Background Limited information is available on the effectiveness of the BBIBP-CorV (Sinopharm, Be... more Background Limited information is available on the effectiveness of the BBIBP-CorV (Sinopharm, Beijing CNBG) vaccine, especially in the elderly, despite the fact that it is approved in more than 50 countries. Methods RBD-specific antibody titres, as a rapidly available and highly predictive surrogate marker, were measured after two doses of the BBIBP-CorV vaccine in 450 subjects. Results were analyzed in a multivariable model accounting for age, sex and time since the administration of the second dose of the vaccine. Results Sex and time since the second dose had little association with the antibody titres. Age, however, was highly relevant: measurable antibody levels were present in about 90% of individuals below the age of 50, but antibody production after BBIBP-CorV vaccination was strongly reduced with increasing age. A large number of elderly subjects, reaching 25% at 60 years, and up to 50% at ages over 80, were found not to produce any protective antibody. Conclusions RBD-spe...
In inside-out hy$n red cell membrane vesicles /IOV/, in th52absence of Mg , the only calcium-indu... more In inside-out hy$n red cell membrane vesicles /IOV/, in th52absence of Mg , the only calcium-induced labelling by g P-ATP occurs in a 140-150 000 molecular weight protein fraction, representing the hydroxylamine-sensitive phosphorylated int?$mediate /EP/ of the calcium pump. In the presence of Mg calcium-induced phosphorylation is accelerated but several other membrane proteins are also phosphorylated through protein kinase action forming hydroxylamine-insensitive bonds. Addition of calmodulin accels+ates EP formation both in the absence and presence of Mg. Treatment of the membrane with SH-group reagents significantly reduces EP formation. Mild trypsin digestion of IOVS, stimulating active calcium transport, eliminates calmodulin action and decreases the steady-state level of %. In trypsin-digested IOVs the molecular weight of the P-1,abelled EP is shifted to lower values / 110-120 000 I We suggest that trypsin digestion cleaves off a 20-40 000 molecular weight calmodulin-binding regulatory subunit of the calcium pump molecule.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1982
In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the diva... more In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the divalent metal ions Mg z+, Mn 2+, Co z+, Ni 2+ and Fe 2+. This activation is based on the formation of Me2+-ATP complexes which can serve as energy-donor substrates for the calcium pump, and probably, satisfy the requirement for free Me z+ in this transport process. Higher Me 2+ concentrations inhibit calcium transport with various efficiencies. Mn 2÷ directly competes with Ca 2+ at the transport site, while other divalent metal ions investigated have no such effect. The formation of the hydroxylamine-sensitive phosphorylated intermediate (EP) of the red cell membrane calcium pump from [T-32p]ATP is induced by Ca 2+ while rapid dephosphorylation requires the presence of Mg 2+. At higher concentrations Mn 2+ and Ni 2+ inhibit predominantly the formation of EP, while Co 2+ and Fe 2+ block dephosphorylation. The possible sites and nature of the divalent metal interactions with the red cell calcium pump are discussed. Hydroxylamine-insensitive membrane phosphorylation in inside-out vesicles from IT-32 P]ATP is significantly stimulated by Mn 2 + and Co 2+, as compared to that produced by Mg 2+, Fe 2+ and Ni 2+. Part of this labelling is found in phospholipids, especially in phosphatidylinositol. The results presented for the metal dependency of protein and lipid phosphorylation in red cell membranes may help in the characterization of ATP consumptions directly related to the calcium pump and those involved in various regulatory processes.
The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distributi... more The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distribution, metabolism, and elimination of a wide range of pharmaceutical compounds. Functional investigation of the ABCB1 expression is also essential in many diseases, including drug-resistant cancer, inflammatory conditions, or Alzheimer disease. In this study, we examined the potential interaction of the ABCB1 multidrug transporter with a group of commercially available viability dyes that are generally considered not to penetrate into intact cells. Here, we demonstrate that the slow cellular accumulation of TO-PRO™-1 (TP1) or TO-PRO™-3 (TP3) was strongly inhibited by ABCB1-dependent dye extrusion. TP1/3 dye accumulation was not affected by the presence of ABCC1 or ABCG2, while this uptake was increased to the level in the ABCB1-negative cells by a specific P-glycoprotein inhibitor, Tariquidar. We suggest that TP compounds can be used as highly sensitive, selective, non-toxic, and stable dye...
