt WST-1 (Roche Molecular Biochemicals). To determine the presence on internucleosomal fragmentation (DNA laddering), cellular DNA was prepared and analysed by agarose gel electrophoresis as described (CanÄ elles et al, 1997). Binding of annexin V to cell surface was carried out by¯ow cytometry with annexin V-FITC kit following the manufacturer's instructions (Genzyme Diagnostics).…”

“…Probe for human c-myc was as described (Delgado et al, 1995); for p53, a 2.1 kb XhoI ± BamHI fragment from pLTRp53cG corresponding to mouse cDNA (provided by M Oren); for p21 WAF1 a 0.6 kb NotI fragment from pBSK-p21 corresponding to human cDNA (provided by M Serrano, Centro Nacional de BiotecnologõÂ a, Madrid); for bax, a 0.5 kb EcoRI fragment form pSSFV-Bax corresponding to human cDNA (provided by S Korsmeyer, Washington University Medical School, St. Louis, USA). For immunoblots, cell lysates were prepared and immunoblotted as described (CanÄ elles et al, 1997). c-Myc and p21 WAF1 were detected with rabbit polyclonal antibodies, p53 with the monoclonal antibody PAb240 (Santa Cruz Biotech.…”

c-Myc Inhibits Ras-Mediated Differentiation of Pheochromocytoma Cells by Blocking c-Jun Up-Regulation

“…Similarly, a Myc mutant carrying and insertion in the basic region and unable to bind DNA (MycIn373; ref. 49) did not repress the Rasinduced activation of the AP-1 reporter (Fig. 8C).…”

Apoptosis and differentiation of human embryonic stem cells induced by sustained activation of c-Myc

“…Deregulated expression of c-Myc promotes proliferation and inhibits a differentiated phenotype in many types of cells such as myeloid and intestinal epithelium (Selvakumaran et al, 1996;Canelles et al, 1997;van de Wetering et al, 2002). Paradoxically, however, c-Myc activation also induces differentiation of epidermal and hematopoietic stem cells (Gandarillas and Watt, 1997;Wilson et al, 2004).…”