An important mechanism by which the tumor suppressor p53 maintains genomic stability is to induce cell cycle arrest through activation of the cyclin-dependent kinase inhibitor p21 WAF1/Cip1 gene. We show that the gene encoding the gut-enriched Krü ppel-like factor (GKLF, KLF4) is concurrently induced with p21 WAF1/Cip1during serum deprivation and DNA damage elicited by methyl methanesulfonate. The increases in expression of both Gklf and p21 WAF1/Cip1 due to DNA damage are dependent on p53. Moreover, during the first 30 min of methyl methanesulfonate treatment, the rise in Gklf mRNA level precedes that in p21, suggesting that GKLF may be involved in the induction of p21 Indeed, GKLF activates p21WAF1/Cip1 through a specific Sp1-like cis-element in the p21 WAF1/Cip1 proximal promoter. The same element is also required by p53 to activate the p21 WAF1/Cip1 promoter, although p53 does not bind to it. Potential mechanisms by which p53 activates the p21 WAF1/Cip1 promoter include a physical interaction between p53 and GKLF and the transcriptional induction of Gklf by p53. Consequently, the two transactivators cause a synergistic induction of the p21 WAF1/Cip1 promoter activity. The physiological relevance of GKLF in mediating p53-dependent induction of p21 WAF1/Cip1 is demonstrated by the ability of antisense Gklf oligonucleotides to block the production of p21 WAF1/Cip1 in response to p53 activation. These findings suggest that GKLF is an essential mediator of p53 in the transcriptional induction of p21 WAF1/Cip1 and may be part of a novel pathway by which cellular responses to stress are modulated.

Tumour metastasis is the principal cause of death for cancer patients. We have identified the nm23 gene, for which RNA levels are reduced in tumour cells of high metastatic potential. In this report we identify the cytoplasmic and nuclear Nm23 protein, and show that it also is differentially expressed in metastatic tumour cells. We also find that the human Nm23 protein has sequence homology over the entire translated region with a recently described developmentally regulated protein in Drosophila, encoded by the abnormal wing discs (awd) gene. Mutations in awd cause abnormal tissue morphology and necrosis and widespread aberrant differentiation in Drosophila, analogous to changes in malignant progression. The metastatic state may therefore be determined by the loss of genes such as nm23/awd which normally regulate development.

The Waf1/Cip1 protein induces cell cycle arrest through inhibition of the activity of cyclin-dependent kinases and proliferating cell nuclear antigen. Expression of the WAF1/CIP1 gene is induced in a p53-dependent manner in response to DNA damage but can also be induced in the absence of p53 by agents such as growth factors, phorbol esters, and okadaic acid. WAF1/CIP1 expression in U937 human leukemic cells is induced by both phorbol ester, a protein kinase C activator, and by okadaic acid, an inhibitor of phosphatases 1 and 2A. Both of these agents induce the differentiation of these leukemic cells toward macrophages. We demonstrate that phorbol esters and okadaic acid stimulate transcription from the WAF1/CIP1 promoter in U937 cells. This transcription is mediated by a region of the promoter between ؊154 and ؉16, which contains two binding sites for the transcription factor Sp1. Deletion or mutation of these Sp1 sites reduces WAF1/CIP1 promoter response to phorbol ester and okadaic acid, while a reporter gene under the control of a promoter containing only multiple Sp1 binding sites and a TATA box is induced by phorbol ester and okadaic acid. The WAF1/ CIP1 promoter is also highly induced by exogenous Sp1 in the Sp1-deficient Drosophila Schnieder SL 2 cell line. These results suggest that phorbol ester and okadaic acid activate transcription of the WAF1/CIP1 promoter through a complex of proteins that includes Sp1 and basal transcription factors.Treatment of the human myeloid leukemic cell line U937 with phorbol esters such as phorbol myristate acetate (PMA), 1 an activator of protein kinase C, leads to macrophage/monocyte-like differentiation over a 72-h period (1, 2). This process involves changes in cell-substrate adherence, growth arrest in late G 1 , and increased expression of monocyte markers (3, 4). Similarly, treatment of U937 cells with okadaic acid, a natural product isolated from the black sponge and a potent inhibitor of protein phosphatases 1 and 2A, also induces differentiation of these cells (5), cell cycle arrest, and eventual (72-h) apoptosis (6). Both PMA and okadaic acid induce expression of the cyclindependent kinase inhibitor, WAF1/CIP1 (7,8).WAF1/CIP1 expression is induced by the p53 protein following irradiation of cells (9, 10), but p53-independent expression of WAF1/CIP1 is associated with differentiation of myocytes (11, 12), of HL 60 leukemic cells (13), and of a number of other cells. WAF1/CIP1 is expressed in a number of tissues over the course of murine development, and expression in most tissues is not dependent on the presence of p53 (14). p53-independent expression of the WAF1/CIP1 gene can be induced in cultured cells by a number of agents, including, besides PMA and okadaic acid, platelet-derived growth factor, fibroblast growth factor, and transforming growth factor ␤ (15, 16). Preliminary analysis of the WAF1/CIP1 promoter suggests that the elements mediating response to serum in fibroblasts are located at least 1.9 kb upstream from the transcription start site (14), while...

