MRP1 not MDR1 gene expression is the predominant mechanism of acquired multidrug resistance in two prostate carcinoma cell lines.

Multidrug resistant prostate cancer cell lines DU 0.03 and PC 0.03 were established from the parental prostate cancer cell lines DU145 and PC-3 respectively by stepwise selection in doxorubicin (DOX) from 0.001 to 0.03 &mgr;g/ml. As cells adapted to each concentration of DOX. the drug concentration was increased by 0.001 &mgr;g/ml. The chemosensitivity of each line was determined by growth inhibition assay. The DU 0.03 and PC 0.03 lines exhibit a 5-10-fold and 1.3-2.8-fold increase in resistance to anthracyclines, vinblastine (VLB) and mitozantrone (Mito), respectively. Verapamil (5 &mgr;M) partially reversed the resistance to the anthracycline and completely reversed the resistance to VLB and Mito. Drug kinetic studies measured by intracellular accumulation of (3)H-daunorubicin demonstrated a 3 fold decrease in the level of intracellular (3)H-daunorubicin in the PC 0.03 and DU 0.03 resistant lines compared with their respective parental line. This effect was partially reversed by 5 &mgr;M verapamil. The expression of MDR1 and MRP genes was analysed by Northern blotting and RT-PCR. P-glycoprotein (Pgp) and MRP protein were tested by immunocytochemistry staining using the monoclonal antibodies J-SB1. C219 and MRK16 (Pgp) and MRPm6 and MRPr1 (MRP). Neither Northern blot analysis nor the more sensitive RT-PCR demonstrated detectable MDR1 transcripts in any of the prostate cancer cell lines and the three Pgp monoclonal antibodies failed to reveal expression of Pgp. A 2-4-fold increase in MRP1 mRNA levels in the drug resistant DU 0.03 and PC 0.03 lines were demonstrated by both Northern blotting and RT-PCR consistent with the findings observed after staining by the two specific monoclonal antibodies, MRPm6 and MRPr1. Southern blot analysis demonstrated a 2-fold increase in the MRP1 gene copy number in the PC 0.03 line but not in the DU 0.03 line, suggesting that the overexpression of the MRP gene was regulated at the level of transcription in the latter line. We conclude that MRP1 not MDR1 overexpression. contributes to acquired drug resistance in these two prostate cancer cell lines. Prostate Cancer and Prostatic Diseases (2000) 3, 66-75

Use of double gradient denaturing gradient gel electrophoresis to detect (AT)n polymorphisms in the UDP-glucuronosyltransferase 1 gene promoter associated with Gilbert's syndrome.

Gilbert's syndrome, due to reduced hepatic bilirubin glucuronidation is associated with the presence of two extra nucleotides (TA) in the promoter region of the UDP-glucuronosyltransferase 1 (UGT1A1) gene. A rapid method was developed to detect this genetic polymorphism, using double gradient denaturing gradient gel electrophoresis (DG-DGGE). The promoter region of the UGT1A1 gene was amplified with a 40-mer GC-clamp attached to the 5'-end of the reverse primer. The polymerase chain reaction (PCR) product was then separated by DG-DGGE using denaturant concentrations of 15-25% and polyacrylamide concentrations of 6-12%. The (TA)6/(TA)6 homozygotes were clearly distinguished from both (TA)7/(TA)7 homozygotes and (TA)6/(TA)7 heterozygotes. The (TA)7 allele frequency was consistent with that previously reported and elevated bilirubin levels correlated with the presence of the (TA)7 allele. The DG-DGGE method described will make detection for this polymorphism fast, simple, nonradioactive and suitable for a clinical routine diagnostic laboratory, helping to establish the role of this polymorphism in individuals with jaundice due to multiple causes.

Altered multidrug resistance phenotype caused by anthracycline analogues and cytosine arabinoside in myeloid leukemia.

