Effects of IKK inhibitor PS1145 on NF-kappaB function, proliferation, apoptosis and invasion activity in prostate carcinoma cells.

A key antiapoptotic transcription factor, nuclear factor kappa-B (NF-kappaB), is known to be critically important for tumor cell growth, angiogenesis and development of metastatic lesions. We and others showed previously that NF-kappaB transcription factor was constitutively activated in androgen-independent prostate carcinoma (PC) cell lines due to the upregulated activity of inhibitor of NF-kappaB kinases (IKK). In this work, using luciferase assay, electrophoretic mobility shift assay and Northern blot analysis of expression of endogenous kappaB-responsive genes, we demonstrate that a novel highly specific small-molecule IKK inhibitor, PS1145, efficiently inhibited both basal and induced NF-kappaB activity in PC cells. We found that PS1145 induced caspase 3/7-dependent apoptosis in PC cells and significantly sensitized PC cells to apoptosis induced by tumor necrosis factor alpha. We also showed that PS1145 inhibited PC cell proliferation. Effects of PS1145 on proliferation and apoptosis correlated with inhibition of interleukin (IL)-6, cyclin D1, D2, inhibitor of apoptosis (IAP)-1 and IAP-2 gene expression and decreased IL-6 protein level. In addition, we found that incubation with PS1145 inhibited the invasion activity of highly invasive PC3-S cells in invasion chamber assay in a dose-dependent manner. Overall, this study provides the framework for development of a novel therapeutic approach targeting NF-kappaB transcription factor to treat advanced PC.

A Nedd8 conjugation pathway is essential for proteolytic targeting of p27Kip1 by ubiquitination.

Temporal control of p27(Kip1) (p27) degradation imposes periodicity in its activity during cell cycle progression and its accumulation during cell cycle exit. Degradation of p27 is initiated by phosphorylation of p27 at Thr-187, which marks the protein for ubiquitination by SCF(Skp2) and subsequent proteolysis by the 26S proteasome. Here we show that the p27 ubiquitination activity in cell extracts depends on the presence of the ubiquitin-like protein Nedd8 and enzymes that catalyze Nedd8 conjugation to proteins. Moreover, we show that reconstitution of the p27 ubiquitination activity of recombinant SCF(Skp2) also requires Nedd8 conjugation pathway components. Inactivation of the Nedd8 conjugation pathway by a dominant negative mutant of the Nedd8-conjugating enzyme Nce1/Ubc12 blocks the ubiquitination and degradation of p27 in cell extracts. Consistent with a role in cell-cycle progression, Nedd8 is expressed in proliferating cells and is itself down-regulated upon cellular differentiation. These results suggest that the Nedd8 conjugation pathway may regulate the turnover of p27(Kip1), independently of p27 phosphorylation, and further establishes the identity of protein components involved in p27 ubiquitination. Finally, these findings provide a direct demonstration of a function for Nedd8 in a biological process.

Nedd8 modification of cul-1 activates SCF(beta(TrCP))-dependent ubiquitination of IkappaBalpha.

Regulation of NF-kappaB occurs through phosphorylation-dependent ubiquitination of IkappaBalpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkappaBalpha is carried out by a ubiquitin-ligase enzyme complex called SCF(beta(TrCP)). Here we show that Nedd8 modification of the Cul-1 component of SCF(beta(TrCP)) is important for function of SCF(beta(TrCP)) in ubiquitination of IkappaBalpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF(beta(TrCP)), phosphorylated IkappaBalpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCF(beta(TrCP)) in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed betaTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkappaBalpha required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCF(beta(TrCP)) containing a K720R mutant of Cul-1 only weakly supported IkappaBalpha ubiquitination compared to SCF(beta(TrCP)) containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF(beta(TrCP)). These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkappaBalpha.

A new model of cancer cachexia: contribution of the ubiquitin-proteasome pathway.

A new model of cachexia is described in which muscle protein metabolism related to the ubiquitin-proteasome pathway was investigated. Cloning of the colon-26 tumor produced a cell line, termed R-1, which induced cytokine (noninterleukin-1beta, interleukin-6 and tumor necrosis factor-alpha)-independent cachexia. Implantation of R-1 cells in mice elicited significant (20-30%) weight loss and decreased blood glucose by 70%, and adipose tissue levels declined by 95% and muscle weights decreased by 20-25%. Food intake was unaffected. The decrease in muscle weight reflected a decline in insoluble, but not soluble, muscle protein that was associated with a significant increase in net protein degradation. The rate of ubiquitin conjugation of proteins was significantly elevated in muscles of cachectic mice. Furthermore, the proteasome inhibitor lactacystin blocked the increase in protein breakdown but had no significant effect on proteolysis. Several markers of the ubiquitin-proteasome pathway, E2(14k) mRNA and E2(14k) protein and ubiquitin-protein conjugates, were not elevated. Future investigations with this new model should gain further insights into the mechanisms of cachexia and provide a background to evaluate novel and more efficacious therapies.

