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Peter M J Burgers - One of the best experts on this subject based on the ideXlab platform.
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atp utilization by yeast replication factor c iii the atp binding domains of rfc2 rfc3 and rfc4 are essential for dna recognition and clamp loading
Journal of Biological Chemistry, 2001Co-Authors: Sonja Gary L Schmidt, Xavier V Gomes, Peter M J BurgersAbstract:The conserved lysine in the Walker A motif of the ATP-binding domain encoded by the yeast RFC1, RFC2, RFC3, and RFC4 genes was mutated to glutamic acid. Complexes of replication factor C with a N-terminal truncation (Delta2-273) of the RFC1 subunit (RFC) containing a single mutant subunit were overproduced in Escherichia coli for biochemical analysis. All of the mutant RFC complexes were capable of interacting with PCNA. Complexes containing a RFC1-K359E mutation were similar to wild type in replication activity and ATPase activity; however, the mutant complex showed increased susceptibility to proteolysis. In contrast, complexes containing either a rfc2-K71E mutation or a rfc3-K59E mutation were severely impaired in ATPase and clamp loading activity. In addition to their defects in ATP hydrolysis, these complexes were defective for DNA binding. A mutant complex containing the rfc4-K55E mutation performed as well as a wild type complex in clamp loading, but only at very high ATP concentrations. Mutant RFC complexes containing rfc2-K71R or rfc3-K59R, carrying a conservative lysine --> arginine mutation, had much milder clamp loading defects that could be partially (rfc2-K71R) or completely (rfc3-K59R) suppressed at high ATP concentrations.
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atp utilization by yeast replication factor c iv rfc atp binding mutants show defects in dna replication dna repair and checkpoint regulation
Journal of Biological Chemistry, 2001Co-Authors: Sonja Gary L Schmidt, Angela L Pautz, Peter M J BurgersAbstract:Abstract Replication factor C is required to load proliferating cell nuclear antigen onto primer-template junctions, using the energy of ATP hydrolysis. Four of the five RFCgenes have consensus ATP-binding motifs. To determine the relative importance of these sites for proper DNA metabolism in the cell, the conserved lysine in the Walker A motif of RFC1,RFC2, RFC3, or RFC4 was mutated to either arginine or glutamic acid. Arginine mutations in allRFC genes tested permitted cell growth, although poor growth was observed for rfc2-K71R. A glutamic acid substitution resulted in lethality in RFC2 andRFC3 but not in RFC1 or RFC4. Most double mutants combining mutations in two RFC genes were inviable. Except for the RFC1-K359R andrfc4-K55E mutants, which were phenotypically similar to wild type in every assay, the mutants were sensitive to DNA-damaging agents. The rfc2-K71R and rfc4-K55R mutants show checkpoint defects, most likely in the intra-S phase checkpoint. Regulation of the damage-inducible RNR3 promoter was impaired in these mutants, and phosphorylation of Rad53p in response to DNA damage was specifically defective when cells were in S phase. No dramatic defects in telomere length regulation were detected in the mutants. These data demonstrate that the ATP binding function ofRFC2 is important for both DNA replication and checkpoint function and, for the first time, that RFC4 also plays a role in checkpoint regulation.
