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1.  Structure and Function of the Reduced Folate Carrier: A Paradigm of A Major Facilitator Superfamily Mammalian Nutrient Transporter 
Vitamins and hormones  2008;79:10.1016/S0083-6729(08)00405-6.
Folates are essential for life and folate deficiency contributes to a host of health problems including cardiovascular disease, fetal abnormalities, neurologic disorders, and cancer. Antifolates, represented by methotrexate, continue to occupy a unique niche among the modern day pharmacopoeia for cancer along with other pathologic conditions. This review focuses on the biology of the membrane transport system termed the “reduced folate carrier” or RFC with a particular emphasis on RFC structure and function. The ubiquitously expressed RFC is the major transporter for folates in mammalian cells and tissues. Loss of RFC expression or function portends potentially profound physiologic or developmental consequences. For chemotherapeutic antifolates used for cancer, loss of RFC expression or synthesis of mutant RFC protein with impaired function results in antifolate resistance due to incomplete inhibition of cellular enzyme targets and low levels of substrate for polyglutamate synthesis. The functional properties for RFC were first documented nearly 40 years ago in murine leukemia cells. Since 1994, when RFC was first cloned, tremendous advances in the molecular biology of RFC and biochemical approaches for studying the structure of polytopic membrane proteins have led to an increasingly detailed picture of the molecular structure of the carrier, including its membrane topology, its N-glycosylation, identification of functionally and structurally important domains and amino acids, and helix packing associations. Although no crystal structure for RFC is yet available, biochemical and molecular studies, combined with homology modeling, based on homologous bacterial Major Facilitator Superfamily transporters such as LacY, now permit the development of experimentally testable hypotheses designed to establish RFC structure and mechanism.
doi:10.1016/S0083-6729(08)00405-6
PMCID: PMC3806226  PMID: 18804694
reduced folate carrier; folate; antifolate; membrane transport
2.  Parameters for individualizing systemic therapy in non-small cell lung cancer 
Rational drug design based on molecular targets is starting to revolutionize cancer care. To maximize its potential for patients, a concomitant leveraging of molecular knowledge for selection of patients to future and current therapeutic options is paramount. The terms “individualized”, “personalized”, or “precision therapy” are currently used to describe these efforts. Here, we summarize current knowledge for selection of systemic targeted and cytotoxic therapy for patients with non-small-cell lung cancer. Based on this knowledge, we present a potential decision algorithm to best select patients for currently available therapies, which include the treatment options single-agent erlotinib or gefitinib, the ALK inhibitor crizotinib, double agent gemcitabine and platinum, double agent platinum and pemetrexed, and as a default option a taxane combined with a non-platinum drug, for instance a vinca alkaloid. The addition of bevacizumab to double-agent chemotherapy is also discussed. Currently available data on predictive biomarkers are largely based on subgroup or companion biomarker analyses of patient cohorts or clinical trials. Current and emerging markers must be incorporated prospectively into the design of clinical trials that test novel and established agents to better understand their clinical utility and to refine selection parameters and marker interactions. Future development will lead to increasing complexity in clinical decision making with substantial anticipated benefits to patients including increased therapeutic efficacy, reduced toxicity, and better quality of life.
doi:10.1016/j.drup.2010.10.001
PMCID: PMC3865930  PMID: 21051275
Lung cancer; ERCC1; RRM1; TS; EGFR; EML4-ALK; Crizotinib; Bevacizumab
3.  Valproic Acid Synergistically Enhances The Cytotoxicity of Clofarabine in Pediatric Acute Myeloid Leukemia Cells 
Pediatric blood & cancer  2012;59(7):1245-1251.
SUMMARY
Background
Acute myeloid leukemia (AML) remains a major therapeutic challenge in pediatric oncology even with intensified cytarabine (ara-C)-based chemotherapy. Therefore, new therapies are urgently needed to improve treatment outcome of this deadly disease. In this study, we evaluated antileukemic interactions between clofarabine (a second-generation purine nucleoside analog) and valproic acid (VPA, a FDA-approved agent for treating epilepsy in both children and adult and a histone deacetylase inhibitor), in pediatric AML.
Methodology
In vitro clofarabine and VPA cytotoxicities of the pediatric AML cell lines and diagnostic blasts were measured by using MTT assays. The effects of clofarabine and VPA on apoptosis and DNA double strand breaks (DSBs) were determined by flow cytometry analysis and Western blotting, respectively. Active form of Bax was measured by Western blotting post immunoprecipitation.
Results
We demonstrated synergistic antileukemic activities between clofarabine and VPA in both pediatric AML cell lines and diagnostic blasts sensitive to VPA. In contrast, antagonism between the two agents could be detected in AML cells resistant to VPA. Clofarabine and VPA cooperate in inducing DNA DSBs, accompanied by Bax activation and apoptosis in pediatric AML cells.
