OBJECTIVETo study the nuclear localization of insulin-like growth factor binding protein-6(IGFBP-6) in PC-3M cells.

METHODSThe two fragments of the nuclear localization sequence (NLS)-deleted IGFBP-6 and the NLS-mutated IGFBP-6 were obtained by overlapping PCR, and then the fragment was inserted into a pEGFP-C1 vector. PC-3M cells were transfected with the expression constructs containing wild-type IGFBP-6 or the two mutants (pEGFP-C1-BP6 Delta NLS and pEGFP-C1-BP6-Mut), and the different distribution of the three EGFP-fusion proteins was observed by confocal laser microscope. The statistical analysis of the ratio of the nuclear fluorescence to the cytoplasmic fluorescence (Fn/c) was performed. Results Confocal microscopic images of transfected cells showed that the green fluorescence of EGFP-IGFBP-6 was concentrated mostly in the nuclei, whereas the control cells expressing EGFP showed green fluorescence distributed uniformly. The results of Fn/c from EGFP and EGFP-IGFBP-6 were significant different (P<0.05). The NLS-deleted IGFBP-6 completely eliminated nuclear accumulation of the green fluorescent signal; in contrast, nuclear accumulation was only slightly reduced for the NLS-mutated IGFBP-6; compared with wild-type IGFBP-6, both mutants were significantly different (P<0.05). Conclusions IGFBP-6 can be translocated to the nucleus in PC-3M cell that is mediated by a putative NLS sequence. Our study provides new evidence for further studies on the insulin-like growth factor-independent activity of IGFBP-6.

Cell Line, Tumor ; Cell Nucleus ; metabolism ; Genetic Vectors ; Humans ; Insulin-Like Growth Factor Binding Protein 6 ; genetics ; metabolism ; Plasmids ; genetics ; Protein Transport ; genetics ; Transfection

OBJECTIVETo investigate the effect of the environmental carcinogenic factor-TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin) on cell apoptosis and gene regulation of insulin-like growth factor binding protein 6 (IGFBP-6) in osteogenic sarcoma (SaOS-2) cells.

METHODSThe SaOS-2 cells were cultured with TCDD (1 x 10(-9), 1 x 10(-8), 1 x 10(-7) mol/L) for 24 hours. The MTT reduction assay and flow cytometry were used to measure the cell proliferation and the cell apoptosis in TCDD-treated SaOS-2 cells. The Nitrophenol phosphate salt method was used to measure activity of alkaline phosphatase (ALP) in SaOS-2 cells. The IGFBP-6 mRNA and protein in SaOS-2 cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and western blotting analysis.

RESULTSSaOS-2 cell proliferation was up-regulated with TCDD (1 x 10(-9), 1 x 10(-8), and 1 x 10(-7) mol/L) about 20%, 47% and 93% (18.4 +/- 4.5, 22.5 +/- 3.6 and 29.4 +/- 4.2), respectively. The synthesis of ALP was up-regulated about 28%, 95%, and 142% (1.12 +/- 0.28, 1.58 +/- 0.14 and 1.96 +/- 0.17), respectively (P < 0.05). The cell apoptosis was down-regulated in dose-dependent biological manner about 5%, 26% and 52%, respectively (P < 0.05). The expression of IGFBP-6 mRNA and protein was decreased in 1 x 10(-7) mol/L TCDD-treated SaOS-2 cells about 76% and 72% (P < 0.05).

CONCLUSIONTCDD at low concentration may have the negative effect on cell apoptosis and down-regulation on gene expression of IGFBP-6 in SaOS-2 cells.

Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Insulin-Like Growth Factor Binding Protein 6 ; genetics ; metabolism ; Osteosarcoma ; metabolism ; pathology ; Polychlorinated Dibenzodioxins ; toxicity ; RNA, Messenger ; genetics

OBJECTIVETo investigate the antiestrogenic effect of environment teratogen on the gene expression of insulin-like growth factors (IGFs) family in osteoblast cells during rat skeleton development.

METHODSThe fetal rat models with congenital skeleton malformation were constructed by treating 20 female Wistar rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on pregnant day 10. The MC-3T3-E1 cells were cultured with estrogen, TCDD, or a combination of the two chemicals for 24 hours. The IGF-II and IGFBP-6 mRNA levels in rat calvaria bone tissue and MC-3T3-E1 cells were detected by reverse transcription-polymerase chain reaction. Flow cytometer was used to determine the cell proliferation.

RESULTSTCDD at the concentration of 5-15 microg/kg induced developmental skeleton defect of fetal rat, and the effect was dose-dependent. The expression of IGF-II mRNA gene was enhanced by estrogen in rat calvaria bone tissue and MC-3T3-E1 cells, whereas IGFBP-6 mRNA was decreased. Estrogen increased the cell proliferation in MC-3T3-E1 cells. TCDD, however, inhibited the effect of estrogen on regulation of IGF-II gene and IGFBP-6 gene as well as MC-3T3-E1 cell proliferation.

