No abstract

The identification and cloning of a segment of a human multidrug resistance gene (mdrl) was reported recently. To examine the molecular basis of one type of multidrug resistance, we have prepared RNA from human tumors and normal tissues and measured their content of mdrl RNA. We find that the mdrl gene is expressed at a very high level in the adrenal gland; at a high level in the kidney; at intermediate levels in the lung, liver, lower jejunum, colon, and rectum; and at low levels in many other tissues. The mdrl gene is also expressed in several human tumors, including many but not all tumors derived from the adrenal gland and the colon. In addition, increased expression was detected in a few tumors at the time of relapse following initial chemotherapy. Although controlled clinical studies will be required, our results suggest that measurement of mdrl RNA may prove to be a valuable tool in the design of chemotherapy protocols.

Intrinsic and acquired multidrug resistance (MDR) is an important problem in cancer therapy. MDR in human KB carcinoma cells selected for resistance to colchicine, vinblastine, or doxorubicin (former generic name adriamycin) is associated with overexpression of the "MDR] " gene, which encodes P-glycoprotein. We previously have isolated an overlapping set of cDNA clones for the (20).DNA and RNA Blot Hybridization. DNA was extracted from transfectants (13) and used for Southern blots (23). Total cellular RNA was isolated from the transfectants (20) and used for RNA blots (23). The 3.4-kilobase (kb) cDNA probe was labeled with 32P to a specific activity of 1-2 x 109 dpm/,ug by nick translation. Hybridization was done at 42°C in 50% formamide containing 5 x NaCl/Cit (lx = 0.15 M NaCl/ 0.015 M sodium citrate, pH 7), 0.2% bovine serum albumin, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.1% NaDodSO4, 50 mM Tris*HC, and 100 ,ug of salmon sperm DNA per ml, followed by washing with 0.2x NaCl/Cit containing 0.2% NaDodSO4 at 60°C for DNA hybridization and with O.1x NaCl/Cit containing 0.1% NaDodSO4 at 70°C for RNA hybridization at high stringency.Ribonuclease Protection Assay. Human MDR] mRNA transcribed from the retroviral promoter was detected with a ribonuclease protection assay using a uniformly labeled SP6 antisense RNA probe (785 nucleotides) derived from a 1-kb genomic fragment containing the MDR] transcription initiation site and the first intron of the MDRJ gene cloned in pGEM3 (Promega Biotec, Madison, WI). Total cellular RNA (10 ,ug) was hybridized with 6 x 105 cpm of probe, and ribonuclease digestion was performed as described (24).

Drug resistance in human cancer is associated with overexpression of the multidrug resistance (MDR1) gene, which confers cross-resistance to hydrophobic natural product cytotoxic drugs. Expression of the MDR1 gene can occur de novo in human cancers in the absence of drug treatment. The promoter of the human MDR1 gene was shown to be a target for the c-Ha-Ras-1 oncogene and the p53 tumor suppressor gene products, both of which are associated with tumor progression. The stimulatory effect of c-Ha-Ras-1 was not specific for the MDR1 promoter alone, whereas a mutant p53 specifically stimulated the MDR1 promoter and wild-type p53 exerted specific repression. These results imply that the MDR1 gene could be activated during tumor progression associated with mutations in Ras and p53.

In the representative gut bacterium Lactobacillus plantarum, we identified genes encoding the enzymes involved in a saturation metabolism of polyunsaturated fatty acids and revealed in detail the metabolic pathway that generates hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and partially saturated transfatty acids as intermediates. Furthermore, we observed these intermediates, especially hydroxy fatty acids, in host organs. Levels of hydroxy fatty acids were much higher in specific pathogen-free mice than in germ-free mice, indicating that these fatty acids are generated through polyunsaturated fatty acids metabolism of gastrointestinal microorganisms. These findings suggested that lipid metabolism by gastrointestinal microbes affects the health of the host by modifying fatty acid composition.biohydrogenation | hydratase | fatty acid isomerase | conjugated linoleic acid | lipid nutrition

Controlled distribution of lipids across various cell membranes is crucial for cell homeostasis and regulation. We developed an imaging method that allows simultaneous in situ quantification of cholesterol in two leaflets of the plasma membrane (PM) using tunable orthogonal cholesterol sensors. Our imaging revealed marked transbilayer asymmetry of PM cholesterol (TAPMC) in various mammalian cells, with the concentration in the inner leaflet (IPM) being ~12-fold lower than that in the outer leaflet (OPM). The asymmetry was maintained by active transport of cholesterol from IPM to OPM and its chemical retention at OPM. Furthermore, the increase in the IPM cholesterol level was triggered in a stimulus-specific manner, allowing cholesterol to serve as a signaling lipid. We found excellent correlation between the IPM cholesterol level and cellular Wnt signaling activity, suggesting that TAPMC and stimulus-induced PM cholesterol redistribution are crucial for tight regulation of cellular processes under physiological conditions.

A full-length cDNA for the human multidrug resistance gene 1 (MDR1) has been inserted into a retroviral vector containing a murine Harvey sarcoma virus from which the viral oncogene was deleted. Ecotropic and amphotropic virus was produced after transfection of this vector into psi-2 and PA-12 packaging cell lines. This virus conferred the full phenotype of multidrug resistance on mouse and human cell lines. Viral titers of up to 2 X 10(5) drug-resistant colonies per ml were observed. Infected cells became resistant to colchicine, vinblastine, doxorubicin, VP16 (etoposide), and puromycin, but not cisplatin, indicating that the presence of the human MDR1 gene is sufficient to cause multidrug resistance. When the dog kidney cell line MDCK was infected with the MDR1 virus, P-glycoprotein was expressed in a polarized manner on the upper surface of the cells, showing that the cloned cDNA also encodes information for polarized expression of P-glycoprotein. The MDR1 virus should be useful for introducing this drug resistance gene into a variety of cell types for biological experiments in vitro and in vivo.

Adaptor proteins, composed of two or more protein-protein interacting modules without enzymatic activity, regulate various cellular functions. Vinexin, CAP/ponsin, and ArgBP2 constitute a novel adaptor protein family. They have a novel conserved region homologous to the active peptide sorbin, as well as three SH3 (src homology 3) domains. A number of proteins binding to this adaptor family have been identified. There is accumulating evidence that this protein family regulates cell adhesion, cytoskeletal organization, and growth factor signaling. This review will summarize the structure and the function of proteins in this family.