Functional characterization of the TSC2 c.3598C>T (p.R1200W) missense mutation that co-segregates with tuberous sclerosis complex in mildly affected kindreds.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by a combination of neurological symptoms and hamartomatous growths, and caused by mutations in the TSC1 and TSC2 genes. Overall, TSC2 mutations are associated with a more severe disease phenotype. We identified the c.3598C>T (R1200W) change in the TSC2 gene in seven different families. The clinical phenotypes in the families were mild, characterized by mild skin lesions, remitting epilepsy and a lack of severe mental retardation or major organ involvement. Functional analysis of the TSC2 R1200W variant, and four other TSC2 missense variants associated with a mild TSC phenotype, confirmed that the changes disrupted the TSC1-TSC2 function. Interestingly however, in each case, the TSC1-TSC2 interaction was not affected by the amino acid substitution.

Mitoxantrone resistance in a small cell lung cancer cell line is associated with ABCA2 upregulation.

The aim of this study was to find factors that could explain the accumulation difference of mitoxantrone in the BCRP1-negative GLC4-MITO cell line compared to GLC4. Comparative genomic hybridisation (CGH) was applied to determine chromosomal differences between GLC4 and GLC4-MITO. Comparative genomic hybridisation analysis revealed gain of 2q, 6p, 9q, 13q, 14q, 15q, 19q and Xp and loss of 1p, 2q, 3p, 3q, 4q, 6q, 8q, 11p, 16p, 17q, 18p, 20p and Xq. In the over-represented chromosomal areas, seven transporter genes were identified: ABCB6, ABCB2 (TAP1), ABCB3 (TAP2), ABCF1 (ABC50), ABCC10 (MRP7), ABCA2 (ABC2) and ABCC4 (MRP4). No RNA or protein upregulation was observed for ABCB6, ABCF1, ABCC10, ABCC4, ABCB2 and ABCB3, but an increased expression was detected for ABCA2 mRNA in GLC4-MITO. ABCA2 is known to be involved in resistance to estramustine. In the MTT assay, GLC4-MITO was two-fold resistant to estramustine compared to GLC4. Coincubation with estramustine and mitoxantrone increased mitoxantrone accumulation in GLC4-MITO, while this was not affected in GLC4. This suggests that estramustine is able to block mitoxantrone efflux in GLC4-MITO cells. These data reveal that cellular reduction of mitoxantrone in a mitoxantrone-resistant cell line is associated with overexpression of ABCA2.

ATP binding cassette transporter gene expression in rat liver progenitor cells.

BACKGROUND AND AIM: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are cytoprotective efflux pumps that may contribute to the preservation of these cells. The aim of this study was to determine the ABC transporter phenotype of HPCs. METHODS: HPC activation was studied in rats treated with 2- acetylaminofluorene (2-AAF) followed by partial hepatectomy (PHx). ABC transporter gene expression was determined by real time detection reverse transcription-polymerase chain reaction in isolated HPCs, hepatocytes, cholangiocytes, and cultured progenitor cell-like RLF phi 13 cells and by immunohistochemistry of total liver samples. ABC transporter efflux activity was studied in RLF phi 13 cells by flow cytometry. RESULTS: 2-AAF/PHx treated animals showed increased hepatic mRNA levels of the genes encoding multidrug resistance proteins Mdr1b, Mrp1, and Mrp3. Immunohistochemistry demonstrated expression of Mrp1 and Mrp3 proteins in periportal progenitor cells and of the Mdr1b protein in periportal hepatocytes. Freshly isolated Thy-1 positive cells and cultured RLF phi 13 progenitor cells highly expressed Mrp1 and Mrp3 mRNA while the hepatocyte specific transporters Mdr2, Bsep, Mrp2, and Mrp6 were only minimally expressed. Blocking Mrp activity by MK-571 resulted in accumulation of the Mrp specific substrate carboxyfluorescein in RLF phi 13 cells. CONCLUSION: HPCs express high levels of active Mrp1 and Mrp3. These may have a cytoprotective role in conditions of severe hepatotoxicity.

Cytotoxicity of rhein, the active metabolite of sennoside laxatives, is reduced by multidrug resistance-associated protein 1.