DiGeorge Syndrome (DGS) Critical Region 8 (DGCR8) is a primary candidate gene in they DGS. The DG... more DiGeorge Syndrome (DGS) Critical Region 8 (DGCR8) is a primary candidate gene in they DGS. The DGCR8 microprocessor complex subunit is an essential cofactor in the canonical miRNA biogenesis which is involved in diverse cellular functions such as cell fate decisions, apoptosis and different signaling pathways. However, the role of DGCR8 in these processes or development of DGS is not fully understood. Here we present a heterozygous DGCR8 mutant human embryonic stem cell line (HuES9 DGCR8+/−) created by the CRISPR/Cas9 system. The generated HuES9 DGCR8+/− cells maintain normal karyotype, morphology, pluripotency and differentiation capacity into all three germ layers.
Human stem cells provide an important novel tool for generating in vitro pharmacological and toxi... more Human stem cells provide an important novel tool for generating in vitro pharmacological and toxicological test systems. In the development of new targeted therapies, as well as in critical safety issues, including hepato-, neuro- and cardio-toxicity, animal-based tests are mostly unsatisfactory, whereas the use of in vitro model systems is limited by the unavailability of relevant human tissues. Human embryonic stem cell lines may fill this gap and offer an advantage over primary cultures as well as tissue-derived (adult) stem cells. Human embryonic stem cells represent an unlimited source for the production of differentiated somatic progenies and allow various stable genetic manipulations. As a new opening in personalized medicine test systems, the generation of induced pluripotent stem cell lines and their derivatives can provide patient- and disease-specific cellular assays for drug development and safety assessments. This article reviews promising human stem cell applications i...
Stem cell-based gene therapy is often unsuccessful because of the relatively low number of geneti... more Stem cell-based gene therapy is often unsuccessful because of the relatively low number of genetically modified cells with repopulating capabilities. To provide a selective advantage for the modified cells we applied the human ABCG2 protein, a resident xenobiotic transporter in stem cells, as a selectable marker. This protein is active as a homodimer, and its relatively small cDNA is an advantage in gene therapy applications. In the present study a mutant form of ABCG2 (R482G), showing drug-pumping activity with an altered substrate specificity, was coexpressed with a therapeutic gene by using a bicistronic vector and an efficient retroviral transduction protocol. Expression of the gp91 phox protein in human gp91 phox knockout hematopoietic progenitor cells corrected the loss-of-function mutation responsible for human chronic granulomatous disease, whereas the mutant ABCG2 protein selectively protected the transduced cells against clinically applicable cytotoxic agents. Overexpression of ABCG2 did not affect hematopoietic cell maturation or the restoration of granulocyte function by gp91 phox. We suggest that the mutant ABCG2 protein is an ideal candidate for human stem cell protection and for use as a selectable marker in gene therapy.
In this study, we evaluated the suitability of the calcein assay as a routine clinical laboratory... more In this study, we evaluated the suitability of the calcein assay as a routine clinical laboratory method for the identification of multidrug-resistant phenotype in acute leukaemia. This study presents the results of the calcein tests obtained in two large haematological centres in Hungary. Assays were performed with blast cells of 93 de novo acute leukaemia patients, including 65 patients with acute myeloid leukaemia (AML). Results were expressed as multidrug resistance activity factor (MAF) values. AML patients were divided into responders and non-responders and MAF values were calculated for each group. In both centres, responder patients displayed significantly lower MAF values than non-responders (P 0´0045 and P 0´0454). Cutoff values were established between the MAF R 1 SEM and MAF NR 2 SEM values. On the basis of these cutoff levels, multidrug resistance (MDR) negativity showed a 72% predictive value for the response to chemotherapy, whereas MDR positivity was found to have an average predictive value of 69% for therapy failure. MDR activity was a prognostic factor for survival rate and the test was suitable for detecting patients at relapse. The calcein assay can be used as a quantitative, standardized, inexpensive screening test in a routine clinical laboratory setting. The assay detects both P-glycoprotein and multidrug resistance-associated protein activities, and identifies AML patients with unfavourable therapy responses.
ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ... more ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2-and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. V
This is an Open Access article distributed under the terms of the Creative Commons Attribution Li... more This is an Open Access article distributed under the terms of the Creative Commons Attribution License
Transforming growth factor-b (TGF-b) principally relays its effects through the Smad pathway howe... more Transforming growth factor-b (TGF-b) principally relays its effects through the Smad pathway however, accumulating evidence indicate that alternative signaling routes are also employed by this pleiotropic cytokine. For instance recently, we have demonstrated that ligand occupied TGF-b receptors can directly trigger the TRAF6-TAK1 signaling module, resulting in MAP kinase activation. Here we report identification of the adaptor molecule T TRAP as a novel component of this noncanonical TGF-b pathway. We show that the protein associates with TGF-b receptors and components of the TRAF6-TAK1 signaling module, resulting in differential regulation of TGF-b activated p38 and NF-kB responses. Modulation of cellular TTRAP level affects cell viability in the presence of TGF-b, suggesting that the protein is an important component of the TGF-b induced apoptotic process.
Single nucleotide polymorphism (SNP) analyses of the ABCG2 gene have revealed three nonsynonymous... more Single nucleotide polymorphism (SNP) analyses of the ABCG2 gene have revealed three nonsynonymous SNPs resulting in the amino acid changes at V12M, Q141K and D620N. To determine whether the SNPs have an effect on drug transport, human embryonic kidney cells (HEK-293) were stably transfected with full length ABCG2 coding wild-type or SNP variants of ABCG2. In 4-day cytotoxicity assays with mitoxantrone, topotecan, SN-38 or diflomotecan, cells transfected with wild-type R482 ABCG2 showed IC 50 values up to 1.2-fold to 5-fold higher than cells expressing comparable levels of Q141K ABCG2, suggesting that the Q141K SNP affects drug transport. FTC-inhibitable mitoxantrone efflux normalized to ABCG2 surface expression as assayed by the anti-ABCG2 antibody 5D3 was significantly lower in cells transfected with Q141K ABCG2 than in those transfected with wild-type R482 ABCG2 (P=0.0048). Values for V12M and D620N ABCG2 were comparable to those for wild-type R482 ABCG2. The vanadate-sensitive ATPase activity of ABCG2 was assayed in Sf9 insect cells infected with wild-type or SNP variants of ABCG2. Basal ATPase activity in cells transfected with Q141K ABCG2 was 1.8-fold lower than in cells transfected with wild-type ABCG2, but was comparable among cells expressing wild-type, V12M or D620N ABCG2. Confocal studies of ABCG2 localization revealed higher intracellular staining in the Q141K transfectants than in cells transfected with wild-type or V12M ABCG2. Decreased transport of Hoechst 33342 was observed in Sf9 cells expressing V12M ABCG2; however, this was not true in HEK-293 cells expressing V12M ABCG2. These results suggest that the Q141K SNP affects the transport efficiency of ABCG2 and may result in altered pharmacokinetics or drug-resistance profiles in clinical oncology.
Rationale: ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseud... more Rationale: ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseudoxanthoma elasticum and general arterial calcification of infancy. To elucidate the role of ABCC6 in cellular physiology and disease, it is crucial to establish the exact subcellular localization of the native ABCC6 protein. Objective: In a recent article in Circulation Research , ABCC6 was reported to localize to the mitochondria-associated membrane and not the plasma membrane. As the suggested mitochondrial localization is inconsistent with published data and the presumed role of ABCC6, we performed experiments to determine the cellular localization of ABCC6 in its physiological environment. Methods and Results: We performed immunofluorescent labeling of frozen mouse and human liver sections, as well as primary hepatocytes. We used several different antibodies recognizing human and mouse ABCC6. Our results unequivocally show that ABCC6 is in the basolateral membrane of hepatocytes and ...