The expression of P-glycoprotein (Pgp) is often increased in acute myeloid leukemia (AML). However, little is known of the regulation of Pgp expression by cytotoxics in AML. We examined whether Pgp expression and function in leukemic blasts was altered after a short exposure to cytotoxics. Blasts were isolated from 19 patients with AML (15 patients) or chronic myeloid leukemia in blastic transformation (BT-CML, 4 patients). Pgp expression and function were analyzed by flow cytometric analysis of MRK 16 binding and Rhodamine 123 retention, respectively. At equitoxic concentrations, ex vivo exposure for 16 hours to the anthracyclines epirubicin (EPI), daunomycin (DAU), idarubicin (IDA), or MX2 or the nucleoside analogue cytosine arabinoside (AraC) differentially upregulated MDR1/Pgp expression in Pgp-negative and Pgp-positive blast cells. In Pgp-negative blasts, all four anthracyclines and AraC significantly increased Pgp expression (P =.01) and Pgp function (P =.03). In contrast, MX2, DAU, and AraC were the most potent in inducing Pgp expression and function in Pgp positive blasts (P <.05). A good correlation between increased Pgp expression and function was observed in Pgp-negative (r =.90, P =.0001) and Pgp-positive blasts (r =.77, P =.0002). This increase in Pgp expression and function was inhibited by the addition of 1 micromol/L PSC 833 to blast cells at the time of their exposure to these cytotoxics. In 1 patient with AML, an increase in Pgp levels was observed in vivo at 4 and 16 hours after the administration of standard chemotherapy with DAU/AraC. Upregulation of Pgp expression was also demonstrated ex vivo in blasts harvested from this patient before the commencement of treatment. In 3 other cases (1 patient with AML and 2 with BT-CML) in which blasts were Pgp negative at the time of initial clinical presentation, serial samples at 1 to 5 months after chemotherapy showed the presence of Pgp-positive blasts. All 3 patients had refractory disease. Interestingly, in all 3 cases, upregulation of Pgp by cytotoxics was demonstrated ex vivo in blasts harvested at the time of presentation. These data suggest that upregulation of the MDR1 gene may represent a normal response of leukemic cells to cytotoxic stress and may contribute to clinical drug resistance.

Induction of MDR1 gene expression by anthracycline analogues in a human drug resistant leukaemia cell line.

The effects of 4-demethoxydaunorubicin (idarubicin, IDA) and MX2, a new morpholino-anthracycline, on up-regulation of the MDR1 gene in the low-level multidrug resistant (MDR) cell line CEM/A7R were compared at similar concentrations (IC10, IC50 and IC90) over a short time exposure (4 and 24 h). The chemosensitivity of each drug was determined by a 3-day cell growth inhibition assay. Compared with epirubicin (EPI), IDA and MX2 were 17- and eightfold more effective in the CEM/A7R line respectively. No cross-resistance to 5-FU was seen in the CEM/A7R line. Verapamil (5 microM) and PSC 833 (1 microM), which dramatically reversed resistance to EPI in the CEM/A7R line, had no sensitizing effect on the resistance of this line to MX2, but slightly decreased resistance to IDA. The sensitivity to 5-FU was unchanged by these modulators. The induction of MDR1 mRNA expression by IDA, MX2 and 5-FU was analysed by Northern blotting and semiquantitatively assessed by scanning Northern blots on a phosphorimager. The relative level of MDR1 expression was expressed as a ratio of MDR1 mRNA to the internal RNA control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). IDA, MX2 and 5-FU differentially up-regulated MDR1 mRNA in the CEM/A7R line in a dose-dependent manner. Both IDA and MX2 induced MDR1 expression within 4 h. 5-FU up-regulated MDR1 expression only when drug exposure was prolonged to 24 h. Based on MRK 16 binding, flow cytometric analysis of P-glycoprotein (Pgp) expression paralleled the increase in MDR1 mRNA levels. For the three anthracyclines, the increase in MDR1 expression was stable in cells grown in the absence of drug for more than 3 weeks after drug treatment. The induction of MDR1 expression by 5-FU was transient, associated with a rapid decrease in the increased Pgp levels which returned to baseline 72 h after the removal of 5-FU. This study demonstrates that MDR1 expression can be induced by analogues of anthracyclines not pumped by Pgp, and that this induction appears to be stable despite a 3-week drug-free period.