Conclusion
Our results document synergistic antileukemic activities of combined VPA and clofarabine in pediatric AML and suggest that this combination could be an alternative treatment option for the disease.
doi:10.1002/pbc.24152
PMCID: PMC3396758  PMID: 22488775
pediatric acute myeloid leukemia; clofarabine; valproic acid; histone deacetylase inhibitor; synergistic antileukemic interaction
4.  Implications of Plasma Protein Binding for Pharmacokinetics and Pharmacodynamics of the γ-Secretase Inhibitor RO4929097 
Purpose
Understanding of plasma protein binding will provide mechanistic insights into drug interactions or unusual pharmacokinetic properties. This study investigated RO4929097 binding in plasma and its implications for the pharmacokinetics and pharmacodynamics of this compound.
Experimental Design
RO4929097 binding to plasma proteins was determined using a validated equilibrium dialysis method. Pharmacokinetics of total and unbound RO4929097 was evaluated in eight patients with breast cancer receiving RO4929097 alone and in combination with the Hedgehog inhibitor GDC-0449. The impact of protein binding on RO4929097 pharmacodynamics was assessed using an in vitro Notch cellular assay.
Results
RO4929097 was extensively bound in human plasma, with the total binding constant of 1.0 × 106 and 1.8 × 104 L/mol for α1-acid glycoprotein (AAG) and albumin, respectively. GDC-0449 competitively inhibited RO4929097 binding to AAG. In patients, RO4929097 fraction unbound (Fu) exhibited large intra- and interindividual variability; GDC-0449 increased RO4929097 Fu by an average of 3.7-fold. Concomitant GDC-0449 significantly decreased total (but not unbound) RO4929097 exposure. RO4929097 Fu was strongly correlated with the total drug exposure. Binding to AAG abrogated RO4929097 in vitro Notch-inhibitory activity.
Conclusions
RO4929097 is highly bound in human plasma with high affinity to AAG. Changes in plasma protein binding caused by concomitant drug (e.g., GDC-0449) or disease states (e.g., ↑AAG level in cancer) can alter total (but not unbound) RO4929097 exposure. Unbound RO4929097 is pharmacologically active. Monitoring of unbound RO4929097 plasma concentration is recommended to avoid misleading conclusions on the basis of the total drug levels.
doi:10.1158/1078-0432.CCR-11-2684
PMCID: PMC3856649  PMID: 22351688
5.  THE ROLE OF THE PROTO-ONCOGENE ETS2 IN ACUTE MEGAKARYOCYTIC LEUKEMIA BIOLOGY AND THERAPY 
Leukemia  2007;22(3):10.1038/sj.leu.2405066.
Acute myeloid leukemia (AML) in Down syndrome (DS) children has several unique features including a predominance of the acute megakaryocytic leukemia (AMkL) phenotype, higher event-free survivals compared to non-DS children using cytosine arabinoside (ara-C)/anthracycline-based protocols, and a uniform presence of somatic mutations in the X-linked transcription factor gene, GATA1. Several chromosome 21-localized transcription factor oncogenes including ETS2 may contribute to the unique features of DS AMkL. ETS2 transcripts measured by real-time RT-PCR were 1.8- and 4.1-fold, respectively, higher in DS and non-DS megakaryoblasts than those in non-DS myeloblasts. In a doxycycline-inducible erythroleukemia cell line, K562pTet-on/ETS2, induction of ETS2 resulted in an erythroid to megakaryocytic phenotypic switch independent of GATA1 levels. Microarray analysis of doxycycline induced and uninduced cells revealed an upregulation by ETS2 of cytokines (e.g. interleukin 1 and CSF2) and transcription factors (e.g. TAL1), which are key regulators of megakaryocytic differentiation. In the K562pTet-on/ETS2 cells, ETS2 induction conferred differences in sensitivities to ara-C and daunorubicin, depending on GATA1 levels. These results suggest that ETS2 expression is linked to the biology of AMkL in both DS and non-DS children, and acts by regulating expression of hematopoietic lineage and transcription factor genes involved in erythropoiesis and megakaryopoiesis, and in chemotherapy sensitivities.
