Javier León

Myc represses differentiation-induced p21CIP1 expression via Miz-1-dependent interaction with the p21 core promoter

Inhibition of cellular differentiation is one of the well-known biological activities of c-Myc-family proteins. We show here that Myc represses differentiation-induced expression of the cyclin-dependent kinase (CDK) inhibitor p21CIP1 (CDKN1A, p21), known to play an important role in cell fate decisions during growth and differentiation, in hematopoietic cells. Our results demonstrate that the c-Myc-responsive region is situated in the p21 core promoter. c-Myc binds to this region in vitro and in vivo through interaction with the initiator-binding Zn-finger transcription factor Miz-1, which associates directly with the promoter. Association of Myc with the promoter in vivo correlates inversely with p21 expression. Using mutants of c-Myc with impaired binding to Miz-1, our results further show that repression of p21 promoter/reporters as well as the endogenous p21 gene by Myc depends on interaction with Miz-1. Expression of Miz-1 increases during hematopoietic differentiation and Miz-1 activates the p21 promoter under conditions of low Myc levels, indicating a positive role for free Miz-1 in this process. In conclusion, repression of differentia-tion-induced p21 expression through Miz-1 may be an important mechanism by which Myc blocks diffe-rentiation.

Contributions of Myc to tumorigenesis

Despite intensive research, the mechanisms by which deregulation of myc gene expression contributes to tumorigenesis are still not fully resolved and many aspects are still enigmatic. Several recent reviews, including one published in this series a few months ago, have summarized recent progress in our understanding of the biochemistry of Myc proteins [Eisenmann, Genes Dev. (2001) in press; Amati et al., Biochim. Biophys. Acta 1471 (2001) 135-145]. Also, the evidence documenting a central role of Myc proteins in human tumorigenesis has been extensively reviewed [Henriksson and Lüscher, Cancer Res. 68 (1996) 109-182]. In this article, we will argue that current progress allows us to present testable hypotheses on how Myc affects specific properties of transformed cells.

Myc and cell cycle control

Soon after the discovery of the Myc gene (c-Myc), it became clear that Myc expression levels tightly correlate to cell proliferation. The entry in cell cycle of quiescent cells upon Myc enforced expression has been described in many models. Also, the downregulation or inactivation of Myc results in the impairment of cell cycle progression. Given the frequent deregulation of Myc oncogene in human cancer it is important to dissect out the mechanisms underlying the role of Myc on cell cycle control. Several parallel mechanisms account for Myc-mediated stimulation of the cell cycle. First, most of the critical positive cell cycle regulators are encoded by genes induced by Myc. These Myc target genes include Cdks, cyclins and E2F transcription factors. Apart from its direct effects on the transcription, Myc is able to hyperactivate cyclin/Cdk complexes through the induction of Cdk activating kinase (CAK) and Cdc25 phosphatases. Moreover, Myc antagonizes the activity of cell cycle inhibitors as p21 and p27 through different mechanisms. Thus, Myc is able to block p21 transcription or to induce Skp2, a protein involved in p27 degradation. Finally, Myc induces DNA replication by binding to replication origins and by upregulating genes encoding proteins required for replication initiation. Myc also regulates genes involved in the mitotic control. A promising approach to treat tumors with deregulated Myc is the synthetic lethality based on the inhibition of Cdks. Thus, the knowledge of the Myc-dependent cell cycle regulatory mechanisms will help to discover new therapeutic approaches directed against malignancies with deregulated Myc. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology.

Myc Roles in Hematopoiesis and Leukemia

Hematopoiesis is a process capable of generating millions of cells every second, as distributed in many cell types. The process is regulated by a number of transcription factors that regulate the differentiation along the distinct lineages and dictate the genetic program that defines each mature phenotype. Myc was first discovered as the oncogene of avian leukemogenic retroviruses; it was later found translocated in human lymphoma. From then on, evidence accumulated showing that c-Myc is one of the transcription factors playing a major role in hematopoiesis. The study of genetically modified mice with overexpression or deletion of Myc has shown that c-Myc is required for the correct balance between self-renewal and differentiation of hematopoietic stem cells (HSCs). Enforced Myc expression in mice leads to reduced HSC pools owing to loss of self-renewal activity at the expense of increased proliferation of progenitor cells and differentiation. c-Myc deficiency consistently results in the accumulation of HSCs. Other models with conditional Myc deletion have demonstrated that different lineages of hematopoietic cells differ in their requirement for c-Myc to regulate their proliferation and differentiation. When transgenic mice overexpress c-Myc or N-Myc in mature cells from the lymphoid or myeloid lineages, the result is lymphoma or leukemia. In agreement, enforced expression of c-Myc blocks the differentiation in several leukemia-derived cell lines capable of differentiating in culture. Not surprising, MYC deregulation is recurrently found in many types of human lymphoma and leukemia. Whereas MYC is deregulated by translocation in Burkitt lymphoma and, less frequently, other types of lymphoma, MYC is frequently overexpressed in acute lymphoblastic and myeloid leukemia, through mechanisms unrelated to chromosomal translocation, and is often associated with disease progression.

