Cooperative functions of Chk1 and Chk2 reduce tumour susceptibility in vivo

Although the linkage of Chk1 and Chk2 to important cancer signalling suggests that these kinases have functions as tumour suppressors, neither Chk1^+/− nor Chk2^−/− mice show a predisposition to cancer under unperturbed conditions. We show here that Chk1^+/−Chk2^−/− and Chk1^+/−Chk2^+/− mice have a progressive cancer-prone phenotype. Deletion of a single Chk1 allele compromises G2/M checkpoint function that is not further affected by Chk2 depletion, whereas Chk1 and Chk2 cooperatively affect G1/S and intra-S phase checkpoints. Either or both of the kinases are required for DNA repair depending on the type of DNA damage. Mouse embryonic fibroblasts from the double-mutant mice showed a higher level of p53 with spontaneous DNA damage under unperturbed conditions, but failed to phosphorylate p53 at S23 and further induce p53 expression upon additional DNA damage. Neither Chk1 nor Chk2 is apparently essential for p53- or Rb-dependent oncogene-induced senescence. Our results suggest that the double Chk mutation leads to a high level of spontaneous DNA damage, but fails to eliminate cells with damaged DNA, which may ultimately increase cancer susceptibility independently of senescence.     

IL-8 promotes cell proliferation and migration through metalloproteinase-cleavage proHB-EGF in human colon carcinoma cells

Interleukin-8 (IL-8) has been reported to promote tumor cell growth in colon cancer cells after binding to its receptors, which are members of the G-protein coupled receptor (GPCR) family. Recent studies demonstrated that stimulation of GPCR can induce shedding of epidermal growth factor (EGF) ligands via activation of a disintegrin and metalloprotease (ADAM), with subsequent transactivation of the EGF receptor (EGFR). In this study, we investigated mechanisms of cell proliferation and migration stimulated by IL-8 in a human colon carcinoma cell line (Caco2). IL-8 increased DNA synthesis of Caco2 in a dose dependent manner and this was inhibited by ADAM, EGFR kinase, and MEK inhibitors. IL-8 transiently induced EGFR tyrosine phosphorylation after 5-90 min and this was completely inhibited by ADAM inhibitor. Neutralizing antibody against HB-EGF as a key ligand for EGFR also blocked transactivation of EGFR and cell proliferation by IL-8. Since IL-8-induced cell migration was further suppressed by the ADAM inhibitor and the HB-EGF neutralizing antibody, our data indicate that IL-8 induces cell proliferation and migration by an ADAM-dependent pathway, and that HB-EGF plays an important role as the major ligand for this pathway.     

Faster and easier chromatin immunoprecipitation assay with high sensitivity

Chromatin immunoprecipitation (ChIP) assays are widely used to investigate where chromatin-binding proteins bind to the genome. The standard assay is very time consuming. We have developed a rapid ChIP assay in which the immunoprecipitates serve directly as PCR templates. This assay eliminates the step to reverse the crosslinking, shortening the assay by 1 day. It also requires a less immunoprecipitating antibody, permits many samples to be tested simultaneously, and is more sensitive than the standard ChIP assay.     

Differentiation of the double membrane system during ascospore-maturation of Arthroderma vanbreuseghemii as revealed by periodic acid-alkaline bismuth staining

Asci of Arthroderma vanbreuseghemii were investigated by electron microscopy using periodic acid-alkaline bismuth (PABi) staining. At the early stage of ascosporogenesis, PABi-reactive substance was present homogeneously on both the inner and outer tracks of the double membrane system. Immediately after ascospore-delimitation, this substance gradually decreased and ultimately disappeared from the outer track. In contrast, the inner membrane was persistently loaded with the PABi-reactive material. These observations suggest that at this stage the outer membrane of the double membrane system begins to differentiate into the ascospore cell wall, utilizing its carbohydrate constituent.     

Angioarchitecture of primary oral malignant melanomas.

Angiogenesis is an essential process in the progression of malignant tumors. However, little is known of the angioarchitecture in primary oral malignant melanoma. We sought to determine this by the use of periodic acid-Schiff (PAS) stain, endothelial markers (CD34, CD105) and laminin, and by transmission electron microscopy in two cases. The results demonstrated that endothelium-lined vessels dominated the tumor microvasculature and these stained positively for PAS, laminin, and endothelial markers. Mosaic and tumor-lined vessels were infrequently encountered. Most PAS-positive patterned networks and loops ultrastructurally represented intratumor microhemorrhages that probably arose secondary to tumor vessel leakiness. Vascular channels of the vasculogenic mimicry type were rare. They stained for laminin but not for endothelial markers.