doi:10.1038/sj.leu.2405066
PMCID: PMC3809919  PMID: 18094719
Down syndrome; acute megakaryocytic leukemia; chromosome 21; ETS2; GATA1; chemotherapy sensitivity
6.  Therapeutic targeting malignant mesothelioma with a novel 6-substituted pyrrolo[2,3-D]pyrimidine thienoyl antifolate via its selective uptake by the proton-coupled folate transporter 
The 5-substituted pyrrolo[2,3-d]pyrimidine antifolate pemetrexed (Pmx) is an active agent for malignant pleural mesothelioma (MPM). Pmx is transported into MPM cells by the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). We tested the notion that a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate (compound 2) might be an effective treatment for MPM, reflecting its highly selective membrane transport by PCFT over RFC. Compound 2 selectively inhibited proliferation of a HeLa subline expressing exclusively PCFT (R1-11-PCFT4) over an isogenic subline expressing only RFC (R1-11-RFC6). By outgrowth, H2452 human MPM cells were highly sensitive to the inhibitory effects of compound 2. By colony-forming assays, following an intermittent (24 h) drug exposure, 2 was cytotoxic. Cytotoxic activity by 2 was due to potent inhibition of glycinamide ribonucleotide formyltransferase (GARFTase) in de novo purine biosynthesis, as confirmed by nucleoside protection and in situ GARFTase assays with [14C]glycine. Assays with [3H]compound 2 and R1-11-PCFT4 or R1-11-RFC6 cells directly confirmed selective membrane transport by PCFT over RFC. PCFT transport was also confirmed for H2452 cells. In R1-11-PCFT4 and H2452 cells, [3H]compound 2 was metabolized to polyglutamates. Potent in vivo efficacy was confirmed toward early- and upstage H2452 xenografts in severe combined immunodeficient mice administered intravenous compound 2. Our results demonstrate potent antitumor efficacy of compound 2 toward H2452 MPM in vitro and in vivo, reflecting its efficient membrane transport by PCFT over RFC, synthesis of polyglutamates, and inhibition of GARFTase. Selectivity for non-RFC cellular uptake processes by novel tumor-targeted antifolates such as compound 2 presents an exciting new opportunity for treating solid tumors.
doi:10.1007/s00280-013-2094-0
PMCID: PMC3769948  PMID: 23412628
proton-coupled folate transporter; mesothelioma; folate; antifolate; pemetrexed
7.  Membrane Transporters and Folate Homeostasis; Intestinal Absorption, Transport into Systemic Compartments and Tissues 
Folates, the generic term for the family of B vitamins, are derived entirely from dietary sources, and are key one-carbon donors required for de novo nucleotide and methionine synthesis. These highly hydrophilic molecules utilize genetically distinct and functionally diverse transport systems to enter cells: the reduced folate carrier (RFC), the proton-coupled folate transporter (PCFT), and the folate receptors. Each plays a unique role in mediating folate transport across epithelia and into systemic tissues. With the recent discovery of the mechanism of intestinal folate absorption, and the clarification of the genetic basis for the autosomal recessive disorder, hereditary folate malabsorption, involving loss-of-function mutations in PCFT protein, it is now possible to piece together how these folate transporters contribute, both individually and collectively, to folate homeostasis in humans. This review focuses on the physiological roles of these major folate transporters with a brief consideration of their impact on the pharmacological activities of antifolates.
doi:10.1017/S1462399409000969
PMCID: PMC3770294  PMID: 19173758
8.  IDENTIFICATION AND CHARACTERIZATION OF NOVEL AML1-ETO FUSION TRANSCRIPTS IN PEDIATRIC t(8;21) ACUTE MYELOID LEUKEMIA: A REPORT FROM THE CHILDREN’S ONCOLOGY GROUP 
Oncogene  2008;27(36):4933-4942.
t(8;21)(q22;q22) results in the AML1-ETO (A1E) fusion gene and is a common cytogenetic abnormality in acute myeloid leukemia (AML). Although insertions at the breakpoint region of the A1E fusion transcripts have been reported, additional structural alterations are largely uncharacterized. By RT-PCR amplifications and DNA sequencing, numerous in-frame and out-of-frame AML1b-ETO and AML1c-ETO transcripts were identified in 13 pediatric t(8;21) AMLs, likely resulting from alternate splicing, internal deletions, and/or breakpoint region insertions involving both the AML1 (RUNX1) and ETO regions. The in-frame A1E fusion transcript forms represented minor forms. These structure alterations were found in AML1c-ETO but not AML1b-ETO transcripts in 2 adult t(8;21) AMLs. Although no analogous alterations were detected in native AML1b transcripts, identical alterations in native ETO transcripts were identified. When transfected into HeLa cells, only AML1b, and not the in-frame A1E forms, transactivated the GM-CSF promoter. In co-transfection experiments, the effects of A1E proteins on GM-CSF transactivation by AML1b ranged from repressive to activating. Our results demonstrate a remarkable and unprecedented heterogeneity in A1E fusion transcripts in t(8;21) myeloblasts and suggest that synthesis of alternate A1E transcript and protein forms can significantly impact the regulation of AML1 responsive genes.
doi:10.1038/onc.2008.134
PMCID: PMC3763903  PMID: 18469864
t(8;21); AML1-ETO; acute myeloid leukemia; fusion transcripts
9.  EFFECTS OF 5’ UNTRANSLATED REGION DIVERSITY ON THE POSTTRANSCRIPTIONAL REGULATION OF THE HUMAN REDUCED FOLATE CARRIER 
Biochimica et biophysica acta  2007;1769(2):131-138.
