Allelic deletions of tumor suppressor genes have been observed frequently in a variety of human tumors. These losses are believed to contribute to the development of human cancer. Three of the most ...frequently deleted chromosomal loci contain the tumor suppressor genes p53, retinoblastoma (Rb), and mcc/apc. In order to detect loss of heterozygosity (LOH) within these genes in dysplastic and cancerous ulcerative colitis, we used an application of the polymerase chain reaction. LOH affecting p53 was observed in 8 of 17 (47%) of heterozygous patients, while LOH of Rb and the mcc/apc locus was observed in 9 of 27 (33%) and 13 of 39 (33%) of heterozygotes, respectively. Among 35 patients heterozygous at 2 or more loci, LOH of p53, Rb, and/or mcc/apc was observed in 18 (51%). LOH was more common in left-sided neoplasms. These data suggest that allelic deletion of p53, Rb, mcc, and/or apc is involved in the pathogenesis and/or progression of at least a subset of colonic dysplasias and carcinomas occurring in the setting of ulcerative colitis.
The transcription factor, signal transducer and activator of transcription (Stat) 6, regulates T(H)2-lymphocyte activity by controlling the expression and responsiveness to interleukin (IL)-4, which ...plays a key role in numerous allergic maladies. Therefore, we sought to use a phosphorothiolate cis-element decoy to target disruption of Stat6 transcriptional activity. Here we showed that the Stat6 decoy potently ablated the messenger RNA expression and production of IL-4, but not of several other cytokines. The Stat6 decoy functionally disrupted IL-4-inducible cell proliferation of murine T(H)2 cells and primary human CD4(+) T lymphocytes. Specificity of the decoy was demonstrated by its ability to directly block Stat6 binding to a cis-element probe and transactivation, but not affect Stat6 tyrosine phosphorylation or expression of the IL-4 receptor chains. Moreover, the decoy failed to inhibit non-Stat6-dependent signaling pathways since IL-2 was competent to induce cell proliferation and activation of Stats 1, 3, and 5a/b. With the use of laser scanning confocal microscopy, fluorescently tagged Stat6 decoy was detectable in the cytoplasm and nucleus; however, greater levels of oligonucleotide were present in the latter following IL-4 treatment. Taken together, these data suggest that IL-4-driven T(H)2 cell activity can be preferentially restricted via targeted disruption of Stat6 by a novel and specific decoy strategy that may possess gene therapeutic potential. (Blood. 2000;95:1249-1257)
Differentiation of metanephric mesenchyme is triggered by an inductive signal(s) from the epithelial ureteric bud. As a result of this induction, most of the metanephric mesenchyme converts into ...epithelium of a nephron. We have developed and characterized an explant culture system, in which metanephric mesenchyme can grow and completely differentiate in vitro in the absence of an inductive tissue. When separated 13 dpc rat metanephric mesenchymes were cultured in serum-free conditioned medium from a rat ureteric bud cell line (RUB1) in the presence of bFGF and TGFalpha, they were induced to differentiate into nephron epithelia and glomeruli-like structures. The nephric type of differentiation was confirmed by both morphological and molecular criteria and paralleled the developmental changes of nephron differentiation in vivo. Expression patterns of brush-border antigen as well as molecular markers of kidney differentiation Wt1, Lim1, Hgf and c-met, c-ret, Shh, Wnt4, Wnt7b, and Wnt11 were analyzed in explants by whole mount and tissue section in situ hybridization following 1â9 days in culture. The expression of secreted patterning molecules Bmp7 and Wnt7b, but not Shh or Wnt11, were demonstrated by RT-PCR and northern blot hybridization with RNA from the RUB1 cells. Our culture system lends itself to examining the relevance of these and other signaling molecules required for nephron differentiation.
Preinvasive lesions of squamous cell carcinoma are well defined morphologically and provide a model for multistage carcinogenesis. Since alterations in the p53 tumor suppressor gene occur frequently ...in invasive esophageal squamous cell carcinoma, we examined a set of preinvasive lesions to investigate the timing of p53 mutation. Surgically resected tissues from nine patients with esophageal squamous cell carcinoma contained precursor lesions which had not yet invaded normal tissues. Immunohistochemistry showed high levels of p53 protein in both preinvasive lesions and invasive carcinomas in six cases; sequence analysis of all invasive tumors identified p53 missense mutations in two cases. Preinvasive lesions from both tumors with mutations plus one wild-type tumor were microdissected and sequenced. In one patient there were different mutations in the invasive carcinoma (codon 282, CGGarg > TGGtrp) and a preinvasive lesion (codon 272, GTGval > T/GTGleu/val). In a second case, an invasive carcinoma had a mutation in codon 175 (CGCarg > CAChis), and adjacent preinvasive lesions contained a wild-type sequence. A carcinoma and preinvasive lesion from the third case contained high levels of protein and a wild-type DNA sequence. Therefore, p53 mutation may precede invasion in esophageal carcinogenesis, and multifocal esophageal neoplasms may arise from independent clones of transformed cells. The timing of p53 protein accumulation is favorable for an intermediate biomarker in multistage esophageal carcinogenesis.
In the CNS, nitric oxide (NO) functions as both neuromodulator and neurotoxic agent. In vivo neuronal expression of NO synthase (NOS) has been attributed to constitutive NOS--both the neuronal and ...the endothelial types. The other class of NOS--the inducible NOS (iNOS)--is known to mediate toxic effects of NO in various tissues. In this study, we show for the first time that direct intracerebellar injection of endotoxin and cytokine (lipopolysaccharide and interferon-gamma) induced in vivo neuronal expression of the iNOS gene, as demonstrated by fluorescent in situ hybridization and immunohistochemical staining analyzed by confocal laser-scanning microscopy. This raises the possibility that neuronal iNOS might contribute significantly to the vulnerability of the brain to various insults.