Anthranoid laxatives, belonging to the anthraquinones as do anthracyclines, possibly increase colorectal cancer risk. Anthracyclines interfere with topoisomerase II, intercalate DNA and are substrates for P-glycoprotein and multidrug resistance-associated protein 1. P-glycoprotein and multidrug resistance-associated protein 1 protect colonic epithelial cells against xenobiotics. The aim of this study was to analyse the interference of anthranoids with these natural defence mechanisms and the direct cytotoxicity of anthranoids in cancer cell lines expressing these mechanisms in varying combinations. A cytotoxicity profile of rhein, aloe emodin and danthron was established in related cell lines exhibiting different levels of topoisomerases, multidrug resistance-associated protein 1 and P-glycoprotein. Interaction of rhein with multidrug resistance-associated protein 1 was studied by carboxy fluorescein efflux and direct cytotoxicity by apoptosis induction. Rhein was less cytotoxic in the multidrug resistance-associated protein 1 overexpressing GLC4/ADR cell line compared to GLC4. Multidrug resistance-associated protein 1 inhibition with MK571 increased rhein cytotoxicity. Carboxy fluorescein efflux was blocked by rhein. No P-glycoprotein dependent rhein efflux was observed, nor was topoisomerase II responsible for reduced toxicity. Rhein induced apoptosis but did not intercalate DNA. Aloe emodin and danthron were no substrates for MDR mechanisms. Rhein is a substrate for multidrug resistance-associated protein 1 and induces apoptosis. It could therefore render the colonic epithelium sensitive to cytotoxic agents, apart from being toxic in itself.

Activity and expression of the multidrug resistance proteins P-glycoprotein, MRP1, MRP2, MRP3 and MRP5 in de novo and relapsed acute myeloid leukemia.

The multidrug resistance proteins (MRPs) MRP1, MRP2, MRP3, MRP5 and P-glycoprotein (P-gp) act in concert with each other to give a net resultant pump function in acute myeloid leukemia (AML). The aim of the present study was to analyze the activity of these proteins, which might be upregulated at relapse as compared with de novo AML due to clonal selection. The mRNA expression and activity of P-gp and the MRPs were determined with RT-PCR and flow cytometry, in conjunction with phenotype, as measured with the monoclonal antibodies CD34, CD38 and CD33, in 30 paired samples of de novo and relapsed AML. P-gp and MRP activity varied strongly between the cases (rhodamine 123 efflux-blocking by PSC833: 5.4+/-7.7, and carboxyfluorescein efflux-blocking by MK-571: 4.3+/-6.7, n = 60). P-gp and MRP activity were increased in 23% and 40% of the relapse samples, and decreased in 30% and 20% of the relapse samples, respectively (as defined by a difference of >2 x standard deviation of the assays). Up- or downregulation of mRNA expression was observed for MDR1 (40%), MRP1 (20%), MRP2 (15%), MRP3 (30%), and MRP5 (5%). Phenotyping demonstrated a more mature phenotype in 23% of the relapsed AML cases, and a more immature phenotype in 23% of the relapses, which was independent of the karyotypic changes that were observed in 50% of the studied cases. P-gp and MRP activity correlated with the phenotypic changes, with higher P-gp and MRP activities in less mature cells (r = -0.66, P < 0.001 and r = -0.31, P = 0.02, n = 58). In conclusion, this study shows that P-gp and MRP activity are not consistently upregulated in relapsed AML. However, P-gp and MRP activities were correlated with the maturation stage as defined by immune phenotype, which was observed to be different in 46% of the relapses.

P-glycoprotein and multidrug resistance protein activities in relation to treatment outcome in acute myeloid leukemia.