The human RFC (hRFC) gene is regulated by five major 5’ non-coding exons, characterized by alternate transcription start sites and splice forms. The result is up to 14 hRFC transcripts for which different 5’ untranslated regions (UTRs) are fused to a common coding sequence. By in vitro translation assays with hRFC constructs corresponding to the major transcript forms, most of the forms were translated poorly. Upon expression of the 5’UTR-hRFC constructs in hRFC-null HeLa cells, a range of steady state hRFC proteins and transcripts were detected that reflected relative transcript stabilities and, to a lesser extent, translation efficiencies. Transcripts including 5’ UTRs derived from non-coding exon A encoded a modified hRFC protein translated from an upstream initiation site. When this modified hRFC protein was expressed in hRFC-null K562 cells, there were only minor differences in surface targeting, stability, or transport function from wild type hRFC. Our results demonstrate an important role for posttranscriptional determinants of cellular hRFC levels and activity.
doi:10.1016/j.bbaexp.2006.12.006
PMCID: PMC1963461  PMID: 17306382
10.  Overexpression of GATA1 Confers Resistance to Chemotherapy in Acute Megakaryocytic Leukemia 
PLoS ONE  2013;8(7):e68601.
It has been previously shown that acute myeloid leukemia (AML) patients with higher levels of GATA1 expression have poorer outcomes. Furthermore, pediatric Down syndrome (DS) patients with acute megakaryocytic leukemia (AMKL), whose blast cells almost universally harbor somatic mutations in exon 2 of the transcription factor gene GATA1, demonstrate increased overall survival relative to non-DS pediatric patients, suggesting a potential role for GATA1 in chemotherapy response. In this study, we confirmed that amongst non-DS patients, GATA1 transcripts were significantly higher in AMKL blasts compared to blasts from other AML subgroups. Further, GATA1 transcript levels significantly correlated with transcript levels for the anti-apoptotic protein Bcl-xL in our patient cohort. ShRNA knockdown of GATA1 in the megakaryocytic cell line Meg-01 resulted in significantly increased cytarabine (ara-C) and daunorubicin anti-proliferative sensitivities and decreased Bcl-xL transcript and protein levels. Chromatin immunoprecipitation (ChIP) and reporter gene assays demonstrated that the Bcl-x gene (which transcribes the Bcl-xL transcripts) is a bona fide GATA1 target gene in AMKL cells. Treatment of the Meg-01 cells with the histone deacetylase inhibitor valproic acid resulted in down-regulation of both GATA1 and Bcl-xL and significantly enhanced ara-C sensitivity. Furthermore, additional GATA1 target genes were identified by oligonucleotide microarray and ChIP-on-Chip analyses. Our findings demonstrate a role for GATA1 in chemotherapy resistance in non-DS AMKL cells, and identified additional GATA1 target genes for future studies.
doi:10.1371/journal.pone.0068601
PMCID: PMC3707876  PMID: 23874683
11.  Synthesis and biological activity of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers as inhibitors of de novo purine biosynthesis with selectivity for cellular uptake by high affinity folate receptors and the proton-coupled folate transporter over the reduced folate carrier 
Journal of Medicinal Chemistry  2012;55(4):1758-1770.
We reported the selective transport of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl-for-benzoyl-substituted side chain and a 3- (3a) and 4-carbon (3b) bridge. Compound 3a was more potent than 3b against tumor cells; While 3b was completely selective for transport by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) over reduced folate carrier (RFC), 3a was not. To determine if decreasing the distance between the bicyclic scaffold and L-glutamate in 3b would preserve transport selectivity and potency against human tumor cells, 3b regioisomers with [1,3] (7 and 8) and [1,2] (4, 5 and 6) substitutions on the thienoyl ring, and with acetylenic insertions in the 4-atom bridge, were synthesized and evaluated. Compounds 7 and 8 were potent nanomolar inhibitors of KB and IGROV1 human tumor cells with complete selectivity for FRα and PCFT over RFC.
doi:10.1021/jm201688n
PMCID: PMC3288238  PMID: 22243528
12.  The human proton-coupled folate transporter 
Cancer Biology & Therapy  2012;13(14):1355-1373.
This review summarizes the biology of the proton-coupled folate transporter (PCFT). PCFT was identified in 2006 as the primary transporter for intestinal absorption of dietary folates, as mutations in PCFT are causal in hereditary folate malabsorption (HFM) syndrome. Since 2006, there have been major advances in understanding the mechanistic roles of critical amino acids and/or domains in the PCFT protein, many of which were identified as mutated in HFM patients, and in characterizing transcriptional control of the human PCFT gene. With the recognition that PCFT is abundantly expressed in human tumors and is active at pHs characterizing the tumor microenvironment, attention turned to exploiting PCFT for delivering novel cytotoxic antifolates for solid tumors. The finding that pemetrexed is an excellent PCFT substrate explains its demonstrated clinical efficacy for mesothelioma and non-small cell lung cancer, and prompted development of more PCFT-selective tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimidine antifolates that derive their cytotoxic effects by targeting de novo purine nucleotide biosynthesis.
doi:10.4161/cbt.22020
PMCID: PMC3542225  PMID: 22954694
folate; antifolate; transport; proton-coupled folate transporter; reduced folate carrier; tumor microenvironment
13.  Synthesis, biological and antitumor activity of a highly potent 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitor with proton-coupled folate transporter and folate receptor selectivity over the reduced folate carrier that inhibits β-glycinamide ribonucleotide formyltransferase 
Journal of medicinal chemistry  2011;54(20):7150-7164.
