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

Abstract

c-myc protooncogene positively regulates cell proliferation and overexpression of c-myc is found in many solid tumors and leukemias. In the present study we used the K562 human myeloid leukemia cell line as a model to study the functional interaction between c-Myc and p53. Using two di erent methods, we generated K562 transfectant cell lines with conditional expression of either c-Myc or p53. The cells expressed the p53 Vall35 mutant, which adopts a wild-type conformation at 328C, while c-Myc induction was achieved with a zinc-inducible expression vector. We found that p53 in wild-type conformation induces growth arrest and apoptosis of K562. Expression of c-Myc signi®cantly attenuated apoptosis and impaired the transcriptional activity of p53 on p21 WAF1 , Bax and cytomegalovirus promoters. The impairment of p21 WAF1 transactivation by c-Myc was con®rmed by transfection of a c-Myc-estrogen receptor fusion protein and by induction of c-myc by zinc in transfected cells. Also, p53-mediated up-regulation of p21 WAF1 mRNA protein were signi®cantly reduced by cMyc, while Bax levels were una ected. Consistently, cMyc increased cyclin-dependent kinase 2 activity in K562 cells expressing p53 in wild-type conformation. These results suggest that c-Myc overexpression may antagonize the pro-apoptotic function of p53, thus providing a molecular mechanism for the frequently observed deregulation of c-myc in human cancer.

Inhibitory effect of c-Myc on p53-induced apoptosis in leukemia cells. Microarray analysis reveals defective induction of p53 target genes and upregulation of chaperone genes

“…The activation of p53 represses endogenous MYC (northern in Figure 1a, compare lanes 1 and 3), but in KmycB/p53 the addition of zinc in the presence of wild-type conformation p53 results in MYC expression levels similar or higher than in absence of p53 (northern in Figure 1a, compare lanes 1 and 4). As we reported earlier (Ceballos et al, 2000), induction of p21 WAF1 mRNA expression was reduced after zinc addition at 321C (northern in Figure 1a, compare lanes 3 and 4). Also, induction of MYC protected K562 cells from p53-mediated apoptosis as shown by the block in PARP proteolysis (immunoblot in Figure 1a, lanes 3 and 4) and the increased fraction of viable cells in KmycB/p53 cells treated with zinc, as assessed by the Trypan blue test ( Figure 1b) and by metabolic activity as measured by a tetrazolium salt reduction assay ( Figure 1c).…”

“…This may be explained by the fact that c-Myc could not reverse the p53-mediated growth arrest (Ceballos et al, 2000). However, despite its inability to promote growth, induction of Myc enhanced expression of D-type cyclins (Figure 8a) and promoted phosphorylation of the retinoblastoma protein, pRB (Figure 8b), in the presence of active p53.…”

“…We used KmycB/p53, a K562-derived cell line, which harbors a temperaturesensitive allele of p53 and a zinc-inducible Myc gene. Activation of p53 and expression of c-Myc can be independently induced by incubating the cells at 321C or adding zinc salts to the media, respectively (Ceballos et al, 2000). The activation of p53 represses endogenous MYC (northern in Figure 1a, compare lanes 1 and 3), but in KmycB/p53 the addition of zinc in the presence of wild-type conformation p53 results in MYC expression levels similar or higher than in absence of p53 (northern in Figure 1a, compare lanes 1 and 4).…”

“…We have previously shown that c-Myc partially protects from p53-mediated apoptosis and that c-Myc impairs p53-mediated transactivation of p21 WAF1 (CDKN1A) in K562 cells (Ceballos et al, 2000). These cells derive from human chronic myeloid leukemia and are p53-ARFand p16 INK4A -null (Delgado et al, 1995Lewis et al, 2001).…”

Functional identification of LZTS1 as a candidate prostate tumor suppressor gene on human chromosome 8p22

“…It would be of interest to investigate the downstream activation of speci®c p53 target genes such as p21 WAF in this cell line. It is also likely that the LZTS1 e ects in these cells are partly counteracted by the presence of myc, which has been shown to antagonize the e ect of TP53 on apoptosis and p21 WAF1 transactivation in K562 leukemia cells and LNCaP cells (Ceballos et al, 2000;Mitchell and El-Deiry, 1999). The TSUPr1 cell line carries an oncogenic mutation in the ras (V12) gene.…”