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atp utilization by yeast replication factor c
2001Co-Authors: Clamp Loading, Sonja Gary L Schmidt, Xavier V Gomes, Peter M J BurgersAbstract:The conserved lysine in the Walker A motif of the ATPbinding domain encoded by the yeast RFC1, RFC2, RFC3, and RFC4 genes was mutated to glutamic acid. Complexes of replication factor C with a N-terminal truncation (2–273) of the RFC1 subunit (RFC) containing a single mutant subunit were overproduced in Escherichia coli for biochemical analysis. All of the mutant RFC complexes were capable of interacting with PCNA. Complexes containing a RFC1-K359E mutation were similar to wild type in replication activity and ATPase activity; however, the mutant complex showed increased susceptibility to proteolysis. In contrast, complexes containing either a rfc2-K71E mutation or a rfc3-K59E mutation were severely impaired in ATPase and clamp loading activity. In addition to their defects in ATP hydrolysis, these complexes were defective for DNA binding. A mutant complex containing the rfc4-K55E mutation performed as well as a wild type complex in clamp loading, but only at very high ATP concentrations. Mutant RFC complexes containing rfc2-K71R or rfc3-K59R, carrying a conservative lysine 3 arginine mutation, had much milder clamp loading defects that could be partially (rfc2-K71R) or completely (rfc3-K59R) suppressed at high ATP concentrations. Replication factor C (RFC) 1,2 uses the energy of ATP hydrolysis to load PCNA onto a primer-template junction. Sequence comparison studies indicate that each of the five Rfc subunits has an ATP-binding domain (reviewed in Ref. 1). The ATPbinding motif present in each of these five subunits represents a structural domain that may also function in ATP binding and/or hydrolysis. The prototypic structure for this domain is the Escherichia coli subunit of the -complex (2, 3). The structure of is C-shaped with the Walker A and B motifs situated at the base near the hinge of the C. Although it has imperfect ATP-binding motifs and does not bind ATP, the protein has high sequence similarity to the subunit, the active ATPase of the -complex, and therefore the structures of these two proteins are expected to be similar. Sequence comparisons between the five RFC subunits and suggest that they may have a similar structure, at least in the base and hinge of the C. From this structure one can easily visualize how ATP binding could cause a conformational change in the entire protein. The top and base of the C clamp are only connected by a small hinge region, giving the overall structure some flexibility. ATP binding could result in an opening or closing of the C, which could be transmitted to the other subunits within the complex, resulting in an overall conformational change in RFC or the -complex (2). The studies in the previous paper (4) indicate that RFC can bind up to four molecules of ATP depending on the presence of
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overproduction in escherichia coli and characterization of yeast replication factor c lacking the ligase homology domain
Journal of Biological Chemistry, 2000Co-Authors: Xavier V Gomes, Sonja L Gary, Peter M J BurgersAbstract:Abstract Eukaryotic replication factor C (RF-C) is a heteropentameric complex that is required to load the replication clamp proliferating cell nuclear antigen onto primed DNA. Saccharomyces cerevisiae RF-C is encoded by the genes RFC1–RFC5. The RFC1 gene was cloned under control of the strong inducible bacteriophage T7 promoter, yet induction did not yield detectable RFC1p. However, a truncated form of RFC1 deleted for the coding region for amino acids 3–273, RFC1-ΔN, did allow overproduction. The other four RFC genes were cloned into the latter plasmid to yield a single plasmid that overproduced RF-C to moderate levels. Overproduction of the complex was further enhanced when the Escherichia coli argU gene encoding the rare arginine tRNA was also overproduced. The enzyme thus produced in E. coli was purified to homogeneity through three column steps, including a proliferating cell nuclear antigen affinity column. This enzyme, as well as the enzyme purified from yeast, is prone to aggregation and inactivation, and therefore, light scattering was used to determine conditions stabilizing the enzyme and preventing aggregation. Broad-range carrier ampholytes at about 0.05% were found to be most effective. In some assays, the RFC1-ΔN containing RF-C from E. coli showed an increased activity compared with the full-length enzyme from yeast, likely because the latter enzyme exhibits significant nonspecific binding to single-stranded DNA. Replacement of RFC1 byRFC1-ΔN in yeast shows essentially no phenotype with regard to DNA replication, damage susceptibility, telomere length maintenance, and intrachromosomal recombination.
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Overproduction in Escherichia coli and Characterization of Yeast Replication Factor C Lacking the Ligase Homology Domain
The Journal of biological chemistry, 2000Co-Authors: Xavier V Gomes, Sonja L Gary, Peter M J BurgersAbstract:Eukaryotic replication factor C (RF-C) is a heteropentameric complex that is required to load the replication clamp proliferating cell nuclear antigen onto primed DNA. Saccharomyces cerevisiae RF-C is encoded by the genes RFC1-RFC5. The RFC1 gene was cloned under control of the strong inducible bacteriophage T7 promoter, yet induction did not yield detectable RFC1p. However, a truncated form of RFC1 deleted for the coding region for amino acids 3-273, RFC1-DeltaN, did allow overproduction. The other four RFC genes were cloned into the latter plasmid to yield a single plasmid that overproduced RF-C to moderate levels. Overproduction of the complex was further enhanced when the Escherichia coli argU gene encoding the rare arginine tRNA was also overproduced. The enzyme thus produced in E. coli was purified to homogeneity through three column steps, including a proliferating cell nuclear antigen affinity column. This enzyme, as well as the enzyme purified from yeast, is prone to aggregation and inactivation, and therefore, light scattering was used to determine conditions stabilizing the enzyme and preventing aggregation. Broad-range carrier ampholytes at about 0.05% were found to be most effective. In some assays, the RFC1-DeltaN containing RF-C from E. coli showed an increased activity compared with the full-length enzyme from yeast, likely because the latter enzyme exhibits significant nonspecific binding to single-stranded DNA. Replacement of RFC1 by RFC1-DeltaN in yeast shows essentially no phenotype with regard to DNA replication, damage susceptibility, telomere length maintenance, and intrachromosomal recombination.