Sentinel lymph node (SLN) metastasis is the first step in the spreading of cancer in many malignancies. Tumor-reactive lymphadenopathy in SLNs has been observed for decades, but alterations of the ...lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain unexplored. Using animal models, we show here that, before the establishment of metastasis in the SLN, there are reorganizations of the lymphatic channels and the vasculature. The node becomes a functional blood vessel-enriched and lymph vessel/sinus-enriched organ before metastasis. The enlargement of the lymph sinuses is correlated with the primary tumor weight. The newly emerged functional blood vessels develop from high endothelial venules (HEV), in which the proliferation rate of the endothelial cells is also significantly increased. Similar alterations of the HEVs are also characterized in the axillary lymph nodes from human breast cancer patients without the evidence of metastasis. These findings support the hypothesis that modification of the microenvironment for a secondary tumor (i.e., vasculature reorganization in the SLN) can be initiated by a primary tumor before and independent of the physical presence of metastatic cancer cells.
Since HIV-1 infection results in severe immunosuppression, and the envelope protein gp120 has been reported to interact with some of the chemokine receptors on human T lymphocytes, we postulated that ...gp120 may also affect monocyte activation by a variety of chemokines. This study shows that human peripheral blood monocytes when preincubated with gp120 either purified from laboratory-adapted strains or as recombinant proteins exhibited markedly reduced binding, calcium mobilization, and chemotactic response to chemokines. The gp-120-pretreated monocytes also showed a decreased response to FMLP. This broad inhibition of monocyte activation by chemoattractants required interaction of gp120 with CD4, since the effect of gp120 was only observed in CD4+ monocytes and in HEK 293 cells only if cotransfected with both chemokine receptors and an intact CD4, but not a CD4 lacking its cytoplasmic domain. Anti-CD4 mAbs mimicked the effect of gp120, and both anti-CD4 Ab and gp120 caused internalization of CXCR4 in HEK 293 cells provided they also expressed CD4. Staurosporine blocked the inhibitory effect of gp120 on monocytes, suggesting that cellular signaling was required for gp120 to inhibit the response of CD4+ cells to chemoattractants. Our study demonstrates a broad suppressive effect of gp120 on monocyte activation by chemoattractants through the down-regulation of cell surface receptors. Thus, gp120 may be used by HIV-1 to disarm the monocyte response to inflammatory stimulation.
P311 is a mouse cDNA originally identified for its high expression in late-stage embryonic brain and adult cerebellum, hippocampus, and olfactory bulb. The protein product of P311, however, had not ...been identified previously, and its function remains unknown. We report here that P311 expression is regulated at multiple levels by pathways that control cellular transformation. P311 mRNA expression was decreased sharply in both neural and smooth muscle cells when the cells were transformed by coexpression of the oncogenic tyrosine kinase receptor Met and its ligand hepatocyte growth factor/scatter factor. The P311 mRNA was found to encode an 8-kDa polypeptide that was subject to rapid degradation by the lactacystin-sensitive ubiquitin/proteasome system and an unidentified metalloprotease, resulting in a protein half-life of about 5 min. These data suggest that P311 expression is dramatically decreased by several pathways that regulate cellular growth.
Hepatocyte growth factor (HGF)-MET signalling in cancer biology has been well characterized in multiple organ systems. Numerous investigations have described an up-regulation of c-met mRNA in human ...colorectal adenomas and carcinomas. However, a quantitative immunohistochemical analysis of MET and HGF protein levels in tumor tissues has not been reported previously. Formalin-fixed and paraffin-embedded tissues from 41 colorectal adenomas and 49 colorectal carcinomas were characterized by immunofluorescent staining using HGF- and MET-specific antibodies. The immunoreactivity was evaluated by confocal laser scanning microscopy, computer-based image analysis and appropriate statistical tests. Normal colorectal mucosa, adenomas and carcinomas exhibited comparable levels of MET and HGF proteins. MET expression in carcinomas, although statistically not significant, demonstrated a tendency to correlate with the grade of differentiation. Correlations of MET and HGF with other clinico-pathological variables including the extent of the mucinous component and the pTNM stage were not observed. The ratio of HGF in carcinoma vs. non-neoplastic tissue was significantly different between high and low carcinoma stage. Alterations of absolute levels of MET and HGF protein during the colorectal adenoma-carcinoma sequence were not significant. The presumed role of MET-HGF interactions in large bowel carcinogenesis may therefore be a result of or depend upon other regulatory factors involved in MET-mediated signalling pathways.
The receptor tyrosine kinase MET is frequently amplified in human tumors, resulting in high cell surface densities and constitutive activation even in the absence of growth factor stimulation by its ...endogenous ligand, hepatocyte growth factor (HGF). We sought to identify mechanisms of signaling crosstalk that promote MET activation by searching for kinases that are coordinately dysregulated with wild-type MET in human tumors. Our bioinformatic analysis identified leucine-rich repeat kinase-2 (LRRK2), which is amplified and overexpressed in papillary renal and thyroid carcinomas. Down-regulation of LRRK2 in cultured tumor cells compromises MET activation and selectively reduces downstream MET signaling to mTOR and STAT3. Loss of these critical mitogenic pathways induces cell cycle arrest and cell death due to loss of ATP production, indicating that MET and LRRK2 cooperate to promote efficient tumor cell growth and survival in these cancers.
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