In recent years, the multi-subunit IKK complex has been shown to be responsible for cytokine-mediated stimulation of genes involved in inflammation and as such represents an attractive target for pharmaceutical intervention. Our finding that parthenolide targets this kinase complex provides a possible molecular basis for the anti-inflammatory properties of parthenolide. In addition, these results may be useful in the development of additional anti-inflammatory agents.

In the standard model of cytokine-induced signal transducer and activator of transcription (STAT) protein family signaling to the cell nucleus, it is assumed that STAT3 is recruited to the cytoplasmic side of the cell surface receptor complex from within a cytosolic monomer pool. By using Superose-6 gel-filtration chromatography, we have discovered that there is little monomeric STAT3 (91 kDa) in the cytosol of liver cells (human hepatoma Hep3B cell line and rat liver). The bulk of STAT3 (and STAT1, STAT5a, and -b) was present in the cytosol as high molecular mass complexes in two broad distributions in the size range 200 -400 kDa ("statosome I") and 1-2 MDa ("statosome II"). Upon treatment of Hep3B cells with interleukin-6 (IL-6) for 30 min (i) cytosolic tyrosine-phosphorylated STAT3 was found to be in complexes of size ranging from 200 -400 kDa to 1-2 MDa; (ii) a small pool of monomeric STAT3 and tyrosinephosphorylated STAT3 eluting at 80 -100 kDa was observed, and (iii) most of the cytoplasmic DNA-binding competent STAT3 (the so-called SIF-A "homodimer") coeluted with catalase at 230 kDa. In order to identify the protein components of the 200 -400-kDa statosome I cytosolic complexes, we used the novel technique of antibody-subtracted differential protein display using anti-STAT3 antibody. Eight polypeptides in the size range from 20 to 114 kDa co-shifted with STAT3; three of these (p60, p20a, and p20b) were co-shifted in an IL-6-dependent manner. In-gel tryptic fragmentation and mass spectroscopy identified the major IL-6-dependent STAT3-coshifted p60 protein as the chaperone GRP58/ER-60/ ERp57. Taken together, these data (i) emphasize the absence of a detectable STAT3 monomer pool in the cytosol of cytokine-free liver cells as posited by the standard model, and (ii) suggest an alternative model for STAT signaling in which STAT3 proteins function in the cytoplasm as heteromeric complexes with accessory scaffolding proteins, including the chaperone GRP58.

KRAS mutation is associated with more rapid and aggressive metastatic behavior of colorectal liver metastases. These data suggest an important role for KRAS activation in colorectal cancer progression and support continued efforts to develop KRAS pathway inhibitors for this disease.

Introduction We examined two genetic markers established early in colorectal tumor development, microsatellite instability (MSI) and mutation of the KRAS proto-oncogene, to see if these genetic changes influence metastatic disease progression and survival. Patients and methods MSI and KRAS mutation status were assessed in 532 primary adenocarcinomas (stage I–IV) from patients treated by colon resection. Median follow-up was 4.1 years (range 0–13.3 years) overall, 5.4 years for survivors. Results MSI and KRAS mutation were detected in 12 and 36% of cases, respectively. MSI was more common in early-stage disease (I, 15%; II, 21%; III, 10%; IV, 2%; P = 0.0001). Prevalence of KRAS mutation did not vary with stage (I, 36%; II, 34%; III, 35%; IV, 40%; P = ns). Disease-specific survival was far superior for MSI tumors than for microsatellite stability (MSS) tumors (5-year survival 92 vs. 59%, P < 0.0001). KRAS mutation was a marker of poor survival (5-year survival 55 vs. 68%, P = 0.0002). Using Cox regression analysis MSI, KRAS mutation, and stage were strong independent predictors of survival in the entire patient population. A high-mortality group with MSS/KRAS-mutant tumors was identified within the stage I and II cohort. Conclusions MSI and KRAS mutation provide fundamental genetic signatures influencing tumor behavior across patient subsets and stages of tumor development.