I. David Goldman - One of the best experts on this subject based on the ideXlab platform.
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Reduced folate carrier transports thiamine monophosphate: an alternative route for thiamine delivery into mammalian cells
American Journal of Physiology-cell Physiology, 2002Co-Authors: Rongbao Zhao, I. David GoldmanAbstract:Although the reduced folate carrier RFC1 and the thiamine transporters THTR-1 and THTR-2 share ∼40% of their identity in protein sequence, RFC1 does not transport thiamine and THTR-1 and THTR-2 do not transport folates. In the present study, we demonstrate that transport of thiamine monophosphate (TMP), an important thiamine metabolite present in plasma and cerebrospinal fluid, is mediated by RFC1 in L1210 murine leukemia cells. Transport of TMP was augmented by a factor of five in cells (R16) that overexpress RFC1 and was markedly inhibited by methotrexate, an RFC1 substrate, but not by thiamine. At a near-physiological concentration (50 nM), TMP influx mediated by RFC1 in wild-type L1210 cells was ∼50% of thiamine influx mediated by thiamine transporter(s). Within 1 min, the majority of TMP transported into R16 cells was hydrolyzed to thiamine with a component metabolized to thiamine pyrophosphate, the active enzyme cofactor. These data suggest that RFC1 may be one of the alternative transport routes available for TMP in some tissues when THTR-1 is mutated in the autosomal recessive disorder thiamine-responsive megaloblastic anemia.
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Role of the C-terminus and the long cytoplasmic loop in reduced folate carrier expression and function.
Biochemical pharmacology, 2002Co-Authors: Iraida G. Sharina, Rongbao Zhao, Yanhua Wang, Solomon Babani, I. David GoldmanAbstract:Abstract The reduced folate carrier (RFC1), a member of the major facilitative superfamily, generates uphill transport of folates into cells through an exchange mechanism with intracellular organic anions. RFC1 has twelve transmembrane domains with N- and C-termini, and the long loop connecting the 6th and 7th transmembrane domains, directed to the cytoplasm. To elucidate the role of the C-terminus and the long cytoplasmic loop in carrier function, mutants with deletion of the entire C-terminus or with progressive deletions of the loop region were constructed and stably transfected into the murine MTX r A cell line, which lacks functional RFC1. While expression of the C-terminus-deleted RFC1 protein could not be detected in the cell lysate, the RFC1 mutant lacking 57 of 66 amino acid residues of the long cytoplasmic loop appeared to be inserted into the cytoplasmic membrane but was not functional. In cell lines in which 17 or 31 amino acids were deleted from the carboxyl half of the loop, there was partial preservation of methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate transport. The loss of 5-formyltetrahydrofolate transport activity in the Δ31 and Δ17 mutants was due primarily to a decrease in substrate binding to the carrier. Mutants with partially truncated internal loops demonstrated an anion responsiveness similar to that of wild-type RFC1, indicating that this region of the carrier does not contain a site(s) that plays a role in anion exchange. This is the first study to describe the important role of the long cytoplasmic loop in substrate binding and the crucial role of the C-terminus in maintaining stability of RFC1.
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Localization of the murine reduced folate carrier as assessed by immunohistochemical analysis.
Biochimica et biophysica acta, 2001Co-Authors: Yanhua Wang, Rongbao Zhao, Robert G. Russell, I. David GoldmanAbstract:The reduced folate carrier (RFC1) is a major route for the transport of folates in mammalian cells. The localization of RFC1 in murine tissues was evaluated by immunohistochemical analysis using a polyclonal antibody to the C-terminus of the carrier. There was expression of RFC1 in the brush-border membrane of the jejunum, ileum, duodenum and colon. RFC1 was localized to the basolateral membrane of the renal tubular epithelium. Carrier was detected on the plasma membrane of hepatocytes but not in bile duct epithelial cells. In the choroid plexus RFC1 was highly expressed at the apical surface. It was also expressed in axons and dendrites and on the apical membrane of cells lining the spinal canal. In spleen, RFC1 was detected only in the cells of the red pulp. These data provide insights into the role that RFC1 plays in folate delivery in a variety of tissues. In particular, the localization of carrier may elucidate the role of RFC1 in the vectorial transport of folates across epithelia. The data also indicate that in kidney tubules and choroid plexus the sites of RFC1 expression are different from what has been reported previously for the folate receptor; and while RFC1 is expressed in small intestine, folate receptor is not.