Background Limited information is available on the effectiveness of the BBIBP-CorV (Sinopharm, Be... more Background Limited information is available on the effectiveness of the BBIBP-CorV (Sinopharm, Beijing CNBG) vaccine, especially in the elderly, despite the fact that it is approved in more than 50 countries. Methods RBD-specific antibody titres, as a rapidly available and highly predictive surrogate marker, were measured after two doses of the BBIBP-CorV vaccine in 450 subjects. Results were analyzed in a multivariable model accounting for age, sex and time since the administration of the second dose of the vaccine. Results Sex and time since the second dose had little association with the antibody titres. Age, however, was highly relevant: measurable antibody levels were present in about 90% of individuals below the age of 50, but antibody production after BBIBP-CorV vaccination was strongly reduced with increasing age. A large number of elderly subjects, reaching 25% at 60 years, and up to 50% at ages over 80, were found not to produce any protective antibody. Conclusions RBD-spe...
In inside-out hy$n red cell membrane vesicles /IOV/, in th52absence of Mg , the only calcium-indu... more In inside-out hy$n red cell membrane vesicles /IOV/, in th52absence of Mg , the only calcium-induced labelling by g P-ATP occurs in a 140-150 000 molecular weight protein fraction, representing the hydroxylamine-sensitive phosphorylated int?$mediate /EP/ of the calcium pump. In the presence of Mg calcium-induced phosphorylation is accelerated but several other membrane proteins are also phosphorylated through protein kinase action forming hydroxylamine-insensitive bonds. Addition of calmodulin accels+ates EP formation both in the absence and presence of Mg. Treatment of the membrane with SH-group reagents significantly reduces EP formation. Mild trypsin digestion of IOVS, stimulating active calcium transport, eliminates calmodulin action and decreases the steady-state level of %. In trypsin-digested IOVs the molecular weight of the P-1,abelled EP is shifted to lower values / 110-120 000 I We suggest that trypsin digestion cleaves off a 20-40 000 molecular weight calmodulin-binding regulatory subunit of the calcium pump molecule.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1982
In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the diva... more In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the divalent metal ions Mg z+, Mn 2+, Co z+, Ni 2+ and Fe 2+. This activation is based on the formation of Me2+-ATP complexes which can serve as energy-donor substrates for the calcium pump, and probably, satisfy the requirement for free Me z+ in this transport process. Higher Me 2+ concentrations inhibit calcium transport with various efficiencies. Mn 2÷ directly competes with Ca 2+ at the transport site, while other divalent metal ions investigated have no such effect. The formation of the hydroxylamine-sensitive phosphorylated intermediate (EP) of the red cell membrane calcium pump from [T-32p]ATP is induced by Ca 2+ while rapid dephosphorylation requires the presence of Mg 2+. At higher concentrations Mn 2+ and Ni 2+ inhibit predominantly the formation of EP, while Co 2+ and Fe 2+ block dephosphorylation. The possible sites and nature of the divalent metal interactions with the red cell calcium pump are discussed. Hydroxylamine-insensitive membrane phosphorylation in inside-out vesicles from IT-32 P]ATP is significantly stimulated by Mn 2 + and Co 2+, as compared to that produced by Mg 2+, Fe 2+ and Ni 2+. Part of this labelling is found in phospholipids, especially in phosphatidylinositol. The results presented for the metal dependency of protein and lipid phosphorylation in red cell membranes may help in the characterization of ATP consumptions directly related to the calcium pump and those involved in various regulatory processes.
The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distributi... more The multidrug transporter ABCB1 (MDR1, Pgp) plays an important role in the absorption, distribution, metabolism, and elimination of a wide range of pharmaceutical compounds. Functional investigation of the ABCB1 expression is also essential in many diseases, including drug-resistant cancer, inflammatory conditions, or Alzheimer disease. In this study, we examined the potential interaction of the ABCB1 multidrug transporter with a group of commercially available viability dyes that are generally considered not to penetrate into intact cells. Here, we demonstrate that the slow cellular accumulation of TO-PRO™-1 (TP1) or TO-PRO™-3 (TP3) was strongly inhibited by ABCB1-dependent dye extrusion. TP1/3 dye accumulation was not affected by the presence of ABCC1 or ABCG2, while this uptake was increased to the level in the ABCB1-negative cells by a specific P-glycoprotein inhibitor, Tariquidar. We suggest that TP compounds can be used as highly sensitive, selective, non-toxic, and stable dye...
