ABSTRACT
The purpose of the study was to develop a test of the multifunctional protein YB-1 in the intraoperative biopsy specimen to predict the course of breast cancer (BC). Its tasks were to use of real-time reverse-transcription polymerase chain reaction (RT-RCR) to substantiate the data previously obtained by semiquantitative RT-PCR and to clarify whether there was a correlation between the amount of YB-1 mRNA in the BC tissue and the status of steroid hormone receptors of these tumors. The determination of the tumor amount of YB-1 mRNA was shown to predict the course of BC: a statistically significant correlation was found between the higher content of YB-1 mRNA and the aggressive course of BC--the emergence of distant metastases. Comparing the content of YB-1 mRNA and the hormonal status of a tumor (the number of estrogen and progesterone receptors) revealed no correlations. The findings indicate that the determination of YB-1 mRNA by both real-time RT-PCR and semiquantitative RT-PCR may be used to predict BC metastases in distant organs.
Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/chemistry , Breast Neoplasms/diagnosis , DNA-Binding Proteins/analysis , Nuclear Proteins/analysis , RNA, Messenger/analysis , Biomarkers, Tumor/genetics , Breast Neoplasms/pathology , DNA-Binding Proteins/genetics , Female , Humans , Middle Aged , Neoplasm Metastasis , Nuclear Proteins/genetics , Prognosis , Y-Box-Binding Protein 1ABSTRACT
The multifunctional mammalian protein YB-1 is involved in multiple DNA- and mRNA-dependent events in the cell and regulates gene expression at different levels. The intracellular localization and relative mRNA content of YB-1 in the breast tumors were studied. The presence of cells with nuclear YB-1 localization in the tumor cell population is a poor predictor that correlates with larger tumors (more than 5 cm). The high YB-1 mRNA content in the breast tumors promotes metastasis of small neoplasms and patients with breast cancer who have high tumor tissue YB-1 mRNA levels may referred to as an early distant metastasis risk group. The findings suggest that combined determination of YB-1 intercellular localization and mRNA levels can ensure a more reliable prognosis of breast cancer.
Subject(s)
Biomarkers, Tumor/metabolism , Breast Neoplasms/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , RNA, Messenger/metabolism , Breast Neoplasms/pathology , Cell Nucleus/metabolism , Cell Nucleus/pathology , Cytoplasm/metabolism , Cytoplasm/pathology , Female , Humans , Immunohistochemistry , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , Y-Box-Binding Protein 1ABSTRACT
The effects of YB-1 gene on the expression level of P-glycoprotein and drug resistance of tumor cells were studied in cultured HCT116 colon cancer cells. Transitory transfection of chimeric YB-1/GFP gene rendered HCT116 cells a selective advantage in a medium with vinblastine, which caused translocation of the chimeric protein into cell nuclei. This was paralleled by an increase in the expression of P-glycoprotein (multiple drug resistance protein).
Subject(s)
Drug Resistance, Multiple/genetics , Drug Resistance, Neoplasm/genetics , Y-Box-Binding Protein 1/physiology , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cell Line, Tumor , Cell Nucleus/metabolism , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Drug Resistance, Multiple/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Microscopy, Fluorescence , Protein Transport/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Vinblastine/pharmacology , Y-Box-Binding Protein 1/genetics , Y-Box-Binding Protein 1/metabolismABSTRACT
Expression of mRNA for the mdr1 gene, cytochrome P450 isoforms 1A1 and 1B1, Ah receptor, and ARNT protein regulating the concentration of cytochrome P450 mRNA was compared in normal and spontaneously transformed mesothelial cells and mesothelioma cells from rats. Expression of cytochrome P450 1A1 and 1B1 mRNA decreased in transformed mesothelial and mesothelioma cells compared to normal mesothelial cells. mRNA for the mdr1 gene was undetected in normal mesothelial cells. Expression of mRNA for the Ah receptor and ARNT protein did not differ in cultured cells.
Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Cytochrome P-450 CYP1A1/genetics , Epithelium/enzymology , Mesothelioma/enzymology , RNA, Messenger/genetics , Animals , Base Sequence , Cells, Cultured , Cytochrome P-450 CYP1B1 , DNA Primers , Epithelium/pathology , Inactivation, Metabolic , Mesothelioma/pathology , RatsABSTRACT
The multifunctional mammalian protein YB-1 is a member of the large DNA- and RNA-binding protein family with an evolutionarily ancient cold-shock domain. YB-1 is involved in multiple DNA- and mRNA-dependent events and regulates gene expression at various levels. It can be found both in the nucleus and the cytoplasm. Bound to DNA in the cell nucleus, YB-1 functions as a transcription factor interacting with inverted CCAAT-box (Y-box) in promoters and enhancers of multiple genes. In particular, YB-1 regulates activity of the multidrug resistance (MDR) genes MDR1 and LRP. In tumors, YB-1 has been suggested to be an early and global marker of MDR. In this study, we compared amounts of YB-1 mRNAs and intracellular localization of YB-1 protein in six pairs of drug sensitive and drug resistant sublines of diverse tumors. We have shown that neither great increase in the level of YB-1 mRNA nor substantial increase in the number of cells with nuclear localization of YB-1 are obligatory traits of drug resistant tumor cell populations. However, the cells with highest amounts of YB-1 mRNA also demonstrated increased quantities of MDR1, MRP1, BCRP, and LRP mRNAs encoding different MDR proteins. Transfection of two different populations of drug-sensitive cells with YB-1 cDNA led to increase in the amount of YB-1 mRNA. The quantities of MRP1 and LRP mRNAs increased in both populations. Introduction of YB-1 small hairpin RNA (shRNA) resulted in decreased amounts of YB-1 mRNA, as well as MRP1, LRP, and MDR1 mRNAs (in three different cell lines). Our data suggest that although YB-1 regulates several MDR genes, it could not be regarded as a global marker of already formed drug resistant tumor cell populations. It is most likely that at the first steps of MDR development YB-1 activity is necessary for propagation of resistant cell populations rather than for maintenance of drug resistance.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Intracellular Fluid/metabolism , Neoplasm Proteins/metabolism , Amino Acid Sequence , Cell Line, Tumor , DNA, Complementary/genetics , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , HCT116 Cells , Humans , Intracellular Fluid/chemistry , K562 Cells , KB Cells , Molecular Sequence Data , Multidrug Resistance-Associated Proteins/biosynthesis , Multidrug Resistance-Associated Proteins/genetics , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Nuclear Proteins , RNA Interference , RNA Polymerase III/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , Vault Ribonucleoprotein Particles/biosynthesis , Vault Ribonucleoprotein Particles/genetics , Y-Box-Binding Protein 1ABSTRACT
We have studied the effect of polycyclic aromatic hydrocarbons (PAH) on gap junction intercellular communications (GJIC) in culture of hepatoma cells Hep G2 and G27. Carcinogenic PAH inhibited GJIC in both cultures in contrast to non-carcinogenic PAH. We showed that both constitutive and inducible expressions of mRNAs of Ah receptor and cytochrome P4501A1 (the main isoform involved in PAH metabolism) were absent in hepatoma G27 cells. We concluded that the initial, non-metabolized molecules of carcinogenic PAH are responsible for changes in GJIC through interaction with an unknown factor in the cellular membrane.
Subject(s)
Cell Communication/drug effects , Gap Junctions/drug effects , Polycyclic Aromatic Hydrocarbons/pharmacology , Base Sequence , Benzo(a)pyrene/metabolism , Benzo(a)pyrene/pharmacology , Cell Line, Tumor , Cells, Cultured , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1A1/metabolism , Humans , Molecular Sequence Data , Polycyclic Aromatic Hydrocarbons/metabolism , Quantitative Structure-Activity Relationship , RNA, Messenger/metabolism , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolismABSTRACT
We studied comparative expression and activity of cytochrome P450 family 1 (CYP1) isoforms in rat embryo cells, both primary and immortalized by Rausher leukemia virus (RLV). In RLV-infected embryonal cells compared with the initial ones the expression levels of CYP1A1 and 1B1 mRNAs and benzo[a]pyrene (BP) hydroxylase activity were higher, regardless of their treatment with the CYP1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin. The sensitivity to BP and 7,12-dimethylbenzo[a]anthracene was higher in the cells immortalized with RLV. The expression level of mRNAs of induction-mediating proteins aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator was the same in both cell cultures tested. Higher sensitivity of cells immortalized with RLV compared with the initial embryo cells to transforming effect of BP, which was described previously, is possibly associated with elevated expression of CYP1 isoforms.