Functional phosphorylation sites in the C-terminal region of the multivalent multifunctional transcriptional factor CTCF.

ABSTRACT CTCF is a widely expressed and highly conserved multi-Zn-finger (ZF) nuclear factor. Binding to various CTCF target sites (CTSs) is mediated by combinatorial contributions of different ZFs. Different CTSs mediate distinct CTCF functions in transcriptional regulation, including promoter repression or activation and hormone-responsive gene silencing. In addition, the necessary and sufficient core sequences of diverse enhancer-blocking (insulator) elements, including CpG methylation-sensitive ones, have recently been pinpointed to CTSs. To determine whether a posttranslational modification may modulate CTCF functions, we studied CTCF phosphorylation. We demonstrated that most of the modifications that occur at the carboxy terminus in vivo can be reproduced in vitro with casein kinase II (CKII). Major modification sites map to four serines within the S604KKEDS609S610DS612E motif that is highly conserved in vertebrates. Specific mutations of these serines abrogate phosphorylation of CTCF in vivo and CKII-induced phosphorylation in vitro. In addition, we showed that completely preventing phosphorylation by substituting all serines within this site resulted in markedly enhanced repression of the CTS-bearing vertebrate c-myc promoters, but did not alter CTCF nuclear localization or in vitro DNA-binding characteristics assayed with c-myc CTSs. Moreover, these substitutions manifested a profound effect on negative cell growth regulation by wild-type CTCF. CKII may thus be responsible for attenuation of CTCF activity, either acting on its own or by providing the signal for phosphorylation by other kinases and for CTCF-interacting protein partners.

Targeting of CTCF to the nucleolus inhibits nucleolar transcription through a poly(ADP-ribosyl)ation-dependent mechanism.

Multiple functions have been reported for the transcription factor and candidate tumour suppressor, CTCF. Among others, they include regulation of cell growth, differentiation and apoptosis, enhancer-blocking activity and control of imprinted genes. CTCF is usually localized in the nucleus and its subcellular distribution during the cell cycle is dynamic; CTCF was found associated with mitotic chromosomes and the midbody, suggesting different roles for CTCF at different stages of the cell cycle. Here we report the nucleolar localization of CTCF in several experimental model systems. Translocation of CTCF from nucleoplasm to the nucleolus was observed after differentiation of K562 myeloid cells and induction of apoptosis in MCF7 breast cancer cells. CTCF was also found in the nucleoli in terminally differentiated rat trigeminal ganglion neurons. Thus our data show that nucleolar localization of CTCF is associated with growth arrest. Interestingly, the 180 kDa poly(ADP-ribosyl)ated isoform of CTCF was predominantly found in the nucleoli fractions. By transfecting different CTCF deletion constructs into cell lines of different origin we demonstrate that the central zinc-finger domain of CTCF is the region responsible for nucleolar targeting. Analysis of subnucleolar localization of CTCF revealed that it is distributed homogeneously in both dense fibrillar and granular components of the nucleolus, but is not associated with fibrillar centres. RNA polymerase I transcription and protein synthesis were required to sustain nucleolar localization of CTCF. Notably, the labelling of active transcription sites by in situ run-on assays demonstrated that CTCF inhibits nucleolar transcription through a poly(ADP-ribosyl)ation-dependent mechanism.

Prolactin induces c-Myc expression and cell survival through activation of Src/Akt pathway in lymphoid cells

