Immunosuppressant inhibition of P-glycoprotein function is independent of drug-induced suppression of peptide-prolyl isomerase and calcineurin activity.

PURPOSE: P-glycoprotein is a 170-kDa plasma membrane multidrug transporter that actively exports cytotoxic substances from cells. Overexpression of P-glycoprotein by tumor cells is associated with a multidrug-resistant phenotype. Immunosuppressive agents such as cyclosporins and macrolides, have been shown to attenuate P-glycoprotein activity. However, the mechanism by which some immunosuppressants inhibit P-glycoprotein function has not been determined. Since cyclosporin and macrolide immunosuppressants inhibit calcineurin (CaN) phosphatase and FKBP12 peptideprolyl isomerase (FKBP12 PPI) activity, studies were conducted to determine if these effects are directly related to the inhibitory effects these immunosuppressants have on P-glycoprotein function. METHODS: Western blot analysis was performed to assess CaN and FKBP12 protein levels in P-glycoprotein-negative (MCF-7) and -positive (MCF-7/Adr) breast cancer cell lines. P-glycoprotein function was determined by intracellular doxorubicin accumulation and/or cytotoxicity assays before and after CaN and FKBP12 were independently inhibited by pharmacological antagonists. RESULTS: CaN and FKBP12 levels were similar in MCF-7 and MCF-7/Adr cells. P-glycoprotein function was not affected by treatment of P-glycoprotein-expressing MCF-7/Adr cells with CaN and FKBP12 antagonists. CONCLUSIONS: These results demonstrate that the inhibitory effects of immunosuppressive agents on P-glycoprotein function are independent of CaN or FKBP12 PPI activity.

Doxorubicin induced expression of P-glycoprotein in a canine osteosarcoma cell line.

Canine and human osteosarcoma are very similar with respect to clinical presentation, radiological and histopathological features, metastatic rate and pattern and response to therapy. For these reasons, canine osteosarcoma is a useful intermediate model for the disease in humans. Overexpression of P-glycoprotein, the product of the MDR1 gene, is the most important predictor of an adverse clinical course in human patients with osteosarcoma. Exposure of canine osteosarcoma cells to doxorubicin resulted in overexpression of MDR1 mRNA and P-glycoprotein. Furthermore, these cells failed to accumulate doxorubicin intracellularly and were less sensitive to vincristine-induced cytotoxicity as compared to parental cells.

Oxidative injury of coronary venular endothelial cells depletes intracellular glutathione and induces HSP 70 mRNA.

Vascular endothelium is one of the first tissues exposed to reactive oxygen species produced during myocardial ischemia-reperfusion. Bovine coronary venular endothelial cells (CVEC) were evaluated for intracellular glutathione (GSH) levels and heat shock protein 70 (HSP 70) mRNA and protein during in vitro oxidative stress. CVEC were incubated with 0.01875 U/ml xanthine oxidase (XO) and 0.5 mM hypoxanthine (HX) for 30 min and then allowed to recover for 0, 1, 2, or 3 h. Relative GSH levels were determined by evaluation of monochlorobimane fluorescence. GSH fluorescence was significantly lower in CVEC treated with XO+HX for 30 min than in controls. GSH fluorescence was also decreased in heat-shocked CVEC. After oxidative stress, GSH levels were higher than in controls at 1 h, but by 2 or 3 h after treatment, GSH fluorescence fell below control values. HSP 70 mRNA was induced in CVEC by a 30-min treatment with XO+HX exposure. These data suggest that CVEC respond to oxidative stress by reducing intracellular GSH levels and inducing HSP 70 mRNA, although significant increases in HSP 70 protein were not detected at the time points tested.

The effect of sterols and brefeldin A on protein degradation in UT-1 cells.

UT-1 cells, a mutant Chinese hamster ovary (CHO) cell line induced to produce an abundance of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), were used to determine the effects of sterols and brefeldin A on the degradation of this enzyme. Brefeldin A has been shown to cause retention of proteins in and relocation of proteins to the endoplasmic reticulum (ER). UT-1 cells were incubated with (a) sterols only (12 micrograms/ml cholesterol and 0.2 microgram/ml 25-hydroxycholesterol), (b) sterols and brefeldin A (0.5 microgram/ml), and (c) brefeldin A only. Western blot analysis showed that incubation with sterols and brefeldin A decreased HMGR levels more slowly than incubation with sterols alone over the first 24-36 h of incubation; however, the rates were not significantly different. By 48 h of incubation, HMGR had decreased to a level comparable to that found when cells were incubated in sterols only. Incubation with brefeldin A alone did not cause a decrease in HMGR over the same 48-h time period. HMGR was undetectable in parental CHO cells under all of the conditions described. Indirect immunofluorescence microscopy revealed a pattern of tight, perinuclear staining with sterol incubation. After 48 h in sterols, HMGR staining was uniformly decreased throughout the cytoplasm. This change in staining pattern is also observed during incubation of UT-1 cells with sterols and brefeldin A. Incubation for 48 h with brefeldin A alone had no effect on the tight perinuclear pattern originally observed. Diffuse, faint staining of CHO cells under all conditions served as a negative control. The results of these experiments indicated that brefeldin A, and therefore retention of proteins in the ER, does not interfere with the degradation of HMG CoA reductase. Despite the presence of brefeldin A, sterol-mediated dispersal and degradation of the crystalloid ER (CER) continued in UT-1 cells. Lack of brefeldin A sensitivity implied that the mechanism for CER dissolution was distinct from previously described mechanisms for ER to Golgi transport.

Low level amplification of c-sis and c-myc in a spontaneous osteosarcoma model.

Canine and human osteosarcoma are very similar clinically, radiologically and pathologically. DNA extracted from canine osteosarcomas (n = 9) and normal canine control tissues (n = 17) was examined for amplification of the c-sis, c-myc, N-myc and c-H-ras protooncogenes. Statistically significant amplification of the c-sis and c-myc protooncogenes was evident in the tumor tissues as compared to the normal control tissues (P less than 0.05). DNA and total cellular RNA from cultured canine and human osteosarcoma and fibroblast cell lines were examined for amplification or enhanced expression of c-sis and c-myc. Very low levels of c-myc and c-sis DNA amplification were noted in canine osteosarcoma cells as compared to canine fibroblasts. Immunostaining of sections of human and canine osteosarcoma for the sis gene product, PDGF B, showed similar levels and patterns of expression in both populations of tumors.

Purified crystalloid endoplasmic reticulum from UT-1 cells contains multiple proteins in addition to 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The crystalloid endoplasmic reticulum (ER) of UT-1 cells is a specialized smooth ER that houses 3-hydroxy-3-methylglutaryl-CoA reductase, a membrane protein that regulates endogenous cholesterol synthesis. The biogenesis of this ER is coupled to the over production of 3-hydroxy-3-methylglutaryl-CoA reductase. To understand better this membrane system and the relationship between the synthesis of a membrane protein and the formation of membrane, we have purified the crystalloid ER. Purified crystalloid ER did not contain significant amounts of membrane derived from the Golgi apparatus, mitochondria, or plasma membrane. Approximately 24% of the protein in this organelle corresponded to 3-hydroxy-3-methylglutaryl-CoA reductase; however, at least eight other proteins were detected by gel electrophoresis. One of these proteins (Mr 73,000) was as abundant as reductase. These results suggest that the biogenesis of this ER involves the coordinate synthesis of multiple membrane and content proteins.