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Mutational analysis of the functional role of conserved arginine and lysine residues in transmembrane domains of the murine reduced folate carrier.
Molecular pharmacology, 2001Co-Authors: Iraida G. Sharina, Rongbao Zhao, Yanhua Wang, Solomon Babani, I. David GoldmanAbstract:The reduced folate carrier (RFC1) plays a major role in the delivery of folates into mammalian cells. RFC1 is an anion exchanger with seven conserved positively charged amino acid residues within 12 predicted transmembrane domains. This article explores the role of these residues in transport function by the development of cell lines in which arginines and lysines in RFC1 were replaced with leucine by site-directed mutagenesis. Three cell lines transfected with R131L, R155L, or R366L all lacked activity, despite high levels of protein expression in the plasma membrane, suggesting the crucial role of these amino acid residues in RFC1 function. In several mutant carriers, R26L, R42L, and K332L, there was little or no change in the influx K t value for MTX or influx K i value for folic acid. However, the R26L, R42L, and K332L carriers had decreased affinity for reduced folates. This was most prominent for K404L, which had 11- and 4-fold increases in influx K i for 5-methyl-THF and 5-formyl-THF, respectively, compared with L1210 cells. The marked influx stimulation observed with wild-type carrier when extracellular chloride was decreased was significantly diminished when influx was mediated by the K404L carrier, but was only slightly decreased with the R26L, R42L, and K332L mutants. This suggested that the K404 residue may be a major site of inhibition by chloride in the wild-type carrier. These studies indicate the important role that some positively charged residues within transmembrane domains of RFC1 play in RFC1 function.
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Rescue of embryonic lethality in reduced folate carrier-deficient mice by maternal folic acid supplementation reveals early neonatal failure of hematopoietic organs.
The Journal of biological chemistry, 2001Co-Authors: Rongbao Zhao, Yanhua Wang, Robert G. Russell, Feng Gao, Laibin Liu, Burkhard Kneitz, Winfried Edelmann, I. David GoldmanAbstract:Abstract The reduced folate carrier (RFC1) is an important route by which the major blood folate, 5-methyltetrahydrofolate, is transported into mammalian cells. In this study we determined the consequences of inactivation of RFC1 in mice by homologous recombination. While RFC1-null embryos died in utero before embryonic day 9.5 (E9.5), near-normal development could be sustained in RFC1 − /−embryos examined at E18.5 by supplementation of pregnantRFC1 +/− dams with 1-mg daily subcutaneous doses of folic acid. About 10% of these animals went on to live birth but died within 12 days. TheseRFC1 − /− mice showed a marked absence of erythropoiesis in bone marrow, spleen, and liver along with lymphoid depletion in the splenic white pulp and thymus. In addition, there was some impairment of renal and seminiferous tubule development. These data indicate that in the absence of RFC1 function, neonatal animals die due to failure of hematopoietic organs.
Fatemeh Aghakhani Moghadam - One of the best experts on this subject based on the ideXlab platform.
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the study of association between reduced folate carrier 1 RFC1 polymorphism and non syndromic cleft lip palate in iranian population
BioImpacts : BI, 2017Co-Authors: Behnoosh Soghani, Asghar Ebadifar, Hamid Reza Khorram Khorshid, Koorosh Kamali, Roya Hamedi, Fatemeh Aghakhani MoghadamAbstract:Introduction: Cleft lip/palate is one of the most common congenital defects and is supposed to have multifactorial etiology, including a complex interaction between genetics and environment. Reduced folate carrier 1 (RFC1) gene takes part in folate transportation within the cells. In this study, the association of A80G polymorphism in the RFC1 gene with the non-syndromic cleft lip/palate (nsCL/P) was investigated in Iranian infants for the first time. Methods: In this case-control survey, 122 Iranian infants with nsCL/P and 164 healthy infants were investigated for RFC1 polymorphism by PCR and RFLP methods. The results were statistically compared with control group, odds ratios with 95% CI were estimated by univariate and multivariate logistic regression model and a P <0.05 was considered statistically significant. Results: The RFC1 G allele was significantly higher (P=0.001; OR=7, 95% CI: 4.7-10.2) in the cases (60.3%) compared with the controls (17.9%). Not only the RFC1 AG genotype was significantly higher (P<0.001; OR=44, 95% CI: 14.6-133) in cases (67.8%) than the controls (27.4%), but also GG genotype (P<0.001; OR=85, 95% CI: 20.5-352) was much higher in cases (26.4%) than the controls (4.3%). Conclusion: Our study indicated that the RFC1 (A80G) polymorphism was associated with the nsCL/P in Iranian population. Moreover, 80GG homozygosity was significant in the cases. The presence of G allele can be considered as a risk factor for the nsCL/P. Infants with the GG and AG genotypes were more prone to cleft lip/palate as compared to the AA ones. This finding emphasizes the role of RFC1 gene and the intracellular levels of folate.