Expression of mdr1 and mrp in the normal B-cell homologue of B-cell chronic lymphocytic leukaemia.

B-cell chronic lymphocytic leukaemia (CLL) cells commonly express the multidrug resistance phenotype. The aim of this study was to establish whether the normal homologue in B-cell ontogeny of B-CLL also expressed the multidrug resistance (mdr) phenotype. Human tonsillar lymphocytes were sorted to yield two B-cell subsets based on the expression of CD19, CD5 and CD10. The normal homologue was represented by a population of B cells that was CD19 positive, CD10 negative and weakly expressed CD5. Based upon functional analysis and the detection of mdr1 mRNA by semi-quantitative PCR, these cells expressed the mdr phenotype. In contrast, functional multidrug resistance could not be demonstrated in CD19-positive CD10-positive cells with strong expression of CD5, nor could mdr1 mRNA be found in these cells. MRP was variably expressed in both B-cell subsets with no discernable differences in the pattern of expression. We conclude that normal B cells with a phenotype resembling that of B-CLL cells express the multidrug resistance phenotype.

Altered methylation of the human MDR1 promoter is associated with acquired multidrug resistance.

One of the most important forms of drug resistance in acute myeloid leukemia is the multidrug resistance (MDR) phenotype, which is characterized by the expression of the MDR1 gene product, P-glycoprotein. Although a number of factors affect MDR1 gene expression, the genetic events that "switch on" the human MDR1 gene in tumor cells that were previously P-glycoprotein negative have remained elusive. Here, we report evidence that the methylation status of the human MDR1 promoter may serve as a basis for this "switch." Based on Southern analysis using methylation-sensitive and methylation-insensitive restriction enzymes, a tight correlation was found between MDR phenotype and demethylation of the 5' region of the MDR1 gene in a human T cell leukemia cell line. Similar results were obtained from the analysis of P-glycoprotein-positive and P-glycoprotein-negative samples of chronic lymphocytic leukemia. Treatment of the cell lines with the demethylating agent 5'-azadeoxycytidine altered the methylation pattern of the MDR1 promoter in P-glycoprotein-negative cells to resemble that of P-glycoprotein-positive cells and activated the promoter such that MDR1 mRNA was now detectable. Treatment also resulted in an increased resistance to epirubicin and decreased daunomycin accumulation, both of which were reversible by verapamil, a characteristic of the classical MDR phenotype in cells expressing P-glycoprotein. These results suggest that the MDR phenotype may be acquired as a result of changes in methylation of the MDR1 promoter.

Cyclosporin A and PSC 833 prevent up-regulation of MDR1 expression by anthracyclines in a human multidrug-resistant cell line.

We have previously demonstrated that within 24 h of exposure of the CEM/A7R cell line to epirubicin (EPI), MDR1 gene expression is induced. The aim of the current study was to investigate the role of cyclosporin A (CyA) and PSC 833, two biochemical modulators of the classical multidrug-resistant phenotype, in this model. CEM/A7R cells were exposed to EPI in the presence or absence of various concentrations of CyA or PSC 833. MDR1 expression was assessed using Northern blot analysis and quantitated using a phosphorimager. P-glycoprotein (P-gp) expression was analyzed by the determination of MRK16 binding using flow cytometry. P-gp function was measured in an assay of [3H]daunomycin accumulation. The coincubation of CyA or PSC 833 with EPI prevented the increase in MDR1 gene expression induced by EPI alone. This effect of the two modulators was dose dependent. Neither modulator alone had any significant effect on the expression of MDR1. In these experiments, changes in MDR1 expression correlated with changes in P-gp levels (based on MRK16 binding) and P-gp function. Thus, both PSC 833 and CyA appear to prevent the induction of MDR1 gene expression caused by the short-term exposure of CEM/A7R cells to EPI.

Inheritance of chromosome 7 is associated with a drug-resistant phenotype in somatic cell hybrids.