Despite treatment with intensive chemotherapy, a considerable number of patients with acute myeloid leukemia (AML) die from their disease due to the occurrence of resistance. Overexpression of the transporter proteins P-glycoprotein (P-gp) and multidrug resistance protein (MRP) 1 has been identified as a major cause of cross-resistance to functionally and structurally unrelated drugs. In the present study, the functional activity of P-gp and MRP was determined in 104 de novo AML patients with a flow cytometric assay using rhodamine 123 (Rh123) in combination with PSC833 and carboxyfluorescein (CF) in combination with MK-571. The results were compared with clinical outcome and with known prognostic factors. The functional activity of P-gp and MRP, expressed as Rh123 efflux blocking by PSC833 and CF efflux blocking by MK-571, demonstrated a great variability in the AML patients. A strong negative correlation was observed between Rh123 efflux blocking by PSC833 and Rh123 accumulation (r(s) = -0.69, P < 0.001) and between CF efflux blocking by MK-571 and CF accumulation (r(s) = -0.59, P < 0.001). A low Rh123 accumulation and a high Rh123 efflux blocking by PSC833 were associated with a low complete remission (CR) rate after the first cycle of chemotherapy (P = 0.008 and P = 0.01, respectively). Patients with both low Rh123 and CF accumulation (n = 16) had the lowest CR rate (6%), whereas patients with both high Rh123 and CF accumulation (n = 11) had a CR rate of 73%. AML patients with French-American-British classification M1 or M2 showed a lower Rh123 accumulation than patients with French-American-British classification M4 or M5 (P = 0.02). No association was observed between the multidrug resistance parameters and overall survival of the AML patients. Risk group was the only predictive parameter for overall survival (P = 0.003).

Deletion of the multidrug resistance protein MRP1 gene in acute myeloid leukemia: the impact on MRP activity.

Deletion of the multidrug resistance gene MRP1 has been demonstrated in acute myeloid leukemia (AML) patients with inversion of chromosome 16 (inv[16]). These AML patients are known to have a relatively favorable prognosis, which suggests that MRP1 might play an important role in determining clinical outcome. This study analyzed MRP1 deletion by fluorescent in situ hybridization (FISH), with a focus on inv(16) AML patients. Functional activity of multidrug resistance protein (MRP) was studied in a flow cytometric assay with the use of the MRP substrate carboxyfluorescein (CF) and the inhibitor MK-571. MRP1, MRP2, and MRP6 messenger RNA (mRNA) expression was determined with reverse transcriptase-polymerase chain reaction (RT-PCR). The results were compared with normal bone marrow cells. MRP1 deletion was detected in 7 AML patients; 2 cases showed no MRP1 FISH signals, and 5 cases had 1 MRP1 signal, whereas in 4 AML patients with inv(16) no MRP1 deletions were observed. A variability in MRP activity, expressed as CF efflux-blocking by MK-571, was observed (efflux-blocking factors varied between 1.2 and 3.6); this correlated with the number of MRP1 genes (r = 0.91, P <. 01). MRP activity in the AML cases was not different from normal hematopoietic cells. MRP1 mRNA was detected in patients with 1 or 2 MRP1 FISH signals, but not in patients with no MRP1 signals. MRP2 and MRP6 mRNA were expressed predominantly in AML samples with 1 MRP1 signal, whereas in normal bone marrow cells no MRP2 and MRP6 mRNA was observed. In conclusion, this study shows that MRP activity varies among inv(16) AML cases and does not differ from that in normal hematopoietic cells; this might be in part due to the up-regulation of other MRP genes.

Breast cancer resistance protein is localized at the plasma membrane in mitoxantrone- and topotecan-resistant cell lines.

Tumor cells may display a multidrug resistant phenotype by overexpression of ATP-binding cassette transporters such as multidrug resistance (MDRI) P-glycoprotein, multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP). The presence of BCRP has thus far been reported solely using mRNA data. In this study, we describe a BCRP-specific monoclonal antibody, BXP-34, obtained from mice, immunized with mitoxantrone-resistant, BCRP mRNA-positive MCF-7 MR human breast cancer cells. BCRP was detected in BCRP-transfected cells and in several mitoxantrone- and topotecan-selected tumor cell sublines. Pronounced staining of the cell membranes showed that the transporter is mainly present at the plasma membrane. In a panel of human tumors, including primary tumors as well as drug-treated breast cancer and acute myeloid leukemia samples, BCRP was low or undetectable. Extended studies will be required to analyze the possible contribution of BCRP to clinical multidrug resistance.

Multidrug resistance protein MRP1, glutathione, and related enzymes. Their importance in acute myeloid leukemia.