CONCLUSIONThese findings provide the evidence that TCDD can induce congenital fetal skeleton malformation under the condition of high estrogen level in pregnant Wistar rats. TCDD has antiestrogenic effect and hence exerts negative influence on the osteoblast cells through target IGF-II and IGFBP-6 of IGFs family.

Abnormalities, Drug-Induced ; etiology ; Animals ; Bone and Bones ; abnormalities ; Dose-Response Relationship, Drug ; Estrogen Receptor Modulators ; toxicity ; Female ; Insulin-Like Growth Factor Binding Protein 6 ; genetics ; Insulin-Like Growth Factor II ; genetics ; Osteoblasts ; drug effects ; metabolism ; Polychlorinated Dibenzodioxins ; toxicity ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar

OBJECTIVETo explore the relationship between the methylation status of CpG islands in the promoter region of 10 genes in breast cancer cells and their sensitivity to 5-fluouracil (5-Fu), and to identify the genes responsible for the 5-Fu resistance in breast cancer.

METHODSThree cell lines (differently resistant to chemotherapy) were used in this study: Bcap-37 (IC(50): 289.77 microg/ml), T47D (IC(50): 134.16 microg/ml) and ZR-75-30 (IC(50): 4.20 microg/ml). The methylation profile of 10 genes (BAG1, C11ORF31, CBR1, CBR4, GJA1, FOXL2, IGFBP6, P4HA1, SRI and TYMS) in the 3 breast cancer cell lines was determined by methylation specific PCR. The steady-state mRNAs of ABCC8, CHFR and IGFBP6 genes were quantified by real-time RT PCR analysis.

RESULTSAmong the 10 genes, only genes IGFBP6 and FOXL2 displayed differential DNA methylation pattern between the 5-Fu-resistant and 5-Fu-sensitive cell lines. The mRNA expression level of genes PRSS21, LOX, IGFBP6, ABCC8 and CHFR was quantified by real-time RT-PCR analysis. Except for CHFR, the expression level of the other 4 genes was correlated with the methylation status of CpG islands, namely, a lower expression level with methylation status and a higher level with demethylation status.

CONCLUSIONThe results of the present study have demonstrated that there are 8 genes with differential methylation status in chemosensitive and chemoresistant breast cancer cell lines, i.e. two genes more than the six genes we reported previously. Our findings provide both mechanistic insights for the drug resistance of breast cancer and the basis for further studies on potential application of the DNA methylation in this set of genes for prediction of chemosensitivity of breast cancer.

Antimetabolites, Antineoplastic ; pharmacology ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; CpG Islands ; genetics ; DNA Methylation ; Drug Resistance, Neoplasm ; Fluorouracil ; pharmacology ; Forkhead Box Protein L2 ; Forkhead Transcription Factors ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Insulin-Like Growth Factor Binding Protein 6 ; genetics ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism

Osteoblasts can synthesize the insulin-like growth factors (IGFs) and the IGF-binding proteins (IGFBPs), which may either enhance or attenuate IGF-stimulated bone cell proliferation. Since estrogen induced osteoblastic differentiation and proliferation through an estrogen-responsive gene in target cells, we investigated the effects of estrogen on IGFBP-6 expression in the human osteoblastic-like cell line SaOS-2. Expressions of IGFBP-6 protein and mRNA increased 2.8 and 2-fold, respectively, in the presence of 17-beta-estradiol (E2) (0.01 to 1 micrometer) and estrogen receptor (ER) in SaOS-2 cells. On the other hand, E2 induced a 2-fold increase in SaOS-2 cell proliferation. To identify genomic sequences associated with estrogen responsiveness, the 5'-promoter region (-44 to +118) of the IGFBP-6 gene was cloned into a chloramphenicol acetyltransferase (CAT) reporter vector. E2 induced a 3-fold increase in CAT activity in SaOS-2 cells transiently transfected with this construct. Identification of the estrogen-responsive element (ERE) [5'-CCTTCA CCTG-3'] (-9 to +1) in this IGFBP-6 gene promoter region was confirmed using electromobility shift assays and deletion analysis. This functional ERE was important for E2-induced trans-activation of the IGFBP-6 gene. These results demonstrate that E2 exhibits a positive effect on IGFBP-6 gene transcription through estrogen-liganded ER binding to the functional ERE in the IGFBP-6 gene promoter in SaOS-2 cells.
Blotting, Western ; Cell Proliferation ; Chloramphenicol O-Acetyltransferase/metabolism ; Electrophoretic Mobility Shift Assay ; Estradiol/*pharmacology ; Estrogen Receptor alpha/genetics/metabolism ; Estrogens/pharmacology ; Humans ; Insulin-Like Growth Factor Binding Protein 6/*genetics/metabolism ; Osteoblasts/*drug effects/metabolism ; Promoter Regions, Genetic/*genetics ; RNA, Messenger/genetics/metabolism ; Response Elements ; Reverse Transcriptase Polymerase Chain Reaction ; *Transcriptional Activation ; Tumor Cells, Cultured