Cause and effect beliefs and self-esteem of overweight children.

In this study, beliefs of the cause and effect of weight were examined in a sample of 9- to 11-year-old clinically overweight children. Lower self-esteem was found in the children who believed they are responsible for their overweight as compared to those who attributed their overweight to an external cause. Lower self-esteem was also found in the children who believed that their overweight hinders their social interaction. Other evidence gathered here lends some support to the view that the overweight child is more vulnerable to low self-esteem. The negative experiences in school and at home that the children reported and the premise that childhood obesity is a stigmatising condition is discussed.

Novel inhibitors of cytokine-induced IkappaBalpha phosphorylation and endothelial cell adhesion molecule expression show anti-inflammatory effects in vivo.

We have identified two compounds that inhibit the expression of endothelial-leukocyte adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. These compounds act by inhibiting tumor necrosis factor-alpha-induced phosphorylation of IkappaB-alpha, resulting in decreased nuclear factor-kappaB and decreased expression of adhesion molecules. The effects on both IkappaB-alpha phosphorylation and surface expression of E-selectin were irreversible and occurred at an IC50 of approximately 10 microM. These agents selectively and irreversibly inhibited the tumor necrosis factor-alpha-inducible phosphorylation of IkappaB-alpha without affecting the constitutive IkappaB-alpha phosphorylation. Although these compounds exhibited other activities, including stimulation of the stress-activated protein kinases, p38 and JNK-1, and activation of tyrosine phosphorylation of a 130-140-kDa protein, these effects are probably distinct from the effects on adhesion molecule expression since they were reversible. One compound was evaluated in vivo and shown to be a potent anti-inflammatory drug in two animal models of inflammation. The compound reduced edema formation in a dose-dependent manner in the rat carrageenan paw edema assay and reduced paw swelling in a rat adjuvant arthritis model. These studies suggest that inhibitors of cytokine-inducible IkappaBalpha phosphorylation exert anti-inflammatory activity in vivo.

Tumor necrosis factor alpha-induced E-selectin expression is activated by the nuclear factor-kappaB and c-JUN N-terminal kinase/p38 mitogen-activated protein kinase pathways.

E-selectin expression by endothelium is crucial for leukocyte recruitment during inflammatory responses. Transcriptional regulation of the E-selectin promoter by tumor necrosis factor alpha (TNFalpha) requires multiple nuclear factor-kappaB (NF-kappaB) binding sites and a cAMP-responsive element/activating transcription factor-like binding site designated positive domain II (PDII). Here we characterize the role of the stress-activated family of mitogen-activated protein (MAP) kinases in induced expression of this adhesion molecule. By UV cross-linking and immunoprecipitation, we demonstrated that a heterodimer of transcription factors ATF-2 and c-JUN is constitutively bound to the PDII site. TNFalpha stimulation of endothelial cells induces transient phosphorylation of both ATF-2 and c-JUN and induces marked activation of the c-JUN N-terminal kinase (JNK1) and p38 but not extracellular signal-regulated kinase (ERK1). JNK and p38 are constitutively present in the nucleus, and DNA-bound c-JUN and ATF-2 are stably contacted by JNK and p38, respectively. MAP/ERK kinase kinase 1 (MEKK1), an upstream activator of MAP kinases, increases E-selectin promoter transcription and requires an intact PDII site for maximal induction. MEKK1 can also activate NF-kappaB -dependent gene expression. The effects of dominant interfering forms of the JNK/p38 signaling pathway demonstrate that activation of these kinases is critical for cytokine-induced E-selectin gene expression. Thus, TNFalpha activates two signaling pathways, NF-kappaB and JNK/p38, which are both required for maximal expression of E-selectin.

Salicylates inhibit I kappa B-alpha phosphorylation, endothelial-leukocyte adhesion molecule expression, and neutrophil transmigration.

The expression of leukocyte adhesion molecules on endothelial cells is induced by TNF-alpha and other inflammatory cytokines. This induction of endothelial-leukocyte adhesion molecule-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 requires the transcription factor nuclear factor-kappa B (NF-kappa B). Recent work has suggested that some nonsteroidal anti-inflammatory agents, including sodium salicylate and aspirin, can inhibit NF-kappa B-dependent gene activation. We studied the effects of salicylates on expression of adhesion molecules in HUVECs. We found that sodium salicylate inhibited activation of NF-kappa B (p50/p65 and p65/p65) by preventing phosphorylation and subsequent degradation of the inhibitor 1 kappa B-alpha. Salicylate treatment had no effect on TNF-alpha-induced phosphorylation of the transcription factor ATF-2. Salicylate blocked the TNF-alpha-induced increase in mRNA levels of adhesion molecules and gave a dose-dependent inhibition of TNF-alpha-induced surface expression of vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 with higher doses required to inhibit endothelial-leukocyte adhesion molecule-1 expression. Indomethacin, a nonsalicylate cyclooxygenase inhibitor, had no effect on surface expression of adhesion molecules, suggesting that the effects were not due to inhibition of cyclooxygenase. Treatment of endothelial cell monolayers with sodium salicylate inhibited transendothelial migration of neutrophils but had no significant effect on neutrophil adhesion under flow conditions. The clinical importance of high-dose salicylates in inflammation may be due, in part, to the ability to prevent expression of inducible adhesion molecules and recruitment of leukocytes.