2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1–3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with L-glutamate diethyl ester, followed by saponification, afforded 1–3. Compound 3 selectively inhibited proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including human tumor cells KB and IGROV1 much more potently than 4. Compound 3 was more inhibitory than 4 toward β-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1, 2 and 4-atom bridge lengths for the activity of this series.
doi:10.1021/jm200739e
PMCID: PMC3209708  PMID: 21879757
14.  MECHANISMS OF SYNERGISTIC ANTILEUKEMIC INTERACTIONS BETWEEN VALPROIC ACID AND CYTARABINE IN PEDIATRIC ACUTE MYELOID LEUKEMIA 
Purpose
To determine the possibility of synergistic anti-leukemic activity and the underlying molecular mechanisms associated with cytarabine combined with valproic acid (VPA) [a histone deacetylase inhibitor (HDACI) and an FDA-licensed drug for treating both children and adults with epilepsy] in pediatric acute myeloid leukemia (AML).
Experimental Design
The type and extent of anti-leukemic interactions between cytarabine and VPA in clinically relevant pediatric AML cell lines and diagnostic blasts from children with AML were determined by MTT assays and standard isobologram analyses. The effects of cytarabine and VPA on apoptosis and cell cycle distributions were determined by flow cytometry analysis and caspase enzymatic assays. The effects of the two agents on DNA damage and Bcl-2 family proteins were determined by Western blotting.
Results
We demonstrated synergistic antileukemic activities between cytarabine and VPA in 4 pediatric AML cell lines and 9 diagnostic AML blast samples. t(8;21) AML blasts were significantly more sensitive to VPA and showed far greater sensitivities to combined cytarabine and VPA than non-t(8;21) AML cases. Cytarabine and VPA cooperatively induced DNA double strand breaks, reflected in induction of γH2AX and apoptosis, accompanied by activation of caspases 9 and 3. Further, VPA induced Bim expression and shRNA knockdown of Bim resulted in significantly decreased apoptosis induced by cytarabine, and by cytarabine plus VPA.
Conclusions
Our results establish global synergistic antileukemic activity of combined VPA and cytarabine in pediatric AML and provide compelling evidence to support the use of VPA in the treatment of children with this deadly disease.
doi:10.1158/1078-0432.CCR-10-1707
PMCID: PMC3018695  PMID: 20889917
15.  Synthesis and biological activity of a novel series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors and the proton-coupled folate transporter over the reduced folate carrier for cellular entry† 
Journal of medicinal chemistry  2010;53(3):1306-1318.
2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl side chain and 4-6 carbon bridge lengths (compounds 1-3) were synthesized as substrates for folate receptors (FRs) and the proton-coupled folate transporter (PCFT). Conversion of acetylene carboxylic acids to α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidines. Sonogashira coupling with (S)-2-[(5-bromo-thiophene-2-carbonyl)-amino]-pentanedioic acid diethyl ester, followed by hydrogenation and saponification, afforded 1-3. Compounds 1 and 2 potently inhibited KB and IGROV1 human tumor cells that express FRα, reduced folate carrier (RFC), and PCFT. The analogs were selective for FR- and PCFT over RFC. Glycinamide ribonucleotide formyltransferase was the principal cellular target. In SCID mice with KB tumors, 1 was highly active against both early (3.5 log kill, 1/5 cures) and advanced (3.7 log kill, 4/5 complete remissions) stage tumors. Our results demonstrate potent in vitro and in vivo antitumor activity for 1 due to selective transport by FRs and PCFT over RFC.
doi:10.1021/jm9015729
PMCID: PMC2836843  PMID: 20085328
16.  Substrate-specific binding and conformational changes involving Ser313 and transmembrane domain 8 of the human reduced folate carrier, as determined by site-directed mutagenesis and protein cross-linking 
Biochemical Journal  2010;430(Pt 2):265-274.