Hepatoma Hep3B cell lines stably expressing a temperature-sensitive p53 species (p53-Val-135) displayed a reduced response to interleukin-6 (IL-6) when cultured at the wild-type (wt) p53 temperature (Wang, L., Rayanade, R., Garcia, D., Patel, K., Pan, H., and Sehgal, P. B. (1995) J. Biol. Chem. 270, 23159 -23165). We now report that in such cultures IL-6 caused a rapid (20 -30 min) and marked loss of cellular immunostaining for STAT3 and STAT5, but not for STAT1. The loss of STAT3 and STAT5 immunostaining was transient (lasted 120 min) and tyrosine kinase-dependent, and even though the loss was blocked by the proteasome inhibitors MG132 and lactacystin it was not accompanied by changes in cellular levels of STAT3 and STAT5 proteins suggesting that IL-6 triggered a rapid masking but not degradation of these transcription factors. STAT3 and STAT5 masking was accompanied by a reduction in IL-6-induced nuclear DNA-binding activity. The data suggest that p53 may influence Jak-STAT signaling through a novel indirect mechanism involving a wt p53-dependent gene product which upon cytokine addition is activated into a "STATmasking factor" in a proteasome-dependent step.The cellular protein p53 has been implicated in the regulation of mammalian cell processes such as proliferation, apoptosis, and DNA repair either directly as a transcription factor which modulates expression of specific regulatory genes or indirectly as a transcription factor which up-regulates the expression of genes whose products, in turn, then regulate other cellular regulatory proteins (1-8). As an example of the latter, the inhibitory effect of p53 on the cell cycle is attributed to the ability of p53 to increase the transcriptional expression of p21, a cellular protein which inhibits cyclin kinases, thus inhibiting the function of cyclins, a class of regulatory proteins required for cell proliferation (5-8). Despite clear evidence of the influence of cytokines upon p53-induced cellular processes, for example the rescue of p53-induced apoptosis in myeloid cells by the cytokine interleukin-6 (IL-6) 1 (9 -14), there is little information concerning the influence, direct or indirect, of p53 upon cytokine-elicited cellular signaling through the Janus kinasesignal transducers and activators of transcription (Jak-STAT) pathway.We previously utilized a constitutive expression vector for the temperature-sensitive p53 mutant (p53-Val-135) (2, 9) to obtain a series of stably transfected human hepatoma Hep3B cell lines that displayed a reduced response to IL-6 at the wild-type (wt) p53 temperature (32.5°C) (14). The temperature dependence of the secretion of plasma proteins such as ␤-fibrinogen and ␣ 1 -antichymotrypsin in response to IL-6 by these p53-Val-135-expressing Hep3B lines, but not by the pSVneo control cell lines or the p53-free parental Hep3B cells, suggested that wt p53 negatively influenced cytokine signaling (14). In as much as the transcriptional response to IL-6 of the ␤-fibrinogen and ␣ 1 -antichymotrypsin genes is primarily through activatio...

Mdm2 is the main regulator of p53 and is amplified in f7% of all human cancers. MDM2 gene amplification as well as expression has been correlated to an increased tumorigenic potential. We have analyzed the prevalence of MDM2 gene amplifications and SNP309 in 284 colorectal tumors using a relatively new highly sensitive PCR/ligase detection reaction method in relation to TP53 mutational status and genomic instability. We found MDM2 to be amplified in 9% of the 284 colorectal cancers analyzed and a significantly higher proportion of tumors with high MDM2 gene amplification retained a wild-type p53 gene (P = 0.058). MDM2 gene amplification was significantly correlated to advanced tumor stage. Several small-molecule MDM2 antagonists have already been identified that either physically inhibit the p53-MDM2 binding or the E3 ligase function of MDM2. Our results suggest that MDM2 is a promising target for this type of cancer therapy in a substantial subgroup of colorectal cancers. (Mol Cancer Res

The multiplex assay identifies colon cancers with MSI-H by assessing three highly accurate microsatellite markers. This assay identifies a smaller MSI-H cohort with more homogeneous clinical features and is superior as a marker of favorable prognosis. It merits prospective evaluation as a marker of prognosis and as a screening test for HNPCC.

Entamoeba histolytica is known to cause intestinal and extra-intestinal disease while the other Entamoeba species are not considered to be pathogenic. However, all Entamoeba spp. should be reported when identified in clinical samples. Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features overlap. E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii are morphologically identical but can be differentiated using molecular tools. We developed a polymerase chain reaction (PCR) procedure followed by DNA sequencing of specific regions of 18S rRNA gene to differentiate the Entamoeba spp. commonly found in human stools. This approach was used to analyze 45 samples from cases evaluated for the presence of Entamoeba spp. by microscopy and a real-time PCR method capable of differential detection of E. histolytica and E. dispar. Our results demonstrated an agreement of approximately 98% (45/44) between the real-time PCR for E. histolytica and E. dispar and the 18S rRNA analysis described here. Five previously negative samples by microscopy revealed the presence of E. dispar, E. hartmanii, or E. coli DNA. In addition, we were able to detect E. hartmanii in a stool sample that had been previously reported as negative for Entamoeba spp. by microscopy. Further microscopic evaluation of this sample revealed the presence of E. hartmanii cysts, which went undetected during the first microscopic evaluation. This PCR followed by DNA sequencing will be useful to refine the diagnostic detection of Entamoeba spp. in stool and other clinical specimens.