Multidrug resistance (MDR), which is cross-resistance to structurally and functionally unrelated drugs such as anthracyclines, epipodophyllotoxins and vinca alkaloids, is a major cause of treatment failure in malignant disorders. Known mechanisms of MDR are overexpression of the ATP-dependent membrane proteins P-glycoprotein (P-gp) and multidrug resistance protein (MRP1), or an increased detoxification of compounds mediated by glutathione (GSH) or GSH related enzymes. MRP1 appeared to transport drugs conjugated to GSH and also unmodified cytostatic agents in presence of GSH. The relation between MRP1, GSH and enzymes involved in GSH metabolism or GSH dependent detoxification reactions recently has drawn a lot of attention. Coordinated induction of MRP1 and GSH related enzymes is reported in malignant cells after exposure to cytostatic agents. Besides MRP1, a number of MRP1 homologs are identified, named MRP2, MRP3, MRP4, MRP5 and MRP6. The relation between MDR and expression of these MRP1 homologs is currently under research.

Activity and expression of the multidrug resistance proteins MRP1 and MRP2 in acute myeloid leukemia cells, tumor cell lines, and normal hematopoietic CD34+ peripheral blood cells.

To develop a functional assay for the activity of the multidrug resistance protein 1 (MRP1), we tested whether carboxyfluorescein (CF) was specifically transported by MRP and whether this transport pump could be specifically blocked by the leukotriene D4 receptor antagonist MK-571. The activity and expression of MRP1 were studied in several tumor cell lines and in leukemic blasts from patients with acute myeloid leukemia (AML). In the MRP1-overexpressing cell line GLC4/ADR and the MRP1-transfected cell line S1(MRP), MK-571 inhibited CF efflux with high factors [45.9+/-5.8 (mean+/-SD) and 14.4+/-3.2, respectively; n=3; efflux-blocking factors are defined as the ratio of the median fluorescence in presence or absence of MK-571] compared with their MRP1 low-expressing counterparts GLC4 (11.5+/-2.7) and S1 (2.8+/-0.4). In 15 AML cases, the CF efflux-blocking factors of MK-571 varied between 1.9 and 5.2. A good correlation was found between MRP1 protein expression and CF efflux-blocking factors of MK-571 (P=0.013, r=0.39). Besides MRP1, MRP2 was demonstrated with reverse transcription-PCR in 40% of the cases. In contrast to the cell lines, MK-571 also inhibited rhodamine 123 (Rh123) efflux in AML samples. On the other hand, PSC833, a P-glycoprotein specific inhibitor, did not modulate the CF efflux but is efficient in blocking Rh123 efflux. This study demonstrates that AML blasts express MRP1 and MRP2 and that MRP1 activity can be determined by a flow cytometric assay using CF and MK-571.

The combined effects of IL-3 and PSC 833 on daunorubicin- and mitoxantrone cytotoxicity in two growth factor-dependent leukemic cell lines.

In the present study it was investigated whether and by which mechanisms the co-administration of interleukin-3 (IL-3) and the P-glycoprotein blocker PSC 833 can augment mitoxantrone (MX) and daunorubicin (DAU) cytotoxicity in two human growth factor dependent P-glycoprotein (P-gp) positive myeloid leukemic cell lines, Mo-7 and GF-D8. Cytotoxicity was determined in MTT assay. Increased cytotoxicity occurred in Mo-7 cells preincubated with 24h IL-3 followed by 1 h MX (cell survival: 85% +/- 6 vs 68% +/- 2, at 0.05 microM MX, P < 0.005) or DAU (79% +/- 8 vs 62% +/- 9 at 0.8 microg/ml DAU, P < 0.05). Similar results were obtained for the GF-D8 cell line. In this cell line, at 0.5 microM MX the cell survival decreased from 84% +/- 13 to 61% +/- 19 (P < 0.05) and at 5.0 microg/ml DAU from 102% +/- 8 to 69% +/- 5, (P < 0.002). The IL-3 administration did not affect the P-gp and bcl-2 protein expression, cellular MX concentration or MX efflux but coincided with an increased percentage of cells in S-phase and topoisomerase II (topo II)-alpha mRNA and topo II activity especially in the Mo-7 cell line. PSC 833 enhanced DAU cytotoxicity in both cell lines. The administration of IL-3 plus PSC 833 in the Mo-7 cell line resulted in an additive effect on DAU cytotoxicity. At 0.8 microg/ml DAU and 2 microg/ml PSC 833, the percentage surviving cells decreased from 62% +/- 9 in the absence of IL-3 to 37% +/- 3 in the presence of IL-3 (P < 0.01). The additive effect of combined treatment was most pronounced in GF-D8 cells which also had the highest P-gp expression. In contrast, PSC 833 did not modulate the MX effects, irrespective of the presence of IL-3. In summary, the results demonstrate that the combined administration of IL-3 and PSC 833 can enhance the cytotoxic effects of DAU but not MX in these P-gp positive cell lines whereas the effects of MX could be modulated by factors which influence topo II activity.