RFC (reduced folate carrier) is the major transporter for reduced folates and antifolates [e.g. MTX (methotrexate)]. RFC is characterized by two halves, each with six TMD (transmembrane domain) α helices connected by a hydrophilic loop, and cytoplasmic N- and C-termini. We previously identified TMDs 4, 5, 7, 8, 10 and 11 as forming the hydrophilic cavity for translocation of (anti)folates. The proximal end of TMD8 (positions 311–314) was implicated in substrate binding from scanning-cysteine accessibility methods; cysteine replacement of Ser313 resulted in loss of transport. In the present study, Ser313 was mutated to alanine, cysteine, phenylalanine and threonine. Mutant RFCs were expressed in RFC-null R5 HeLa cells. Replacement of Ser313 with cysteine or phenylalanine abolished MTX transport, whereas residual activity was preserved for the alanine and threonine mutants. In stable K562 transfectants, S313A and S313T RFCs showed substantially decreased Vmax values without changes in Kt values for MTX compared with wild-type RFC. S313A and S313T RFCs differentially impacted binding of ten diverse (anti)folate substrates. Cross-linking between TMD8 and TMD5 was studied by expressing cysteine-less TMD1–6 (N6) and TMD7–12 (C6) half-molecules with cysteine insertions spanning these helices in R5 cells, followed by treatment with thiol-reactive homobifunctional cross-linkers. C6–C6 and N6–N6 cross-links were seen for all cysteine pairs. From the N6 and C6 cysteine pairs, Cys175/Cys311 was cross-linked; cross-linking increased in the presence of transport substrates. The results of the present study indicate that the proximal end of TMD8 is juxtaposed to TMD5 and is conformationally active in the presence of transport substrates, and TMD8, including Ser313, probably contributes to the RFC substrate-binding domain.
doi:10.1042/BJ20100181
PMCID: PMC2947195  PMID: 20557288
antifolate; cross-linking; folate; major facilitator superfamily; mutagenesis; oligomer; reduced folate carrier; transporter; BMH, 1,6-bis(maleimido)hexane; C6, transmembrane domains 7–12; cl, cysteine-less; HA, haemagglutinin; MFS, major facilitator superfamily; MTSES, 2-sulfonatoethyl methanethiosulfonate; MTX, methotrexate; N6, transmembrane domains 1–6; p-PDM, p-phenylenedimaleimide; RFC, reduced folate carrier; hRFC, human RFC; TMD, transmembrane domain; wt, wild-type
17.  The impact of NOTCH1, FBW7 and PTEN mutations on prognosis and downstream signaling in pediatric T- cell acute lymphoblastic leukemia: A report from the Children's Oncology Group 
We explored the impact of mutations in the NOTCH1, FBW7 and PTEN genes on prognosis and downstream signaling in a well-defined cohort of 47 pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. In T-ALL lymphoblasts, we identified high frequency mutations in NOTCH1 (n=16), FBW7 (n=5) and PTEN (n=26). NOTCH1 mutations resulted in 1.3-3.3-fold increased transactivation of a HES1 reporter construct over wild-type NOTCH1; mutant FBW7 resulted in further augmentation of reporter gene activity. NOTCH1 and FBW7 mutations were accompanied by increased median transcripts for NOTCH1 target genes (HES1, DELTEX1, cMYC). However, none of these mutations were associated with treatment outcome. Elevated HES1, DELTEX1 and cMYC transcripts were associated with significant increases in transcript levels of several chemotherapy relevant genes, including MDR1, ABCC5, reduced folate carrier, asparagine synthetase, thiopurine methyltranserase, Bcl-2 and dihydrofolate reductase. PTEN transcripts positively correlated with HES1 and cMYC transcript levels. Our results suggest that (1) multiple factors should be considered with attempting to identify molecular-based prognostic factors for pediatric T-ALL, and (2) depending on the NOTCH1 signaling status, modifications in the types or dosing of standard chemotherapy drugs for T-ALL, or combinations of agents capable of targeting NOTCH1, AKT and/or mTOR with standard chemotherapy agents may be warranted.
doi:10.1038/leu.2009.64
PMCID: PMC2726275  PMID: 19340001
acute lymphoblastic leukemia; NOTCH1; FBW7; PTEN; HES1; DELTEX1; cMYC; chemotherapy; T-cell
18.  Synthesis and Discovery of High Affinity Folate Receptor-Specific Glycinamide Ribonucleotide Formyltransferase Inhibitors With Antitumor Activity 
Journal of medicinal chemistry  2008;51(16):5052-5063.
A series of 6-substituted classical pyrrolo[2,3-d]pyrimidine antifolates with a 3- to 6-carbon bridge between the heterocycle and the benzoyl-L-glutamate (compounds 2, 3, 4 and 5, respectively) was synthesized starting from methyl 4-formylbenzoate and a Wittig reaction with the appropriate triphenylphosphonium bromide, followed by reduction and conversion to the α-bromomethylketones. Cyclocondensation of 2,4-diamino-4-oxopyrimidine with the α-bromoketones, coupling with diethyl-L-glutamate and saponification afforded 2–5. Compounds 2–5 had negligible substrate activity for RFC but showed variably potent (nanomolar) and selective inhibitory activities toward Chinese hamster ovary cells that expressed FRα or FRβ, and toward FRα-expressing KB and IGROV1 human tumor cells. Inhibition of KB cell colony formation was also observed. Glycinamide ribonucleotide formyl transferase (GARFTase) was identified as the primary intracellular target of the pyrrolo[2,3-d]pyrimidines. The combined properties of selective FR targeting, lack of RFC transport, and GARFTase inhibition resulting in potent antitumor activity are unprecedented and warrant development of these analogs as antitumor agents.
doi:10.1021/jm8003366
PMCID: PMC2748117  PMID: 18680275
19.  Synthesis and biological activity of a novel series of 6-substituted thieno[2,3-d]pyrimidine antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors over the reduced folate carrier and proton-coupled folate transporter for cellular entry 
Journal of medicinal chemistry  2009;52(9):2940-2951.
