Colorectal tumor DNA was examined for somatic instability at (CA)n repeats on human chromosomes 5q, 15q, 17p, and 18q. Differences between tumor and normal DNA were detected in 25 of the 90 (28 percent) tumors examined. This instability appeared as either a substantial change in repeat length (often heterogeneous in nature) or a minor change (typically two base pairs). Microsatellite instability was significantly correlated with the tumor's location in the proximal colon (P = 0.003), with increased patient survival (P = 0.02), and, inversely, with loss of heterozygosity for chromosomes 5q, 17p, and 18q. These data suggest that some colorectal cancers may arise through a mechanism that does not necessarily involve loss of heterozygosity.

The atypical protein kinase C (PKC) isotypes (lambda/iotaPKC and zetaPKC) have been shown to be critically involved in important cell functions such as proliferation and survival. Previous studies have demonstrated that the atypical PKCs are stimulated by tumor necrosis factor alpha (TNF-alpha) and are required for the activation of NF-kappaB by this cytokine through a mechanism that most probably involves the phosphorylation of IkappaB. The inability of these PKC isotypes to directly phosphorylate IkappaB led to the hypothesis that zetaPKC may use a putative IkappaB kinase to functionally inactivate IkappaB. Recently several groups have molecularly characterized and cloned two IkappaB kinases (IKKalpha and IKKbeta) which phosphorylate the residues in the IkappaB molecule that serve to target it for ubiquitination and degradation. In this study we have addressed the possibility that different PKCs may control NF-kappaB through the activation of the IKKs. We report here that alphaPKC as well as the atypical PKCs bind to the IKKs in vitro and in vivo. In addition, overexpression of zetaPKC positively modulates IKKbeta activity but not that of IKKalpha, whereas the transfection of a zetaPKC dominant negative mutant severely impairs the activation of IKKbeta but not IKKalpha in TNF-alpha-stimulated cells. We also show that cell stimulation with phorbol 12-myristate 13-acetate activates IKKbeta, which is entirely dependent on the activity of alphaPKC but not that of the atypical isoforms. In contrast, the inhibition of alphaPKC does not affect the activation of IKKbeta by TNF-alpha. Interestingly, recombinant active zetaPKC and alphaPKC are able to stimulate in vitro the activity of IKKbeta but not that of IKKalpha. In addition, evidence is presented here that recombinant zetaPKC directly phosphorylates IKKbeta in vitro, involving Ser177 and Ser181. Collectively, these results demonstrate a critical role for the PKC isoforms in the NF-kappaB pathway at the level of IKKbeta activation and IkappaB degradation.

The interleukin-2 (IL-2) promoter consists of several independent T cell receptor (TcR) responsive elements. The induction of promoters dependent on these elements is inhibitable by the immunosuppressants cyclosporin A (CsA) and tacrolimus . Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is the FK-506-and CsA-sensitive enzyme required for TcR mediated activation of the LL-2 promoter. We report that a constitutively active form of calcineurin partially substitutes for the Ca2+ co-stimulus required to activate the IL-2 promoter elements IL-2A (which binds the factors OAP and Oct-i) and LL-2E (which binds NF-AT), and completely substitutes for the Ca2+ co-stimulus required to stimulate an NF-xB-dependent element. Calcineurin stimulates the NF-xB element by enhancing inactivation of IxB/MAD3, an inhibitor of NF-xB, thereby increasing the amount of nuclear NF-xB DNA binding activity. These data provide the first demonstration in vivo that activation of a protein phosphatase can inactivate IxB, and suggest one possible explanation for mechanism-based toxicities associated with FK-506 and CsA by demonstrating that these drugs can inhibit the calcineurin-dependent activation of a virtually ubiquitous transcription factor.

RelA and RelB are two members of the NF-kB family that dier structurally and functionally. While RelA is regulated through its cytosolic localization by inhibitor proteins or IkB and not through transcriptional mechanisms, the regulation of RelB is poorly understood. In this study we demonstrate that stimuli (TNF or LPS) lead within minutes to the nuclear translocation of RelA, but require hours to result in the nuclear translocation of RelB. The delayed nuclear translocation of RelB correlates with increases in its protein synthesis which are secondary to increases in RelB gene transcription. RelA is alone sucient to induce RelB gene transcription and to mediate the stimuli-driven increase in RelB transcription. Cloning and characterization of the RelB 5' untranslated gene region indicates that RelB transcription is dependent on a TATA-less promoter containing two NF-kB binding sites. One of the NF-kB sites is primarily involved in the binding of p50 while the other one in the binding and transactivation by RelA and also RelB. Lastly, it is observed that p21, a protein involved in cell cycle control and oncogenesis known to be regulated by NF-kB, is upregulated at the transcriptional level by RelB. Thus, RelB is regulated at least at the level of transcription in a RelA and RelB dependent manner and may exert an important role in p21 regulation. Oncogene (2001) 20, 7722 ± 7733.