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The study of association between reduced folate carrier 1 (RFC1) polymorphism and non-syndromic cleft lip/palate in Iranian population.
BioImpacts : BI, 2017Co-Authors: Behnoosh Soghani, Asghar Ebadifar, Hamid Reza Khorram Khorshid, Koorosh Kamali, Roya Hamedi, Fatemeh Aghakhani MoghadamAbstract:Introduction: Cleft lip/palate is one of the most common congenital defects and is supposed to have multifactorial etiology, including a complex interaction between genetics and environment. Reduced folate carrier 1 (RFC1) gene takes part in folate transportation within the cells. In this study, the association of A80G polymorphism in the RFC1 gene with the non-syndromic cleft lip/palate (nsCL/P) was investigated in Iranian infants for the first time. Methods: In this case-control survey, 122 Iranian infants with nsCL/P and 164 healthy infants were investigated for RFC1 polymorphism by PCR and RFLP methods. The results were statistically compared with control group, odds ratios with 95% CI were estimated by univariate and multivariate logistic regression model and a P
Rongbao Zhao - One of the best experts on this subject based on the ideXlab platform.
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Reduced folate carrier transports thiamine monophosphate: an alternative route for thiamine delivery into mammalian cells
American Journal of Physiology-cell Physiology, 2002Co-Authors: Rongbao Zhao, I. David GoldmanAbstract:Although the reduced folate carrier RFC1 and the thiamine transporters THTR-1 and THTR-2 share ∼40% of their identity in protein sequence, RFC1 does not transport thiamine and THTR-1 and THTR-2 do not transport folates. In the present study, we demonstrate that transport of thiamine monophosphate (TMP), an important thiamine metabolite present in plasma and cerebrospinal fluid, is mediated by RFC1 in L1210 murine leukemia cells. Transport of TMP was augmented by a factor of five in cells (R16) that overexpress RFC1 and was markedly inhibited by methotrexate, an RFC1 substrate, but not by thiamine. At a near-physiological concentration (50 nM), TMP influx mediated by RFC1 in wild-type L1210 cells was ∼50% of thiamine influx mediated by thiamine transporter(s). Within 1 min, the majority of TMP transported into R16 cells was hydrolyzed to thiamine with a component metabolized to thiamine pyrophosphate, the active enzyme cofactor. These data suggest that RFC1 may be one of the alternative transport routes available for TMP in some tissues when THTR-1 is mutated in the autosomal recessive disorder thiamine-responsive megaloblastic anemia.
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Role of the C-terminus and the long cytoplasmic loop in reduced folate carrier expression and function.
Biochemical pharmacology, 2002Co-Authors: Iraida G. Sharina, Rongbao Zhao, Yanhua Wang, Solomon Babani, I. David GoldmanAbstract:Abstract The reduced folate carrier (RFC1), a member of the major facilitative superfamily, generates uphill transport of folates into cells through an exchange mechanism with intracellular organic anions. RFC1 has twelve transmembrane domains with N- and C-termini, and the long loop connecting the 6th and 7th transmembrane domains, directed to the cytoplasm. To elucidate the role of the C-terminus and the long cytoplasmic loop in carrier function, mutants with deletion of the entire C-terminus or with progressive deletions of the loop region were constructed and stably transfected into the murine MTX r A cell line, which lacks functional RFC1. While expression of the C-terminus-deleted RFC1 protein could not be detected in the cell lysate, the RFC1 mutant lacking 57 of 66 amino acid residues of the long cytoplasmic loop appeared to be inserted into the cytoplasmic membrane but was not functional. In cell lines in which 17 or 31 amino acids were deleted from the carboxyl half of the loop, there was partial preservation of methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate transport. The loss of 5-formyltetrahydrofolate transport activity in the Δ31 and Δ17 mutants was due primarily to a decrease in substrate binding to the carrier. Mutants with partially truncated internal loops demonstrated an anion responsiveness similar to that of wild-type RFC1, indicating that this region of the carrier does not contain a site(s) that plays a role in anion exchange. This is the first study to describe the important role of the long cytoplasmic loop in substrate binding and the crucial role of the C-terminus in maintaining stability of RFC1.