A major form of drug resistance in tumour cells known as classical multidrug resistance (MDR) is associated with the overexpression of the mdr1 gene product, the membrane protein P-glycoprotein (P-gp), which acts as an energy-dependent drug efflux pump. In this study the inheritance of P-gp expression was examined using hybrids formed after somatic cell fusion between a drug-sensitive human T-cell leukaemia cell line, CEM/CCRF, and a drug-resistant derivative, CEM/A7, which is characterized by a clonal chromosomal duplication dup(7)(q11.23q31.2). Fourteen hybrids, chosen at random, were analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) and by binding studies involving the monoclonal antibody MRK16, which recognises an external P-gp epitope. Only two hybrids were positive for both MRK16 antibody labelling and mdr1 mRNA. Partial karyotypic analysis of all hybrids revealed that only the MRK16-positive hybrids contained the duplication in chromosome 7 seen in the CEM/A7 parental MDR line. Therefore, P-gp overexpression in the MRK16-positive hybrids may be linked to the inheritance of chromosome 7 from CEM/A7 and possibly associated with the chromosome 7 abnormality.

Skin necrosis following subcutaneous heparin injection.

Heparin-induced skin necrosis is a rare but serious complication of subcutaneously administered heparin. Previous reports indicate that the skin necrosis is often accompanied by thrombocytopenia and occasionally by lethal thromboembolism. It thus shows features similar to the heparin-induced thrombocytopenia (HIT) syndrome and probably represents a localized form of this condition. Caution is required in the event of skin necrosis; heparin therapy should be ceased immediately and not used again if the complications of HIT are to be avoided.

Rapid up-regulation of mdr1 expression by anthracyclines in a classical multidrug-resistant cell line.

Studies were carried out in a variant human multidrug-resistant (MDR) cell line CEM/A7R, which expresses very low levels of mdr1 mRNA and P-glycoprotein (P-gp). The induction of mdr1 RNA expression by three anthracyclines, (doxorubicin, daunorubicin, epirubicin), VP-16 and two vinca alkaloids (vincristine, vinblastine) was semiquantitatively assessed by scanning Northern blots on a phosphorimager. The relative level of mdr1 expression was expressed as ratio of mdr1 to the internal RNA (actin). A significant increase (P < 0.02) in expression of mdr1 was noted within 4 hrs of exposure to 1.5 micrograms ml-1 daunorubicin or epirubicin. Neither vinblastine nor vincristine had any effect on mdr1 levels after an 8 h exposure. With increasing concentrations of daunorubicin or epirubicin in a fixed 24 h time period, mdr1 expression increased, although a biphasic response was seen. Based on MRK 16 binding, an increase in P-gp levels was seen in the CEM/A7R line after a 24 h exposure to 1 microgram ml-1 daunorubicin or epirubicin. The rapid increase in mdr1 expression after a short period of exposure to doxorubicin, daunorubicin or epirubicin suggests that induction of mdr1 expression may have an important role in the development of drug-resistant tumours.

Cellular and karyotypic characterization of two doxorubicin resistant cell lines isolated from the same parental human leukemia cell line.

Separate mechanisms underlying the multidrug resistant (MDR) phenotype were identified in 2 independent approaches to select tumour cells resistant to low concentrations of doxorubicin (Dox) from the sensitive T cell leukemia cell line CCRF-CEM. The CEM/A7 cell line was selected at an initial concentration of 0.005 microgram/ml of Dox and maintained at 0.07 microgram/ml. In contrast, the CEM/A5 line was selected using an initial concentration of 0.01 microgram/ml and maintained in Dox at a concentration of 0.05 microgram/ml. P-glycoprotein expression was demonstrated in the CEM/A7 line but not the CEM/A5 line. Amplification of the mdrI gene was not observed in the CEM/A7 cell line. Both cell lines showed cross-resistance to a number of structurally unrelated cytotoxic drugs including anthracyclines and etoposide (VP-16), although only the CEM/A7 line was cross resistant to Vinca alkaloids. Immunoblots of total cell lysates of the CEM/A5 line have revealed almost undetectable levels of topoisomerase II alpha and beta in this line. Cytogenetic analyses of both lines revealed numerous karyotypic abnormalities which were present in the parental cell line as well as both resistant cell lines. The CEM/A7 line also demonstrated a duplication of part of the long arm of chromosome 7 which included the region containing the mdrI gene, a finding not seen in the parental or CEM/A5 line. CEM/A5, however, demonstrated an abnormality of chromosome 7, outside the region of the mdrI gene, and it also contained a deletion of the short arm of chromosome 2. Abnormalities in this latter region of genome have been associated with non-P-glycoprotein-mediated MDR.

