Expression of PTEN and Akt phosphorylation in lipopolysaccharide-treated NIH3T3 cells.

PTEN is a tumor suppressor gene encoding a phosphatase, and it negatively regulates cell survival mediated by the phosphoinositol 3-kinase (PI3-Kinase)-Akt pathway. To elucidate PTEN expression and its effect on the PI3-kinase-Akt pathway in fibroblasts and macrophages, we investigated the expression of PTEN and the phosphorylation status of Akt in NIH3T3 and RAW264.7 cells treated with LPS. Phosphorylation of Akt was induced by LPS treatment in a dose-dependent manner in RAW264.7 cells, but not in NIH3T3 cells. LPS induced the expression of PTEN in a dose and time-dependent manner in NIH3T3 cells (0-1 microg/ml, 0-6h). However, LPS did not stimulate PTEN expression in RAW264.7 cells. These data indicate the existence of diverse mechanisms for PTEN expression and Akt activation in fibroblasts and macrophages. RNA interference using double-stranded RNA specific for the PTEN gene reduced both mRNA and protein levels of PTEN in NIH3T3 cells treated or not with LPS. The phosphorylation status of Akt in NIH3T3 cells stimulated with LPS did not change when the PTEN expression had been inhibited by RNA interference. The present results suggest that the up-regulation of PTEN expression by LPS is not involved in the activation of Akt in NIH3T3 cells. PTEN expression might be involved in the diverse inflammatory responses to LPS in fibroblasts and macrophages.

Transcription factor NF-Y regulates mdr1 expression through binding to inverted CCAAT sequence in drug-resistant human squamous carcinoma cells.

In this study, the expression and transcriptional regulation of the multidrug resistance-1 (MDR1) gene in multidrug-resistant SCCTF cells and -sensitive SCCKN cells derived from human squamous carcinoma were investigated. RT-PCR revealed that mdr1 mRNA was highly expressed in SCCTF cells while it was under the limit of detection in SCCKN cells. With an electrophoretic mobility shift assay using the mdr1 promoter region, a DNA-protein complex was detected strongly in SCCTF cells, but weakly in SCCKN cells. Incubation of the DNA-protein complex with an anti-NF-Y antibody caused a supershift in the migration to a position near the origin of the gel. Chromatin immunoprecipitation assay with an anti-NF-Y antibody showed that NF-Y binds to mdr1 promoter in SCCTF cells. The mdr1 promoter region including its NF-Y binding sequence was cloned into the luciferase reporter plasmid pGL3-basic vector, and this vector was used to transfect SCCTF and SCCKN cells. The luciferase assay showed that the inverted CCAAT sequence in the mdr1 promoter region is involved in the positive regulation of mdr1 promoter activity. NF-YA protein was expressed at higher levels in SCCTF cells than that in SCCKN cells. Hoechst dye staining also showed that MDR1 protein acts more effectively as an efflux pump in SCCTF cells than that in SCCKN cells.

Transblot and cytochemical identification of avidin-interacting proteins in mitochondria of cultured cells.

Cell lysates prepared from 3T3-L1 cells were analyzed by western blotting using the avidin-biotin complex system and anti-Bax antibody. The antibody interacted with bands of proteins with estimated molecular weights of 120, 74, 72, and 25 kDa. However, only the 25-kDa band was detected with the anti-Bax antibody when the direct immunoblotting method was used. Peroxidase-conjugated avidin interacted with the 120-, 74-, and 72-kDa bands. This interaction was not limited to 3T3-L1 cells, because peroxidase-avidin also interacted with these three proteins in MC3T3-E1, YROS, Saos-2, MG63, SCCKN, and SCCTF cells although the staining intensity was different in each cell type. Avidin-peroxidase also interacted with these three proteins in the mitochondria-containing fractions prepared from 3T3-L1 cells. FITC-streptavidin was also localized in mitochondria in the cultured cells. The localization of avidin/streptavidin-interacting proteins in mitochondria was confirmed by using double staining with FITC-streptavidin and Mito-tracker.