A series of seven 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines, with bridge length variations (from 2-8 carbon atoms) were synthesized as selective folate receptor (FR) α and β substrates and as antitumor agents. The syntheses were accomplished from appropriate allylalcohols and 4-iodobenzoate to afford the aldehydes which were converted to the appropriate 2-amino-4-carbethoxy-5-substituted thiophenes 23-29. Cyclization with chlorformamidine afforded the thieno[2,3-d]pyrimidines 30-36 which were hydrolyzed and coupled with diethyl-L-glutamate, followed by saponification to give the target compounds 2-8. Compounds 3-6 were potent growth inhibitors (IC50 4.7 to 334 nM) of human tumor cells (KB and IGROV1) that express FRs. In addition, compounds 3-6 inhibited the growth of Chinese hamster ovary (CHO) cells that expressed FRs but not the reduced folate carrier (RFC) or proton-coupled folate transporter (PCFT). However, the compounds were inactive toward CHO cells that lacked FRs but contained either the RFC or PCFT. By nucleoside and 5-amino-4-imidazole carboxamide (AICA) protection studies, along with in vitro and in situ enzyme activity assays, the mechanism of antitumor activity was identified as the dual inhibition of glycinamide ribonucleotide formyltransferase and, likely, AICA ribonucleotide formyltransferase. The dual inhibitory activity of the active thieno[2,3-d]pyrimidine antifolates and the FR specificity represent unique mechanistic features for these compounds distinct from all other known antifolates. The potent inhibitory effects of compounds 3-6 toward cells expressing FRs but not PCFT provide direct evidence that cellular uptake of this series of compounds by FRs does not depend on the presence of PCFT and argues that direct coupling between these transporters is not obligatory.
doi:10.1021/jm8011323
PMCID: PMC2730022  PMID: 19371039
20.  A Humanized Mouse Model for the Reduced Folate Carrier 
The ubiquitously expressed reduced folate carrier (RFC) or SLC19A1 is recognized to be an essential transport system for folates in mammalian cells and tissues. In addition to its generalized role as a folate transporter, RFC provides specialized tissue functions including absorption across intestinal/colonic epithelia, transport across the basolateral membrane of renal proximal tubules, transplacental transport of folates, and folate transport across the blood-brain barrier. The human RFC (hRFC) gene is regulated by 5 major upstream non-coding regions (designated A1/A2, A, B, C, and D), each transcribed from a unique promoter. Altogether, at least 14 distinct hRFC transcripts can be envisaged in which different 5’ untranslated regions (UTRs) are fused to a common splice acceptor region (positions -1 to -49) within the first coding exon with a common 1776 bp coding sequence. The 5’ non-coding regions are characterized by alternate transcription start sites, multiple splice forms, and selective tissue distributions. Alternate 5’UTRs impact mRNA stabilities and translation efficiencies, and result in synthesis of modified hRFC proteins translated from upstream AUGs. In this report, we describe production and characterization of transgenic mice (TghRFC1) containing a functional hRFC gene and of humanized mice in which the mRFC gene is inactivated and an active hRFC gene has been introduced. The mice appear to be healthy and to breed well. Analysis of tissue specificity of expression in both the TghRFC1 and humanized hRFC mice by real-time RT-PCR demonstrates that the hRFC gene is expressed with a specificity closely resembling that seen in human tissues. For the humanized hRFC mice, levels of B and A1/A2 5’UTRs predominated in all mice/tissues, thus resembling results in normal human tissues. Lower levels of A and C 5’UTRs were also detected. The availability of humanized mouse models for hRFC will permit investigators to address critical unanswered questions pertinent to human health and disease. These include the ability to analyze the hRFC gene in vivo, to control dietary and other environmental conditions that may impact levels of gene expression, and to control the genetics of the mice in order to assess the effects of hRFC gene alterations on tissue folate uptake and distribution, none of which can be easily achieved in human populations.
doi:10.1016/j.ymgme.2007.09.014
PMCID: PMC2271060  PMID: 17983788
reduced folate carrier; mouse models; folate homeostasis; promoter usage
21.  Role of Rat Organic Anion Transporter 3 (Oat3) in the Renal Basolateral Transport of Glutathione 
Chemico-biological interactions  2007;170(2):124-134.