P-glycoprotein expression in classical multi-drug resistant leukaemia cells does not correlate with enhanced chloride channel activity.

1. P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump responsible for classical multi-drug resistance (MDR). 2. Pgp is part of a supergene family of membrane transport proteins that includes the cystic fibrosis gene product. 3. Transfection of cells with the MDR1 gene has been previously shown to generate volume-regulated chloride channel activity in association with Pgp expression. 4. We have used whole-cell patch clamping to examine the drug-sensitive T lymphoblastic cell line CEM-CCRF and its classical MDR derivative CEM/VLB100. The results suggest that expression of Pgp is not associated with increased chloride channel activity in this multi-drug resistant cell line. 5. We were unable to confirm previously reported results in MDR1 transfected cell lines that suggested that Pgp was associated with the presence of volume-regulated chloride channels.

Clinical application of a rapid, functional assay for multidrug resistance based on accumulation of the fluorescent dye, fluo-3.

A rapid and simple functional assay for P-glycoprotein (Pgp) using flow cytometry to measure the accumulation of the flurophore fluo-3 has been applied to samples from patients with B-cell chronic lymphocytic leukaemia (B-CLL). Peripheral blood lymphocytes from 37 patients with B-CLL were studied for Pgp. Pgp expression, using MRK-16, a monoclonal antibody recognising an external surface epitope of Pgp, was detected in 92% of patients with B-CLL. The functional assays for Pgp expression were positive in 78 and 59% of patients using the fluo-3 and doxorubicin (dox) assays, respectively. When compared with the MRK-16 assay, the fluo-3 assay had a sensitivity of 82% compared to a sensitivity of 56% for the dox assay (P = 0.004). The specificity of the fluo-3 and dox assays could not be evaluated because of the low number of MRK-16 negative CLL cells.

Prophylaxis against venous thrombosis after total hip arthroplasty.

Venous thrombosis rates were compared in 200 patients undergoing total hip arthroplasty and randomized to receive either fixed mini-dose warfarin (1 mg daily) or adjusted-dose warfarin to maintain an international normalized prothrombin ratio (INR) of 2.0-4.0. Bilateral lower limb venography was performed between days 11 and 13 inclusive. Fixed mini-dose warfarin was associated with a significantly higher rate of total thrombosis (P less than 0.05). General anaesthesia was associated with a significantly higher rate of thrombosis than spinal anaesthesia (P less than 0.05). Adjusted-dose warfarin was associated with more bleeding complications than mini-dose warfarin although these were not attributable to excessive anticoagulation. A single death from pulmonary embolus occurred in the early postoperative period in a patient receiving adjusted-dose warfarin.

Mild bleeding disorders. A clinical and laboratory study.

OBJECTIVE: To investigate in-vitro haemostasis in subjects with symptoms suggesting a mild bleeding disorder. DESIGN: A prospective study in which an extensive range of in-vitro tests were applied unselectively. SETTING: Patients were referred from community-based practices and hospital outpatient services. PATIENTS: Ninety-three consecutive patients were examined. Hospital patients with severe illness were excluded. CLINICAL FEATURES: Patients presented with easy bruising (68%), epistaxis (12%), excessive operative bleeding (7%), menorrhagia (4%), haematuria (3%), dental bleeding (1%) and bleeding from other sites (5%). In no instance was the bleeding life threatening. OUTCOME MEASURES: Results of laboratory tests for patients presenting with the symptoms of a mild bleeding disorder were compared with the results for a healthy reference group. RESULTS: Abnormal results of in-vitro tests were found in 53% of the subjects. Thirteen per cent had a prolonged bleeding time, of whom the majority had abnormal results of other in-vitro tests. Von Willebrand's disease was diagnosed in 7% of patients, although only half of these had a prolonged bleeding time. CONCLUSIONS: Abnormal results of in-vitro tests were prevalent among subjects with symptoms of mild bleeding disorder. Easy bruising was as powerful a clue as any other bleeding manifestation to the presence of an abnormal in-vitro test result.