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Localization of the murine reduced folate carrier as assessed by immunohistochemical analysis.
Biochimica et biophysica acta, 2001Co-Authors: Yanhua Wang, Rongbao Zhao, Robert G. Russell, I. David GoldmanAbstract:The reduced folate carrier (RFC1) is a major route for the transport of folates in mammalian cells. The localization of RFC1 in murine tissues was evaluated by immunohistochemical analysis using a polyclonal antibody to the C-terminus of the carrier. There was expression of RFC1 in the brush-border membrane of the jejunum, ileum, duodenum and colon. RFC1 was localized to the basolateral membrane of the renal tubular epithelium. Carrier was detected on the plasma membrane of hepatocytes but not in bile duct epithelial cells. In the choroid plexus RFC1 was highly expressed at the apical surface. It was also expressed in axons and dendrites and on the apical membrane of cells lining the spinal canal. In spleen, RFC1 was detected only in the cells of the red pulp. These data provide insights into the role that RFC1 plays in folate delivery in a variety of tissues. In particular, the localization of carrier may elucidate the role of RFC1 in the vectorial transport of folates across epithelia. The data also indicate that in kidney tubules and choroid plexus the sites of RFC1 expression are different from what has been reported previously for the folate receptor; and while RFC1 is expressed in small intestine, folate receptor is not.
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Mutational analysis of the functional role of conserved arginine and lysine residues in transmembrane domains of the murine reduced folate carrier.
Molecular pharmacology, 2001Co-Authors: Iraida G. Sharina, Rongbao Zhao, Yanhua Wang, Solomon Babani, I. David GoldmanAbstract:The reduced folate carrier (RFC1) plays a major role in the delivery of folates into mammalian cells. RFC1 is an anion exchanger with seven conserved positively charged amino acid residues within 12 predicted transmembrane domains. This article explores the role of these residues in transport function by the development of cell lines in which arginines and lysines in RFC1 were replaced with leucine by site-directed mutagenesis. Three cell lines transfected with R131L, R155L, or R366L all lacked activity, despite high levels of protein expression in the plasma membrane, suggesting the crucial role of these amino acid residues in RFC1 function. In several mutant carriers, R26L, R42L, and K332L, there was little or no change in the influx K t value for MTX or influx K i value for folic acid. However, the R26L, R42L, and K332L carriers had decreased affinity for reduced folates. This was most prominent for K404L, which had 11- and 4-fold increases in influx K i for 5-methyl-THF and 5-formyl-THF, respectively, compared with L1210 cells. The marked influx stimulation observed with wild-type carrier when extracellular chloride was decreased was significantly diminished when influx was mediated by the K404L carrier, but was only slightly decreased with the R26L, R42L, and K332L mutants. This suggested that the K404 residue may be a major site of inhibition by chloride in the wild-type carrier. These studies indicate the important role that some positively charged residues within transmembrane domains of RFC1 play in RFC1 function.
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Rescue of embryonic lethality in reduced folate carrier-deficient mice by maternal folic acid supplementation reveals early neonatal failure of hematopoietic organs.
The Journal of biological chemistry, 2001Co-Authors: Rongbao Zhao, Yanhua Wang, Robert G. Russell, Feng Gao, Laibin Liu, Burkhard Kneitz, Winfried Edelmann, I. David GoldmanAbstract:Abstract The reduced folate carrier (RFC1) is an important route by which the major blood folate, 5-methyltetrahydrofolate, is transported into mammalian cells. In this study we determined the consequences of inactivation of RFC1 in mice by homologous recombination. While RFC1-null embryos died in utero before embryonic day 9.5 (E9.5), near-normal development could be sustained in RFC1 − /−embryos examined at E18.5 by supplementation of pregnantRFC1 +/− dams with 1-mg daily subcutaneous doses of folic acid. About 10% of these animals went on to live birth but died within 12 days. TheseRFC1 − /− mice showed a marked absence of erythropoiesis in bone marrow, spleen, and liver along with lymphoid depletion in the splenic white pulp and thymus. In addition, there was some impairment of renal and seminiferous tubule development. These data indicate that in the absence of RFC1 function, neonatal animals die due to failure of hematopoietic organs.
Kenneth H. Cowan - One of the best experts on this subject based on the ideXlab platform.