Optimization of a classical aromatase activity assay and application in normal, adenomatous and malignant breast parenchyma.

The tritium water release assay, originally described for the analysis of aromatase activity in placental tissue, was used to estimate aromatase activity in breast tissue samples. The lower activity in this tissue necessitates longer incubation times and thus optimization of the assay conditions. To prevent oxidative and proteolytic inactivation of aromatase, dithiothreitol and albumin were added to the incubation mixture. Extra NADPH, cofactor in the aromatase reaction, also improved reaction rate in placental incubations, but after approximately 120 min activity rapidly decreased. Inhibitors gradually produced during the incubation could explain this phenomenon. Quantitative gas chromatography-mass spectrometry (GC-MS) analyses of testosterone, oestradiol, oestrone and androstenedione after incubation with non-labelled androstenedione proved that a substantial amount of the substrate is converted into testosterone. Qualitative GC-MS steroid profiling of the incubation mixture demonstrated the presence of hydroxylated oestradiol and hydroxylated testosterone, produced during incubation, which could have caused partial aromatase inhibition. The adjusted assay was used to analyse 84 breast tissue samples, histologically classified as normal, adenoma or carcinoma. Aromatase activity was found in 56% of all samples and ranged from 0.6 to 26 pmol oestrogen/g protein per hour. Aromatase positivity was found in 80% of the normal samples, 56% of the adenoma samples and 48% of the carcinoma samples. Although carcinoma samples were less often aromatase positive than normal tissue samples (chi2 = 4.80; P < 0.050) there was no difference in absolute aromatase activity. Because no less than approximately 50% of the carcinomas contained aromatase activity and because of the non-routine character of the assay we conclude that it is justified to start aromatase inhibition therapy without previous knowledge of the aromatase status.

VP-16-mediated cytotoxicity is modulated by interleukin-3 in a growth factor-dependent leukemic cell line.

A growth factor-dependent cell line (TF-1) was treated with interleukin-3 (IL-3) or medium in combination with variable doses of VP-16 to test whether the latter's cytotoxicity can be modulated by IL-3. The results demonstrated that an augmented cell death occurred with TF-1 cells when pre-incubated for 24 h with IL-3 followed by treatment with VP-16 (10 micrograms/ml) for 1 h. The increased cell death could not be ascribed to an increased number of apoptotic cells as measured with the acridine orange method. However, the IL-3 treatment coincided with an upregulation of DNA topoisomerase II alpha (Topo II alpha) at mRNA and protein level after 24 h, which was preceded by an upregulation of c-myc mRNA. In contrast, Topo II beta did not demonstrate an upregulation at mRNA level in response to IL-3 stimulation. In addition, it was shown that cells treated with IL-3 followed by VP-16 demonstrated a higher number of cleavable DNA complexes which was not due to an increased uptake of VP-16, since cellular concentrations of VP-16 in the presence of IL-3 or medium were 16.8 +/- 7.8 ng/10(6) cells and 19.8 +/- 7.8 ng/10(6) cells (mean +/- SD, n = 3), respectively. These data indicate that IL-3 pretreatment followed by VP-16 results in an increased cell death due to cytotoxicity which may be ascribed to an upregulation of Topo II alpha.