A comparison of five devices for the bedside monitoring of heparin therapy.

Five instruments were tested for their capacity to monitor heparin therapy on whole blood at the bedside. The instruments were 512 Coagulation Monitor (Ciba-Corning), Thrombotrack (Nycomed), Automated Coagulation Timer (Hemotec), Hemochron-ACT and Hemochron-APTT (International Technidyne Corporation). Fifty subjects with various levels of heparinisation were tested on each instrument and were also assayed for antithrombin III, fibrinogen, haematocrit, platelet count and plasma heparin level. The results were compared with a reference APTT performed on the Automated Coagulation Laboratory 300R (Instrumentation Laboratories). The Hemochron-ACT correlated least well. The Hemotec and Thrombotrack were unsuitable in a clinical setting because of pipetting requirements, although the Thrombotrack did correlate well with the reference parameters. The 512 Coagulation Monitor was the simplest to use, but its maximum response corresponded to the midpoint of the reference APTT therapeutic range. The Hemochron-APTT was simple to use, had an adequate response range and correlated well with reference parameters.

Neutropenia due to low-dose methotrexate therapy for psoriasis and rheumatoid arthritis may be fatal.

OBJECTIVE: To review experience with neutropenia related to low-dose methotrexate therapy in patients with psoriasis and rheumatoid arthritis. DESIGN: Retrospective review of medical records. SETTING: A 509-bed Melbourne teaching hospital. PATIENTS: Five patients admitted in 1987 and 1988, with neutrophil counts of less than 1 x 10(9)/L, given low doses of methotrexate for psoriasis or rheumatoid arthritis. MAIN OUTCOME MEASURES: Death, or length of hospital admission. FINDINGS: Four patients were women, and one a man; three had been treated for psoriasis, and two for rheumatoid arthritis. Ages ranged from 56 to 91 years. The eldest patients, aged 77, 81 and 91 years, died. The other two were discharged after 43 and 48 days. Prior to or shortly after admission, four patients were treated with penicillin antibiotics which may have interfered with methotrexate excretion. CONCLUSIONS: Methotrexate clearances (related to creatinine clearance rates and presumably low) were probably reduced sufficiently by concomitant therapy to result in neutropenia. Practitioners using methotrexate should be aware of drug interactions resulting in delayed methotrexate excretion. Blood counts should be monitored after changes in therapy, especially in patients with impaired renal function, such as the elderly.

Autoantibodies in systemic vasculitis.

We have studied 495 sera that were referred to us from patients suspected on clinical and/or histological grounds to have a small vessel vasculitis. These sera were tested for antibodies against neutrophil cytoplasm antigens (anti-neutrophil cytoplasm antibodies, ANCA) using assays based on neutrophil acid extract, myeloperoxidase and elastase. Such antibodies are commonly found in Wegener's granulomatosis (WG) and microscopic polyarteritis (MPA), and sometimes in other small vessel vasculitides. One hundred and twenty-six of these sera (25%) were positive in the acid extract ELISA, 68 (14%) in the assay for anti-myeloperoxidase antibodies and 35 (16%) in the assay for anti-elastase antibodies. A total of 166 sera (34%) were positive for antibodies against neutrophil cytoplasm constituents. No ANCA, anti-myeloperoxidase or anti-elastase antibodies were detected in 26 convalescent sera from patients either with WG or MPA, or who had previously been positive. The mean time between positive and negative sera was eight weeks (range three weeks to six months) and three out of three who relapsed again developed ANCA of the same specificity as the original sera. Of the 228 sera also tested for anti-GBM antibodies, 13 (5.7%) were positive. All these contained antibodies against neutrophil cytoplasm constituents (three against the acid extract, eight against myeloperoxidase and two against elastase). Forty-nine of the 74 sera (66%) tested for ANA were positive. Twenty-nine (39%) had a speckled and 20 (27%) had a homogeneous pattern.(ABSTRACT TRUNCATED AT 250 WORDS)