Body size, parental appraisal, and self-esteem in blind children.

This study of self-esteem, body size, and parental views in 9-11-year-old blind children found positive views about self-presentation with no sex or weight differences. Lower self-esteem emerged in children who thought they were judged by parents as too thin but being fat, being appraised as fat, or believing they are thought of as fat by parents, showed no effect on self-esteem. Their responses to questions about the causes, characteristics, and psychosocial functioning of obesity suggest an innate desire and possible need for a more robust stature, a bigger presence, and a feeling of weight which appeared to supercede any acquired negative attitudes to fatness.

Activation of IL-2 receptor alpha-chain gene by individual members of the rel oncogene family in association with serum response factor.

Expression of the IL-2R alpha gene is regulated by members of the c-Rel/NF-kappa B family of transcription factors binding to the kappa B site in the promoter. Previous work has not defined the role of individual members of the c-Rel family in the activation of the IL-2R alpha gene. Using the COS cell system, we were able to reconstitute the regulation of the IL-2R alpha promoter by expressing cloned Rel family members with serum response factor (SRF). We found that c-rel alone activated the IL-2R alpha promoter only weakly but worked with the p50 subunit of NF-kappa B (NFKB1) to give a higher level of expression. We showed that c-rel heterodimerizes with p50 and the amount of this heterodimer correlated with the level of IL-2R alpha gene expression. Our results provide evidence that c-rel/p50 heterodimers activate gene expression in the context of a cellular promoter. We show that c-rel or p65 can cooperate with SRF in the activation of this promoter and the transactivation by c-rel with SRF was enhanced by p50. Synergistic activation required both kappa B and CArG sites, and binding studies show that these adjacent sites can be occupied simultaneously. The transactivation observed with cloned transcription factors mimics the physiologic induction of the IL-2R alpha gene since multiple sequence elements cooperate to give gene activation. The data support the model that c-rel/p50 or p65 can cooperate with SRF to specifically target the expression of the IL-2R alpha gene in activated T cells.

Expression and induction of nuclear factor-kappa B-related proteins in thymocytes.

Thymocytes mature in response to the cues from the thymic micro-environment, which regulate stage-specific gene expression during development. We find that several proteins that bind the kappa B sequence in vitro are constitutively activated in freshly isolated thymocytes. These include the rel-related p50 homodimers, p50/p65 heterodimers, low levels of c-rel, and two other factors that may be thymus specific. Disruption of the thymic micro-environment resulted in loss of DNA-binding, suggesting that lymphocyte-stromal cell interactions induce and maintain these proteins in a DNA-binding form. Phorbol ester and ionomycin treatment induced p50, p65, and p68 c-rel kappa B DNA-binding activity. Expression of p68 c-rel protein, but not p50 or p65, was suppressed by the immunosuppressive drug FK506. Because FK506 specifically inhibits the appearance of mature single-positive thymocytes, gene expression regulated by p68 c-rel may play a role in selection and maturational signals involved in the double-positive to single-positive transition.

Differential regulation of the c-myc oncogene promoter by the NF-kappa B rel family of transcription factors.

The murine c-myc gene contains two elements responsive to the rel-oncogene-related family of NF-kappa B factors. Previously we have shown that factor binding to these two NF-kappa B elements mediates induction of transcription of the c-myc promoter upon interleukin-1 treatment of human dermal fibroblasts and human T-cell leukemia virus type I tax gene expression in T cells (D. J. Kessler, M. P. Duyao, D. B. Spicer, and G. E. Sonenshein, J. Exp. Med. 176:787-792, 1992; M. P. Duyao, D. J. Kessler, D. B. Spicer, C. Bartholomew, J. L. Cleveland, M. Siekevitz, and G. E. Sonenshein, J. Biol. Chem. 267:16288-16291, 1992). To begin to delineate the specific roles of the individual members of the NF-kappa B family, here we have tested their effects on activation of a c-myc promoter/exon 1-CAT construct in NIH 3T3 cells. Classical NF-kappa B (p65/p50) was a potent transcriptional activator of the c-myc promoter. Cotransfection with either p65 alone or p65 in combination with p50 mediated significant induction. In contrast, expression of either v-rel or chicken c-rel failed to transactivate, while murine c-rel induced c-myc promoter activity only slightly. Furthermore, induction by classical NF-kappa B was inhibited by coexpression of either v-rel or chicken c-rel. Thus, individual members of the rel family have differential effects of the c-myc promoter, which can modulate overall transcriptional activity and allow for precise regulation of this oncogene under diverse physiologic conditions.

