The human brm protein is cleaved during apoptosis: the role of cathepsin G.

The human brm (hbrm) protein (homologue of the Drosophila melanogaster brahma and Saccharomyces cervisiae SNF-2 proteins) is part of a polypeptide complex believed to regulate chromatin conformation. We have shown that the hbrm protein is cleaved in NB4 leukemic cells after induction of apoptosis by UV-irradiation, DNA damaging agents, or staurosporine. Because hbrm is found only in the nucleus, we have investigated the nature of the proteases that may regulate the degradation of this protein during apoptosis. In an in vitro assay, the hbrm protein could not be cleaved by caspase-3, -7, or -6, the "effector" caspases generally believed to carry out the cleavage of nuclear protein substrates. In contrast, we find that cathepsin G, a granule enzyme found in NB4 cells, cleaves hbrm in a pattern similar to that observed in vivo during apoptosis. In addition, a peptide inhibitor of cathepsin G blocks hbrm cleavage during apoptosis but does not block activation of caspases or cleavage of the nuclear protein polyADP ribose polymerase (PARP). Although localized in granules and in the Golgi complex in untreated cells, cathepsin G becomes diffusely distributed during apoptosis. Cleavage by cathepsin G removes a 20-kDa fragment containing a bromodomain from the carboxyl terminus of hbrm. This cleavage disrupts the association between hbrm and the nuclear matrix; the 160-kDa hbrm cleavage fragment is less tightly associated with the nuclear matrix than full-length hbrm.

Regulation of dual-specificity phosphatases M3/6 and hVH5 by phorbol esters. Analysis of a delta-like domain.

Treatment of leukemic cells with phorbol 12-myristate 13-acetate (PMA) induces a short-lived phosphorylation and activation of stress-activated protein kinase (SAPK) and cellular differentiation. To investigate whether the rapid deactivation of SAPK results from dephosphorylation by dual-specificity phosphatases (DSPs), we studied regulation of the DSP hVH5 and its murine orthologue M3/6 in K562 human leukemia cells. PMA treatment rapidly induced hVH5 transcripts in these cells, and induced expression of M3/6 completely inhibited PMA-stimulated phosphorylation of SAPK, suggesting a feedback loop to control SAPK activity. Using both stable cell lines and transient transfection we demonstrate that activation of SAPK rapidly stimulated phosphorylation of M3/6. This phosphorylation did not regulate the half-life of total cellular M3/6. hVH5 and M3/6 shares with all sequenced mammalian DSPs an amino acid motif, XILPXLXL, located approximately 80 amino acids from the active site. The hVH5-M3/6 sequence, RILPHLYL, shares significant homology with the SAPK binding site of the c-Jun protein, called the delta domain. This motif was found to be important for DSP function, because deletion of RILPHLYL inhibits SAPK-mediated phosphorylation of M3/6, and deletion of this sequence or mutation of the LYL portion blocks the ability of this phosphatase to dephosphorylate SAPK.

The gut-enriched Kruppel-like factor (Kruppel-like factor 4) mediates the transactivating effect of p53 on the p21WAF1/Cip1 promoter.

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 Krüppel-like factor (GKLF, KLF4) is concurrently induced with p21(WAF1/Cip1) during 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(WAF1/Cip1), suggesting that GKLF may be involved in the induction of p21(WAF1/Cip1). Indeed, GKLF activates p21(WAF1/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.

The role of the Smad3 protein in phorbol ester-induced promoter expression.