DiGeorge Syndrome (DGS) Critical Region 8 (DGCR8) is a primary candidate gene in they DGS. The DG... more DiGeorge Syndrome (DGS) Critical Region 8 (DGCR8) is a primary candidate gene in they DGS. The DGCR8 microprocessor complex subunit is an essential cofactor in the canonical miRNA biogenesis which is involved in diverse cellular functions such as cell fate decisions, apoptosis and different signaling pathways. However, the role of DGCR8 in these processes or development of DGS is not fully understood. Here we present a heterozygous DGCR8 mutant human embryonic stem cell line (HuES9 DGCR8+/−) created by the CRISPR/Cas9 system. The generated HuES9 DGCR8+/− cells maintain normal karyotype, morphology, pluripotency and differentiation capacity into all three germ layers.
Human stem cells provide an important novel tool for generating in vitro pharmacological and toxi... more Human stem cells provide an important novel tool for generating in vitro pharmacological and toxicological test systems. In the development of new targeted therapies, as well as in critical safety issues, including hepato-, neuro- and cardio-toxicity, animal-based tests are mostly unsatisfactory, whereas the use of in vitro model systems is limited by the unavailability of relevant human tissues. Human embryonic stem cell lines may fill this gap and offer an advantage over primary cultures as well as tissue-derived (adult) stem cells. Human embryonic stem cells represent an unlimited source for the production of differentiated somatic progenies and allow various stable genetic manipulations. As a new opening in personalized medicine test systems, the generation of induced pluripotent stem cell lines and their derivatives can provide patient- and disease-specific cellular assays for drug development and safety assessments. This article reviews promising human stem cell applications i...
Stem cell-based gene therapy is often unsuccessful because of the relatively low number of geneti... more Stem cell-based gene therapy is often unsuccessful because of the relatively low number of genetically modified cells with repopulating capabilities. To provide a selective advantage for the modified cells we applied the human ABCG2 protein, a resident xenobiotic transporter in stem cells, as a selectable marker. This protein is active as a homodimer, and its relatively small cDNA is an advantage in gene therapy applications. In the present study a mutant form of ABCG2 (R482G), showing drug-pumping activity with an altered substrate specificity, was coexpressed with a therapeutic gene by using a bicistronic vector and an efficient retroviral transduction protocol. Expression of the gp91 phox protein in human gp91 phox knockout hematopoietic progenitor cells corrected the loss-of-function mutation responsible for human chronic granulomatous disease, whereas the mutant ABCG2 protein selectively protected the transduced cells against clinically applicable cytotoxic agents. Overexpression of ABCG2 did not affect hematopoietic cell maturation or the restoration of granulocyte function by gp91 phox. We suggest that the mutant ABCG2 protein is an ideal candidate for human stem cell protection and for use as a selectable marker in gene therapy.
In this study, we evaluated the suitability of the calcein assay as a routine clinical laboratory... more In this study, we evaluated the suitability of the calcein assay as a routine clinical laboratory method for the identification of multidrug-resistant phenotype in acute leukaemia. This study presents the results of the calcein tests obtained in two large haematological centres in Hungary. Assays were performed with blast cells of 93 de novo acute leukaemia patients, including 65 patients with acute myeloid leukaemia (AML). Results were expressed as multidrug resistance activity factor (MAF) values. AML patients were divided into responders and non-responders and MAF values were calculated for each group. In both centres, responder patients displayed significantly lower MAF values than non-responders (P 0´0045 and P 0´0454). Cutoff values were established between the MAF R 1 SEM and MAF NR 2 SEM values. On the basis of these cutoff levels, multidrug resistance (MDR) negativity showed a 72% predictive value for the response to chemotherapy, whereas MDR positivity was found to have an average predictive value of 69% for therapy failure. MDR activity was a prognostic factor for survival rate and the test was suitable for detecting patients at relapse. The calcein assay can be used as a quantitative, standardized, inexpensive screening test in a routine clinical laboratory setting. The assay detects both P-glycoprotein and multidrug resistance-associated protein activities, and identifies AML patients with unfavourable therapy responses.
ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ... more ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2-and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. V
This is an Open Access article distributed under the terms of the Creative Commons Attribution Li... more This is an Open Access article distributed under the terms of the Creative Commons Attribution License
Transforming growth factor-b (TGF-b) principally relays its effects through the Smad pathway howe... more Transforming growth factor-b (TGF-b) principally relays its effects through the Smad pathway however, accumulating evidence indicate that alternative signaling routes are also employed by this pleiotropic cytokine. For instance recently, we have demonstrated that ligand occupied TGF-b receptors can directly trigger the TRAF6-TAK1 signaling module, resulting in MAP kinase activation. Here we report identification of the adaptor molecule T TRAP as a novel component of this noncanonical TGF-b pathway. We show that the protein associates with TGF-b receptors and components of the TRAF6-TAK1 signaling module, resulting in differential regulation of TGF-b activated p38 and NF-kB responses. Modulation of cellular TTRAP level affects cell viability in the presence of TGF-b, suggesting that the protein is an important component of the TGF-b induced apoptotic process.
Single nucleotide polymorphism (SNP) analyses of the ABCG2 gene have revealed three nonsynonymous... more Single nucleotide polymorphism (SNP) analyses of the ABCG2 gene have revealed three nonsynonymous SNPs resulting in the amino acid changes at V12M, Q141K and D620N. To determine whether the SNPs have an effect on drug transport, human embryonic kidney cells (HEK-293) were stably transfected with full length ABCG2 coding wild-type or SNP variants of ABCG2. In 4-day cytotoxicity assays with mitoxantrone, topotecan, SN-38 or diflomotecan, cells transfected with wild-type R482 ABCG2 showed IC 50 values up to 1.2-fold to 5-fold higher than cells expressing comparable levels of Q141K ABCG2, suggesting that the Q141K SNP affects drug transport. FTC-inhibitable mitoxantrone efflux normalized to ABCG2 surface expression as assayed by the anti-ABCG2 antibody 5D3 was significantly lower in cells transfected with Q141K ABCG2 than in those transfected with wild-type R482 ABCG2 (P=0.0048). Values for V12M and D620N ABCG2 were comparable to those for wild-type R482 ABCG2. The vanadate-sensitive ATPase activity of ABCG2 was assayed in Sf9 insect cells infected with wild-type or SNP variants of ABCG2. Basal ATPase activity in cells transfected with Q141K ABCG2 was 1.8-fold lower than in cells transfected with wild-type ABCG2, but was comparable among cells expressing wild-type, V12M or D620N ABCG2. Confocal studies of ABCG2 localization revealed higher intracellular staining in the Q141K transfectants than in cells transfected with wild-type or V12M ABCG2. Decreased transport of Hoechst 33342 was observed in Sf9 cells expressing V12M ABCG2; however, this was not true in HEK-293 cells expressing V12M ABCG2. These results suggest that the Q141K SNP affects the transport efficiency of ABCG2 and may result in altered pharmacokinetics or drug-resistance profiles in clinical oncology.
Rationale: ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseud... more Rationale: ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseudoxanthoma elasticum and general arterial calcification of infancy. To elucidate the role of ABCC6 in cellular physiology and disease, it is crucial to establish the exact subcellular localization of the native ABCC6 protein. Objective: In a recent article in Circulation Research , ABCC6 was reported to localize to the mitochondria-associated membrane and not the plasma membrane. As the suggested mitochondrial localization is inconsistent with published data and the presumed role of ABCC6, we performed experiments to determine the cellular localization of ABCC6 in its physiological environment. Methods and Results: We performed immunofluorescent labeling of frozen mouse and human liver sections, as well as primary hepatocytes. We used several different antibodies recognizing human and mouse ABCC6. Our results unequivocally show that ABCC6 is in the basolateral membrane of hepatocytes and ...
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