Stimulation of resting W53 cells (lymphoid murine cells expressing prolactin (PRL) receptor) by PRL induced expression of growth-related immediate-early genes (IEG), and proliferation through activation of the Src kinases. Since IEG are essential for cell cycle progression, we have studied how PRL controls expression of c-Myc mRNA and c-Fos. Stimulation of W53 cell proliferation by PRL required activation of MAPK, as the Mek1/2 inhibitor PD184352 eliminated Erk1/2 stimulation, cell proliferation, and expression of c-Fos mRNA. In contrast, PD184352 did not alter PRL activation of c-Myc mRNA expression or stimulation of p70S6K, Akt, and the Jak2/Stat5 pathway. Activation of the PI3K by PRL was necessary for the expression of c-MycmRNA and W53 cell proliferation, as the PI3K inhibitor LY294002 abolished them. However, it did not modify PRL stimulation of c-Fos mRNA expression or activation of Erk1/2 and Stat5. Furthermore, rapamycin, an inhibitor of mTOR and consequently of p70S6K, did not alter PRL stimulation of c-Myc and c-Fos mRNA expression and it had a very minor inhibitory effect on PRL stimulation of W53 cell proliferation. In addition, rapamycin did not affect PRL stimulation of Akt or Stat5. However, it reinforced PRL activation of Erk1/2. Overexpression of a constitutively activated Akt (myristoylated Akt) in W53 cells overcame the inhibitory effect of LY294002 on c-Myc expression, as well as cell death upon PRL deprivation. Consistently, inducible expression of Akt-CAAX Box in W53 cells caused inhibition of c-Myc expression. PRL stimulation of W53 cells resulted in Akt translocation to the nucleus, phosphorylation of FKHRL1 transcription factor, and its nuclear exclusion. In contrast, induced expression of Akt-CAAX Box caused inhibition of FKHRL1 phosphorylation. Furthermore, transient expression of nonphosphorylatable FKHRL1-A3 mutant impaired PRL-induced activation of the c-Myc promoter. Akt activation also resulted in phosphorylation and inhibition of glycogen synthetase kinase 3 (GSK3), which in turn promoted c-Myc stability. Consistently, treatment of W53 with selective inhibitors of GSK3 such as SB415286 and lithium salts resulted in increased levels of c-Myc. Also, overexpression of c-Myc in W53 cells overcame the decrease in cell proliferation induced by LY294002. These findings defined a PRL-signalling cascade in W53 cells, involving Src kinases/PI3K/Akt/FKHRL1-GSK3, that mediates stimulation of c-Myc expression.

SKP2 Oncogene Is a Direct MYC Target Gene and MYC Down-regulates p27KIP1 through SKP2 in Human Leukemia Cells

SKP2 is the ubiquitin ligase subunit that targets p27KIP1 (p27) for degradation. SKP2 is induced in the G1-S transit of the cell cycle, is frequently overexpressed in human cancer, and displays transformation activity in experimental models. Here we show that MYC induces SKP2 expression at the mRNA and protein levels in human myeloid leukemia K562 cells with conditional MYC expression. Importantly, in these systems, induction of MYC did not activate cell proliferation, ruling out SKP2 up-regulation as a consequence of cell cycle entry. MYC-dependent SKP2 expression was also detected in other cell types such as lymphoid, fibroblastic, and epithelial cell lines. MYC induced SKP2 mRNA expression in the absence of protein synthesis and activated the SKP2 promoter in luciferase reporter assays. With chromatin immunoprecipitation assays, MYC was detected bound to a region of human SKP2 gene promoter that includes E-boxes. The K562 cell line derives from human chronic myeloid leukemia. In a cohort of chronic myeloid leukemia bone marrow samples, we found a correlation between MYC and SKP2 mRNA levels. Analysis of cancer expression databases also indicated a correlation between MYC and SKP2 expression in lymphoma. Finally, MYC-induced SKP2 expression resulted in a decrease in p27 protein in K562 cells. Moreover, silencing of SKP2 abrogated the MYC-mediated down-regulation of p27. Our data show that SKP2 is a direct MYC target gene and that MYC-mediated SKP2 induction leads to reduced p27 levels. The results suggest the induction of SKP2 oncogene as a new mechanism for MYC-dependent transformation.

H-, K- and N-Ras inhibit myeloid leukemia cell proliferation by a p21WAF1-dependent mechanism

Mutated ras genes are frequently found in human cancer. However, it has been shown that oncogenic ras inhibits growth of primary cells, through pathways involving p53 and the cell cycle inhibitors p16INK4a and p19ARF. We have analysed the effect of the ectopic expression of the three mammalian ras genes on the proliferation of K562 leukemia cells, which are deficient for p53, p16INK4a, p15INK4b and p19ARF genes. We have found that high expression levels of both wild-type and oncogenic H-, K- and N-ras inhibit the clonogenic growth of K562 cells. Induction of H-rasV12 expression in K562 transfectants retards growth and this effect is accompanied with an increase of p21WAF1 mRNA and protein levels. Furthermore, p21WAF1 promoter is activated potently by oncogenic ras and less pronounced by wild-type ras. This induction is p53-independent since a p21WAF1 promoter devoid of the p53 responsive elements is still activated by Ras. Finally, inhibition of p21WAF1 expression by an antisense construct partially overcomes the growth inhibitory action of oncogenic H-ras. Altogether, these results indicate that the antiproliferative effect of ras in myeloid leukemia cells is associated to the induction of p21WAF1 expression and suggest the existence of p19ARF and p16INK4a-independent pathways for ras-mediated growth inhibition.

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

We have used the human leukemia cell line K562 as a model to study the role of c-myc in differentiation 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 differentiate towards erythroid or myelomonocytic lineages. K562 transfected with c-myc mutants showed a higher expression of erythroid differentiation markers, without any detectable effects in the myelomonocytic differentiation. We also transfected K562 cells with a zinc-inducible max gene. Ectopic Max overexpression resulted in an increased erythroid differentiation, thus reproducing the effects 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 undifferentiated state and to undergo drug-mediated apoptosis.