The Sp1 transcription factor plays an important role in mediating the p53-independent activation of the p21(WAF1) (WAF1) promoter by phorbol 12-myristate13-acetate (PMA) in hematopoietic cells. Using GAL4-Sp1 fusion proteins and a luciferase reporter, PMA is shown to activate the transcriptional activity of Sp1 independent of the WAF1 promoter. This activation does not require the Ser/Thr-rich region of Sp1 and can be mediated by 41 amino acids (152-193) of Sp1 that are important for the interaction with human TAF130. Because transforming growth factor-beta enhances WAF1 promoter activity through both Sp1 and Smad proteins, the role of Smads in PMA transcriptional activation was examined. PMA addition to hematopoietic cells was found to activate a GAL4/Smad-dependent promoter and the transforming growth factor-beta-responsive promoter, p3TP-lux. Immunofluorescence data demonstrate that PMA addition to hematopoietic cells induces the translocation of Smad3 to the nucleus. However, Smad3 does not stimulate the WAF1 promoter, but rather slightly inhibits the PMA-mediated induction of transcription from this upstream region. Additionally, transfection of Smad3 did not enhance the activation of GAL4/Sp1 by PMA. These results demonstrate that, while PMA can activate Smad-mediated transcription, Smad proteins do not appear to play a major role in the PMA induction of the WAF1 promoter.

Radionuclides in deer and elk from Los Alamos National Laboratory and the doses to humans from the ingestion of muscle and bone.

This paper summarizes radionuclide concentrations (3H, 90Sr, 137Cs, 238Pu, 239,240Pu, 241Am, and totU) in muscle and bone tissue of mule deer (Odocoileus hemionus) and Rocky Mountain elk (Cervus elaphus) collected from Los Alamos National Laboratory (LANL), Los Alamos, New Mexico, lands from 1991 through 1998. Also, the committed effective dose equivalent (CEDE) and the risk of excess cancer fatalities (RECF) to people who ingest muscle and bone from deer and elk collected from LANL lands were estimated. Most radionuclide concentrations in muscle and bone from individual deer (n = 11) and elk (n = 22) collected from LANL lands were either at less than detectable quantities (where the analytical result was smaller than two counting uncertainties) and/or within upper (95%) level background (BG) concentrations. As a group, most radionuclides in muscle and bone of deer and elk from LANL lands were not significantly higher (p < 0.10) than in similar tissues from deer (n = 3) and elk (n = 7) collected from BG locations. Also, elk that had been radio collared and tracked for two years and spent an average time of 50% of LANL lands were not significantly different in most radionuclides from road kill elk that have been collected as part of the environmental surveillance program. Overall, the upper (95%) level net CEDEs (the CEDE plus two sigma for each radioisotope minus background) at the most conservative ingestion rate (50 lbs of muscle and 13 lbs of bone) were as follows: deer muscle = 0.22 mrem y-1 (2.2 microSv y-1), deer bone = 3.8 mrem y-1 (38 microSv y-1), elk muscle = 0.12 mrem y-1 (1.2 microSv y-1), and elk bone = 1.7 mrem y-1 (17 microSv y-1). All CEDEs were far below the International Commission on Radiological Protection guideline of 100 mrem y-1 (1000 microSv y-1), and the highest muscle plus bone net CEDE corresponded to a RECF of 2E-06, which is far below the Environmental Protection Agency upper level guideline of 1E-04.

Functional nonequivalence of Drosophila actin isoforms.

We show that different Drosophila actin isoforms are not interchangeable. We sequenced the six genes that encode conventional Drosophila actins and found that they specify amino acid replacements in 27 of 376 positions. To test the significance of these changes we used directed mutagenesis to introduce 10 such conversions, independently, into the Act88F flight muscle-specific actin gene. We challenged these variant actins to replace the native protein by transforming germline chromosomes of a Drosophila strain lacking flight muscle actin. Only one of the 10 reproducibly perturbed myofibrillar function, demonstrating that most isoform-specific amino acid replacements are of minor significance. In order to establish the consequences of multiple amino acid replacements, we substituted portions of the Drosophila Act88F actin gene with corresponding regions of genes encoding other isoforms. Only one of five constructs tested engendered normally functioning flight muscles, and the severity of myofibrillar defects correlated with the number of replacements within the chimeric genes. Finally, we completely converted the flight muscle actin-encoding gene to one specifying a nonmuscle isoform, a change entailing a total of 18 amino acid replacements. Transformation of flies with this construct resulted in disruption of flight muscle structure and function. We conclude that actin isoform sequences are not equivalent and that effects of the amino acid replacements, while minor individually, collectively confer unique properties.

Activation of the mitogen-activated protein kinase pathway in U937 leukemic cells induces phosphorylation of the amino terminus of the TATA-binding protein.