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reduced folate carrier gene RFC1 expression and anti folate resistance in transfected and non selected cell lines
International Journal of Cancer, 1997Co-Authors: Jeffrey A. Moscow, Tatiana Connolly, Timothy G. Myers, C. C. Cheng, Kenneth D. Paull, Kenneth H. CowanAbstract:Methotrexate transport deficiency due to decreased reduced folate carrier (RFC) activity has been observed in several cell lines selected for resistance to methotrexate (MTX). Since MTX resistance is multifactorial, however, it is difficult to quantify the relative importance of changes in RFC activity in selected cell lines and even more so to determine the relative contribution of naturally occurring RFC activity in the MTX sensitivity of non-selected cell lines. We examined the role of RFC in MTX resistance by studying a transport-deficient cell line transfected with the gene for human RFC, RFC1, and by correlating relative RFC1 expression with MTX and trimetrexate (TMTX) growth inhibition (GI50) in a panel of cell lines used in the NCI Anticancer Drug Screen. Clones of transport-deficient, MTX-resistant ZR-75-1 human breast cancer cells (MTXR ZR-75-1) transfected with RFC1 were 250-fold more sensitive to MTX and 300-fold more resistant to TMTX than control cell clones, showing that restoration of RFC activity has a significant impact on MTX and TMTX cytotoxicity. We also surveyed 40 of the 60 cell lines in the NCI drug screen panel for RFC1 RNA levels by a quantitative RT-PCR assay. RFC1 RNA levels varied over a range of 15-fold, with only 1 cell line found to be null in expression. Using data from the 6-day drug exposure assay, RFC1 correlated positively with MTX and negatively with TMTX cytotoxicity. As predicted by transfection studies, the calculated difference between MTX and TMTX potency was even more strongly correlated with RFC1 RNA levels of the cell lines. In addition, compounds in the NCI Anticancer Drug Screen database with cytotoxicity profiles which correlated with RFC1 RNA levels or with the calculated difference in MTX-TMTX potency were examined for MTX uptake inhibition and cytotoxicity in the RFC1-transfected MTXR ZR-75-1 cell line. Overall, our data demonstrate the importance of RFC1 in MTX resistance both as a transgene and as a constitutively expressed gene in non-selected cell lines. Int. J. Cancer 72:184–190, 1997. © 1997 Wiley-Liss Inc.
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Reduced folate carrier gene (RFC1) expression and anti‐folate resistance in transfected and non‐selected cell lines
International journal of cancer, 1997Co-Authors: Jeffrey A. Moscow, Tatiana Connolly, Timothy G. Myers, C. C. Cheng, Kenneth D. Paull, Kenneth H. CowanAbstract:Methotrexate transport deficiency due to decreased reduced folate carrier (RFC) activity has been observed in several cell lines selected for resistance to methotrexate (MTX). Since MTX resistance is multifactorial, however, it is difficult to quantify the relative importance of changes in RFC activity in selected cell lines and even more so to determine the relative contribution of naturally occurring RFC activity in the MTX sensitivity of non-selected cell lines. We examined the role of RFC in MTX resistance by studying a transport-deficient cell line transfected with the gene for human RFC, RFC1, and by correlating relative RFC1 expression with MTX and trimetrexate (TMTX) growth inhibition (GI50) in a panel of cell lines used in the NCI Anticancer Drug Screen. Clones of transport-deficient, MTX-resistant ZR-75-1 human breast cancer cells (MTXR ZR-75-1) transfected with RFC1 were 250-fold more sensitive to MTX and 300-fold more resistant to TMTX than control cell clones, showing that restoration of RFC activity has a significant impact on MTX and TMTX cytotoxicity. We also surveyed 40 of the 60 cell lines in the NCI drug screen panel for RFC1 RNA levels by a quantitative RT-PCR assay. RFC1 RNA levels varied over a range of 15-fold, with only 1 cell line found to be null in expression. Using data from the 6-day drug exposure assay, RFC1 correlated positively with MTX and negatively with TMTX cytotoxicity. As predicted by transfection studies, the calculated difference between MTX and TMTX potency was even more strongly correlated with RFC1 RNA levels of the cell lines. In addition, compounds in the NCI Anticancer Drug Screen database with cytotoxicity profiles which correlated with RFC1 RNA levels or with the calculated difference in MTX-TMTX potency were examined for MTX uptake inhibition and cytotoxicity in the RFC1-transfected MTXR ZR-75-1 cell line. Overall, our data demonstrate the importance of RFC1 in MTX resistance both as a transgene and as a constitutively expressed gene in non-selected cell lines. Int. J. Cancer 72:184–190, 1997. © 1997 Wiley-Liss Inc.