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The third-chromosome mutation Killer of prune (K-pn) causes no phenotype by itself, but causes lethality in individuals homozygous for the nonlethal X-chromosome mutation prune (pn). We have recovered 12 gammaray-induced revertants of Killer o[prune. All of the revertants fail to complement a recessive cell lethal mutation in the abnormal wing ddscs (awd) gene. We present evidence that Killer o[prune is a mutation in the awd gene. First, revertant awd r's14 leads to reduced accumulation of the awd gene product, but does not affect flanking genes. Second, when a copy of the awd gene is cloned from Killer o[prtme homozygous flies and injected into embryos, transformants express the lethal interaction with prune. In individuals of the genotype pn; awdX'~°/awd+ the awd mRNA is present at normal levels but the awd polypeptide fails to accumulate. The absence of the awd gene product in such individuals is the cause of death. Although the awd polypeptide is a subunit of a cytoplasmic protein, its sequence is similar to subunit V of yeast cytochrome oxidase.

AML1 (RUNX1) regulates hematopoiesis, angiogenesis, muscle function, and neurogenesis. Previous studies have shown that phosphorylation of AML1, particularly at serines 276 and 303, affects its transcriptional activation. Here, we report that phosphorylation of AML1 serines 276 and 303 can be blocked in vivo by inhibitors of the cyclin-dependent kinases (CDKs) Cdk1 and Cdk2. Furthermore, these residues can be phosphorylated in vitro by purified Cdk1/cyclin B and Cdk2/cyclin A. Mutant AML1 protein which cannot be phosphorylated at these sites (AML1-4A) is more stable than wild-type AML1. AML-4A is resistant to degradation mediated by Cdc20, one of the substrate-targeting subunits of the anaphase-promoting complex (APC). However, Cdh1, another targeting subunit used by the APC, can mediate the degradation of AML1-4A. A phospho-mimic protein, AML1-4D, can be targeted by Cdc20 or Cdh1. These observations suggest that both Cdc20 and Cdh1 can target AML1 for degradation by the APC but that AML1 phosphorylation may affect degradation mediated by Cdc20-APC to a greater degree.The AML1 proteins, including AML1a, AML1b, and AML1c (AML1c is also known as AML1B), are generated from one gene by alternative splicing (22). This gene has been given the names RUNX1, AML1, CBFA2, and PEBP2␣B (37). The AML1 protein is composed of a DNA binding runt homology domain located in the amino terminus followed by a transcriptional activation domain and a negative regulatory C-terminal domain (15,20,29). AML1 was initially identified during the study of breakpoint t(8;21), which is a common chromosomal translocation in acute myeloid leukemia (23). The association of AML1 with blood cell development is shown by the disruption of the AML1 gene through multiple chromosomal translocations, deletions, point mutations, or amplification in approximately 30% of human myeloid leukemias and myelodysplastic syndrome patients and a significant number of lymphoid leukemia patients (21,24,27,32). Furthermore, no detectable definitive hematopoiesis is observed in Aml1 knockout mice (26,40). The importance of AML1 in nonhematopoietic cells has also been recognized in angiogenesis, muscle function, and neurogenesis (6,12,14,35,41).AML1 is detected as a serine and threonine phosphorylated protein (9). Previous work has suggested that AML1 activity may be regulated by phosphorylation (34,45). Phosphorylation at specific serine-proline or threonine-proline sites in AML1 appears to be necessary for normal activity (45). It has also been suggested that phosphorylation releases AML1 from an association with the nuclear matrix mediated by sin3A, in turn leading to both increased activity and an increased rate of degradation (11).We have now shown that AML1 phosphorylation by cyclindependent kinases (CDKs) affects the overall stability of AML1 as well as the ability of certain ubiquitin ligase complexes, such as Cdc20-anaphase-promoting complex (APC), to target AML1 for degradation. MATERIALS AND METHODSCell culture and treatment. 293T and NIH 3T3 cells were grown...

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