Phorbol ester treatment of U937 leukemic cells results in the activation of numerous protein kinase pathways, followed by cell cycle arrest and differentiation into macrophage-like cells. Because major changes in gene transcription are associated with this process, the role of general transcription factors in the cell response to phorbol esters was examined. Experiments demonstrate that phorbol ester treatment of U937 cells stimulates the phosphorylation of the TATA-binding protein (TBP); this phosphorylation occurs within 30 min and is still apparent, although greatly reduced, at 4 h. The following results demonstrate that TBP phosphorylation occurs as a result of activation of an extracellular signal-regulated kinase (ERK) protein kinase: (a) overexpression of mitogen-activated protein kinase phosphatase-1 blocks phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of TBP both in vitro and in vivo; (b) pretreatment with the ERK kinase kinase inhibitor PD098059 also blocks PMA-induced phosphorylation of TBP both in vitro and in vivo; and (c) phosphorylation of TBP is observed when serum-starved NIH 3T3 cells are stimulated with fresh serum, another activator of the ERK pathway. TBP can be phosphorylated in vitro by extracts of U937 cells or by bacterially expressed activated ERK2; the phosphorylation sites were mapped to ERK kinase consensus sites in the TBP amino-terminal domain. Using glutathione S-transferase-TBP fusion proteins, cellular proteins that bind specifically to the TBP amino terminus have been identified. These observations suggest that ERK-mediated phosphorylation of TBP occurs during the PMA-induced differentiation of U937 cells and the stimulation of the G0-G1 transition in fibroblasts and could play a role in the regulation of TBP protein interactions and thus regulate gene transcription during these two processes.

The role of the transcription factor Sp1 in regulating the expression of the WAF1/CIP1 gene in U937 leukemic cells.

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 okaidaic 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.

Inhibitors of cyclin-dependent kinase and cancer.

Recent research has yielded a dramatic increase in the number of connections between oncogenesis and the proteins which regulate the cell cycle. Three classes of protein which inhibit the activity of cyclin-dependent kinases (CDKs) have emerged as potential targets for oncogenic inactivation. p16 and related proteins inhibit the cyclin/CDK complexes which regulate the transition from G1 to S phase; numerous studies have revealed that p16 is mutated in most tumor cell lines and in some types of primary tumor. p21/WAF1/Cip 1 and the related p27Kip protein inhibit a broader range of cyclin/CDK complexes than p16. Although the absence of p21/WAF1/Cip1 from cyclin/CDK complexes is correlated with cellular transformation, no mutations in this gene have been found in tumors or tumor-derived cell lines. A third class of genes which are potential targets for oncogenic inactivation are the kinases and phosphatases which regulate the activity of cyclin/CDK complexes by phosphorylation and dephosphorylation of the CDK proteins. Disruption of any of these genes would result in loss of normal regulation of cell growth.

Repression of the Id2 (inhibitor of differentiation) gene promoter during exit from the cell cycle.

The Id2 gene is one of several "Id-like" genes which encode helix-loop-helix proteins which dimerize with basic helix-loop-helix proteins and inhibit binding to the DNA enhancer element known as an E box. By repressing the DNA binding activity of basic helix-loop-helix proteins, Id proteins inhibit transcription of tissue-specific genes in myoblasts, hematopoietic precursor cells, and other types of undifferentiated cells. Serum starvation results in the disappearance of Id gene transcripts in most types of cultured cells, and often induces differentiation of these cells. In order to gain some insight into this process, we have analyzed Id2 promoter function in the glioma cell line U87Y. We have isolated 300 base pairs of Id2 promoter sequence which is sufficient to repress the activity of a reporter gene in serum-starved U87Y cells, but induces the activity of the reporter gene when the cells are stimulated with fresh serum. Two regions within this 300 base pair sequence contain repressor elements; deletion of either region results in increased promoter activity. Both repressor regions serve as binding sites for a protein present in extracts from serum-starved U87Y cells but not in serum-stimulated cells.

Reduced Nm23/Awd protein in tumour metastasis and aberrant Drosophila development.

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.