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Molecular mechanism of antifolate transport-deficiency in a methotrexate resistant MOLT-3 human leukemia cell line
Blood, 1997Co-Authors: Maokai Gong, James Yess, Tatiana Connolly, S. Percy Ivy, Takao Ohnuma, Kenneth H. Cowan, Jeffrey A. MoscowAbstract:Ohnuma et al reported a series of methotrexate-resistant MOLT-3 human T-cell acute lymphoblastic leukemia cell lines that showed decreasing methotrexate (MTX) uptake as the sublines acquired increasing MTX resistance ( Cancer Res 45:1815, 1985). The alteration of MTX uptake kinetics in these cells, the intermediately resistant MOLT-3/MTX 200 and the highly resistant MOLT-3/MTX 10,000 cell lines, was attributed to a change in Vmax for methotrexate transport, without an apparent change in affinity of the transporter for MTX. We studied these cell lines to determine whether alteration of transcription or translation of the recently isolated reduced folate carrier gene ( RFC1 ) was the cause of MTX transport deficiency in these cell lines. Reconstitution of RFC activity in MOLT-3/MTX 10,000 cells by transduction with a murine RFC retroviral vector reversed MTX resistance and trimetrexate sensitivity. Although RFC1 RNA levels were unchanged in the resistant cell lines, FACS analysis using a polyclonal anti-RFCl antibody showed no detectable RFCl protein in the MOLT-3/MTX 10,000 cells. Determination of the nucleotide sequence of RFC1 genes from MOLT-3/MTX 10,000 cells revealed that this cell line contained 3 RFC1 alleles: a wild-type allele, an allele containing the premature stop codon at codon 40 and a third allele containing another mutation, which resulted in a premature stop codon at codon 25. We examined the relative expression of these alleles by determining the nucleotide sequence of 24 RFC1 cDNA subclones from MOLT-3/MTX 10,000 cells and found that only one-third of these clones contained the wild-type sequence. Determination of the genomic sequence of RFC1 in MOLT-3/MTX 200 cells demonstrated that these cells were heterozygous for a mutation at codon 40, but were homozygous for the wild-type sequence at codon 25. Thus, the acquisition of MTX transport-deficiency in MOLT-3/MTX 10,000 cells results from inactivating mutations of RFC1 gene alleles.
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Isolation of a gene encoding a human reduced folate carrier (RFC1) and analysis of its expression in transport-deficient, methotrexate-resistant human breast cancer cells.
Cancer research, 1995Co-Authors: Jeffrey A. Moscow, Maokai Gong, Magdalene K. Sgagias, Katharine H. Dixon, Sarah L. Anzick, Paul S. Meltzer, Kenneth H. CowanAbstract:Abstract Our laboratory has previously reported the isolation of a murine cDNA which restores reduced folate carrier (RFC) activity and methotrexate (MTX) sensitivity to a MTX-resistant, transport-deficient human breast cancer cell line (MTXR ZR-75-1) (K. H. Dixon et al., J. Biol. Chem., 269: 17–20, 1994). Using this murine cDNA as a probe, we have isolated two homologous overlapping partial cDNAs from a human testis cDNA library. In addition, using human cDNA as a probe, we have isolated a 20-kb human genomic fragment which contains RFC coding regions. Analysis of the nucleotide sequence of these clones revealed that the human RFC gene, RFC1, is approximately 65% homologous to the murine and hamster genes. Using a human genomic P1 plasmid clone containing RFC1, we mapped the location of RFC1 by fluorescence in situ hybridization to the end of the long arm of chromosome 21 (21q22.2–q22.3). Fluorescence in situ hybridization analysis also showed that two copies of RFC1 were present in MTXR ZR-75-1 cells, and showed no evidence of rearrangement of this gene. Northern blot analysis of MTXR ZR-75-1 cells demonstrated a marked decrease in the level of the 3-kb RFC1 transcript relative to the parental cell line, and Western blot analysis using a polyclonal antibody raised against a peptide generated from the RFC1 sequence showed decreased expression of an approximately Mr 56,000 protein in MTXR ZR-75-1 cells. Finally, MTXR ZR-75-1 cells transfected with an RFC1 gene showed increased MTX uptake, which was more sensitive to competition by folinic acid than by folic acid. Therefore, decreased RFC1 expression appears to be the molecular mechanism of decreased MTX uptake in this MTX-resistant cell line.