RelB mediates the constitutive nuclear pool of NF-B transcriptional activity in myeloid and lymphoid cells, which is believed to be secondary to its weak interaction with the classical NF-B inhibitor proteins, the IBs. In other cell types, RelB is located in the cytosol, thus suggesting that RelB is also regulated by an inhibitory protein(s). In this study, it is demonstrated that RelB is associated in the cytosol with p100 but not with IB␣, IB␤, IB⑀, nor p105. Its cytosolic control is not affected by stimuli that lead to RelA nuclear translocation, and RelB nuclear localization is prevented by p100, but not by p105 or IB␣. Structure function analysis p100-RelB interactions indicates that p100 amino acids 623-900 are required for effective interaction and repression of nuclear translocation and RelB driven NF-B-dependent transcription. Moreover, this carboxyl-portion of p100 contains a nuclear export signal(s), which is required for effective retrieval of RelB from the nucleus. Finally, overexpression of NF-B-inducing kinase, a kinase that has recently been shown to induce p100 processing, possibly through IKK␣ activation, causes nuclear translocation of RelB protein. Thus, these studies indicate that p100 is a bone fide inhibitor of RelB and that this transcription factor may be regulated by NF-B-inducing kinase and/or IKK␣.

The phosphoprotein IB␣ exists in the cytoplasm of resting cells bound to the ubiquitous transcription factor NF-B (p50-p65). In response to specific cellular stimulation, IB␣ is further phosphorylated and subsequently degraded, allowing NF-B to translocate to the nucleus and transactivate target genes. NF-B is an inducible transcription factor involved in the regulation of numerous genes (5, 43). The classic NF-B complex consists of a heterodimer of p50 (NF-B1) and p65 (RelA) (38,54,68). These two subunits are members of a family of factors with homology to the c-rel proto-oncogene (70). In response to a variety of stimuli, combinations of these cytoplasmic c-rel-like factors translocate to the nucleus and transactivate specific target genes (4).In most resting cells, NF-B is anchored in the cytoplasm by its association with inhibitory molecules known as IBs (43). This family of ankyrin-containing inhibitors includes IB␣ (2,3,19,27,31), IB␤ (76), IB␥ (36), the p105 precursor of p50 (44,61,72), and the p100 precursor of p52 (66). The cloning of the cDNA of IB␣ (MAD3) (31) demonstrated that aside from the IB hallmark ankyrin repeat region, consensus sites for tyrosine phosphorylation and a phosphatidylinositol-3-kinase binding site are present. In addition, the C-terminal domain contains consensus phosphorylation sites for both protein kinase C (PKC) and casein kinase II (CKII) and a region rich in proline, glutamic acid, serine, and threonine (PEST) residues, which has been associated with rapid protein turnover (63). IB␣ protein specifically inhibits the DNA binding of the p50-p65 heterodimer (22, 31) by binding to p65 and physically masking its nuclear localization signal, resulting in the cytoplasmic retention of the heterodimeric complex (8). IB␣ in resting cells is a phosphoprotein with a short half-life (30 min to 3 h) (34,48,60,71). Upon activation of cells with cytokines or mitogens, IB␣ is further phosphorylated prior to proteasome-mediated IB␣ degradation and NF-B translocation (7,10,13,18,21,25,26,33,42,56,(71)(72)(73), resulting in a half-life of less than 5 min (71). While it is known that the stimulusinduced phosphorylation event is necessary for rapid 12), the identity of the stimulus-induced kinase(s) remains elusive. Moreover, the identity and functional relevance of the kinase(s) that constitutively phosphorylates IB␣ need to be determined.Although in vitro IB␣ can be phosphorylated by many different kinases, including PKC (27, 69), heme-regulated eIF2␣ kinase (27), protein kinase A (PKA) (27, 69), p38 (30), Raf-1 (40), a PKC--associated kinase (20), and CKII (6), the significance of this phosphorylation in vivo is unknown. Recent studies demonstrate that Ser-32 and Ser-36 are targets of inducible phosphorylation and are necessary for the subsequent degradation of this molecule in response to different stimuli (11,12,15,74,75). In addition, the carboxy-terminal region, which contains several CKII consensus motifs and the PEST sequence, is not necessary for the inducible phosphorylation of IB␣ bu...

In the absence of HO-1, expression of MCP-1 is significantly and consistently enhanced in unstressed and stressed conditions. We speculate that the protective effects of HO-1 in injured tissue may involve, at least in part, the capacity of HO-1 to restrain up-regulation of MCP-1.

Human immunodeficiency virus type 1 (HIV-1) infection causes apoptosis of infected CD4 T cells as well as uninfected (bystander) CD4 and CD8 T cells. It remains unknown what signals cause infected cells to die.We demonstrate that HIV-1 protease specifically cleaves procaspase 8 to create a novel fragment termed casp8p41, which independently induces apoptosis. casp8p41 is specific to HIV-1 protease-induced death but not other caspase 8-dependent death stimuli. In HIV-1-infected patients, casp8p41 is detected only in CD4 ؉ T cells, predominantly in the CD27 ؉ memory subset, its presence increases with increasing viral load, and it colocalizes with both infected and apoptotic cells. These data indicate that casp8p41 independently induces apoptosis and is a specific product of HIV-1 protease which may contribute to death of HIV-1-infected cells.