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.

The e ects of HIV-1 Tat protein on mitochondria membrane permeability and apoptosis were analysed in lymphoid cells. In this report we show that stabletransfected HIV-Tat cells are primed to undergo apoptosis upon serum withdrawal. This e ect was observed in both the Jhan T cell line and the K562 cells, the latter expressing the bcr-abl chimeric gene, which confers resistance to apoptosis induced by di erent stimuli. Using a cyto¯uorimetric approach we have determined that serum withdrawal induces a disruption of the transmembrane mitochondrial potential (Dc m ) followed by an increase of reactive oxygen species (ROS) and the subsequent DNA nuclear loss in K562-Tat cells but not in the K562-pcDNA cell line. These pre-apoptotic events were associated with the cleavage of the caspase-3, while the expression of Bcl-2, Bcl-X L and Bax proteins was not a ected by the presence of Tat. Regardless of the steady state of the Bax protein, we found that in both K562 and K562-Tat cells, this protein is located in the nucleus, but after serum withdrawal its localization was mainly in the cytoplasm. The activity of caspase-3 detected in K562-Tat cells after serum withdrawal paralleled with the mitochondria permeability transition. Nevertheless, in Jhan-Tat cells the inhibition of this caspase with the speci®c inhibitor, z-DEVD-cmk, did not a ect the disruption of the mitochondria potential induced by serum withdrawal. Interestingly, we found that HIV-Tat protein accumulates at the mitochondria in the K562-Tat cells cultured under low serum conditions, and this mitochondrial localization correlated with the Dc m disruption detected in these cells. In addition, HIV-1 Tat protein synergies with protoporphyrin IX (PPIX), a ligand of the mitochondrial benzodiazepine receptor, in the induction of apoptosis in both Jhan and K562 cells. Thus, HIV-1 Tat protein may induce apoptosis by a mechanism that involves mitochondrial PT and may contribute to the lymphocyte depletion seen in AIDS patients.

We have previously demonstrated that c-Myc impairs p53-mediated apoptosis in K562 human leukemia cells, which lack ARF. To investigate the mechanisms by which c-Myc protects from p53-mediated apoptosis, we used K562 cells that conditionally express c-Myc and harbor a temperature-sensitive allele of p53. Gene expression profiles of cells expressing wild-type conformation p53 in the presence of either uninduced or induced c-Myc were analysed by cDNA microarrays. The results show that multiple p53 target genes are downregulated when c-Myc is present, including p21 WAF1 , MDM2, PERP, NOXA, GADD45, DDB2, PIR121 and p53R2. Also, a number of genes that are upregulated by c-Myc in cells expressing wild-type conformation p53 encode chaperones related to cell death protection as HSP105, HSP90 and HSP27. Both downregulation of p53 target genes and upregulation of chaperones could explain the inhibition of apoptosis observed in K562 cells with ectopic c-Myc. Myc-mediated impairment of p53 transactivation was not restricted to K562 cells, but it was reproduced in a panel of human cancer cell lines derived from different tissues. Our data suggest that elevated levels of Myc counteract p53 activity in human tumor cells that lack ARF. This mechanism could contribute to explain the c-Myc deregulation frequently found in cancer.

The mitogen-activated protein kinase ERK1/2 pathway is essential in the control of cell proliferation and differentiation in most cellular systems. As such, it has been considered a potential target for antineoplastic therapy. For this purpose, we have examined the role of ERK activation in myeloid leukemia cell growth and differentiation. Using a representative set of myeloid leukemia cell lines, we show that cell proliferation was not accompanied by increases on ERK1/2 activation, and mitogenic stimulation did not enhance ERK activity. Moreover, abolition of ERK function by the inhibitor PD98059 or by a dominant inhibitory mutant ERK2 had no significant effects on proliferation. With the aid of various differentiation inducers, we found that within the same cell line, differentiation to a given lineage could occur with and without ERK1/2 activation, depending on the stimulus. Also, a differentiator could have the same effect in the presence or absence of ERK stimulation, depending on the cell line. ERK inhibition did not affect the differentiation elicited by stimuli whose effects were accompanied by ERK activation. Finally, constitutive ERK activity was also ineffective on proliferation and differentiation. Thus, our results indicate that ERK1/2 activation is not an essential requirement for leukemic cell growth and differentiation.