Self-esteem, parental appraisal and body size in children.

This study investigates the influence of body size, parental appraisal of body size, and children's beliefs about parental appraisal, on self-esteem in children from 9 to 11 years old. Parents' and children's responses to a matched question about body size suggest that children are accurate predictors of parental evaluation and that their self-esteem scores are influenced both by actual parental dissatisfaction and beliefs about parental dissatisfaction. For boys, lower self-esteem is associated both with thinness and being perceived as too thin. For girls, lower self-esteem is more associated with fatness.

Silencing of the expression of the immunoglobulin kappa gene in non-B cells.

Although the activating factor NF-kappa B can be present in the nucleus of many cell types, transcription and rearrangement of the immunoglobulin kappa chain gene is restricted to cells of the B lineage. Part of this specificity is determined by sequences within the major intron of the kappa gene that specifically silence gene expression in non-B cells (T cells and HeLa cells). These sequences are found in a 232-bp fragment located 5' of the NF-kappa B binding sequence of the enhancer. When this fragment is added back upstream of an active NF-kappa B site, it specifically decreases the expression of a linked gene by more than 10-fold in activated T cells but it has no effect on expression in B cells. The kappa silencer region acts in an orientation- and distance-independent manner and appears to be composed of multiple negative elements. The kappa silencer may act to restrict transcription and rearrangement of the C kappa locus to cells of the B lineage.

Oligonucleotide that binds nuclear factor NF-kappa B acts as a lymphoid-specific and inducible enhancer element.

The immunoglobulin kappa light chain gene contains a lymphoid-specific enhancer that includes several short protein-binding sequences. The sequence that binds the nuclear factor NF-kappa B was tested for its ability to act independently as an enhancer element by inserting it into test plasmids containing the chloramphenicol acetyltransferase gene. When analyzed for activity by transient transfection into lymphoid and nonlymphoid cells, a single copy of the NF-kappa B binding site could act as a tissue-specific upstream activating element. Two copies (dimer) showed 10-fold higher activity than did one copy and could act as an enhancer element 2.5 kilobases downstream of the transcriptional start site. The enhancer activity of this sequence was correlated with the presence of the cognate binding protein, NF-kappa B. This sequence acted as an inducible enhancer under conditions that induce NF-kappa B binding activity. Thus, the NF-kappa B binding site acts by itself as a tissue-specific and inducible enhancer element, and two copies show cooperative interaction.

Protein-binding sites in Ig gene enhancers determine transcriptional activity and inducibility.

Individual protein-binding sites within the mouse immunoglobulin heavy chain and kappa light chain gene enhancers were altered, making it possible to examine the functional role of the sites during transcription. The E motifs, which bind factors that are present in many if not all cells, mostly behave as transcriptional activating sites. The only known heavy chain enhancer site that binds a lymphocyte-specific factor, the "octamer" site, plays a critical role in transcription but only in a truncated form of the enhancer. In the full enhancer, no one site is crucial because of an apparent functional redundancy. The site in the kappa enhancer that binds a factor specific to mature B cells, kappa B, was crucial to the constitutive activity of the enhancer in B cells. This factor is also inducible in pre-B cells, and the site was necessary for inducibility of the kappa enhancer. Thus, the sites defined by protein binding are important for the functional activity of immunoglobulin enhancers, with the sites that bind proteins restricted in their cellular distribution playing the most important roles.

The role of a novel VH sequence (V11) in the formation of anti-phosphocholine antibodies.

The immune response to phosphocholine (PC) in mice is highly restricted. Most anti-PC antibodies use heavy-chain variable-region (VH) sequences derived from single VH gene segment, V1. In order to investigate whether a highly homologous VH gene segment, V11, could contribute to the formation of PC-binding antibodies, we carried out chain recombination experiments with M47A, a non-PC binding myeloma protein whose H-chain is encoded by the V11 gene segment, and two PC-binding antibodies, HP101.6G6 (HP6G6) and M511. The H-chains from the non-PC-binding myeloma protein, M47A, formed a functional PC-binding site when paired with L-chains from both PC-binding antibodies. These results suggest that a second VH gene segment, V11, could theoretically be used to form PC-binding antibodies. In addition, these results provide direct evidence that a single H-chain can be used in combinatorial association with different L-chains to form antibodies of differing specificities.