The tripeptide GSH is important in maintenance of renal redox status and defense against reactive electrophiles and oxidants. Previous studies showed that GSH is transported across the basolateral plasma membrane (BLM) into the renal proximal tubule by both sodium-coupled and sodium-independent pathways. Substrate specificity and inhibitor studies suggested the function of several carriers, including organic anion transporter 3 (Oat3). To test the hypothesis that rat Oat 3 can function in renal GSH transport, the cDNA for rat Oat3 was expressed as a His6-tagged protein in E. coli, purified from inclusion bodies and by Ni2+-affinity chromatography, and reconstituted into proteoliposomes. cDNA-expressed and reconstituted Oat3 transported both GSH and p-aminohippurate (PAH) in exchange for 2-oxoglutarate (2-OG) and 2-OG and PAH in exchange for GSH, and PAH uptake was inhibited by both probenecid and furosemide, consistent with function of Oat3. mRNA expression of Oat3 and several other potential carriers was detected by semiquantitative RT-PCR in rat kidney cortex but was absent from NRK-52E cells, a rat proximal tubular cell line. Basolateral uptake of GSH in NRK-52E cells showed little PAH- or 2-OG-stimulated uptake. We conclude that Oat3 can function in GSH uptake and that NRK-52E cells possess a low background rate of GSH uptake, making these cells a good model for overexpression of specific, putative GSH carriers.
doi:10.1016/j.cbi.2007.07.004
PMCID: PMC2080658  PMID: 17719021
Glutathione; Transport; Rat Kidney; Basolateral Plasma Membrane; Organic Anion Transporters; mRNA Expression
22.  Modulation of Expression of Rat Mitochondrial 2-Oxoglutarate Carrier in NRK-52E Cells Alters Mitochondrial Transport and Accumulation of Glutathione and Susceptibility to Chemically Induced Apoptosis 
We previously showed that two anion carriers of the mitochondrial inner membrane, the dicarboxylate carrier (DIC; Slc25a10) and oxoglutarate carrier (OGC; Slc25a11), transport glutathione (GSH) from cytoplasm into mitochondrial matrix. In the previous study, NRK-52E cells, derived from normal rat kidney proximal tubules, were transfected with the wild-type cDNA for the DIC expressed in rat kidney; DIC transfectants exhibited increased mitochondrial uptake and accumulation of GSH and were markedly protected from chemically induced apoptosis. In the present study, cDNAs for both wild-type (WT) and a double-cysteine mutant of rat OGC (rOGC and rOGC-C221,224S, respectively) were expressed in Escherichia coli, purified, and reconstituted into proteoliposomes to assess their function. Although both WT rOGC and rOGC-C221,224S exhibited transport properties for GSH and 2-oxoglutarate that were similar to those found in mitochondria of rat kidney proximal tubules, rates of transport and mitochondrial accumulation of substrates were reduced by > 75% in rOGC-C221,224S compared with the WT carrier. NRK-52E cells were stably transfected with the cDNA for WT-rOGC and exhibited 10- to 20-fold higher GSH transport activity than nontransfected cells and were markedly protected from apoptosis induced by tert-butyl hydroperoxide (tBH) or S-(1,2-dichlorovinyl)-l-cysteine (DCVC). In contrast, cells stably transfected with the cDNA for rOGC-C221,224S were not protected from tBH- or DCVC-induced apoptosis. These results provide further evidence that genetic manipulation of mitochondrial GSH transporter expression alters mitochondrial and cellular GSH status, resulting in markedly altered susceptibility to chemically induced apoptosis.
doi:10.1124/jpet.105.094599
PMCID: PMC1408317  PMID: 16291728
GSH, glutathione; DIC, dicarboxylate carrier; 2-OG, 2-oxoglutarate; OGC, oxoglutarate carrier; rDIC, rat dicarboxylate carrier; tBH, tert-butyl hydroperoxide; WT, wild-type; rOGC, rat 2-oxoglutarate carrier; DCVC, S-(1; 2-dichlorovinyl)-l-cysteine; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s); Mes, 4-morpholineethanesulfonic acid; TMD, transmembrane domain; PLP, pyridoxal 5′-phosphate; PhSucc, phenylsuccinate
23.  Role of Lysine 411 in Substrate Carboxyl Group Binding to the Human Reduced Folate Carrier, as Determined by Site-directed Mutagenesis and Affinity Inhibition 
Molecular pharmacology  2008;73(4):10.1124/mol.107.043190.
Reduced folate carrier (RFC) is the major membrane transporter for folates and antifolates in mammalian tissues. Recent studies used radioaffinity labeling with N-hydroxysuccinimide (NHS)-3H-methotrexate (MTX) to localize substrate binding to residues in transmembrane domain (TMD) 11 of human RFC. To identify the modified residue(s), seven nucleophilic residues in TMD11 were mutated to Val or Ala and mutant constructs expressed in RFC-null HeLa cells. Only Lys411Ala RFC was not inhibited by NHS-MTX. By radioaffinity labeling with NHS-3H-MTX, wild type (wt) RFC was labeled; for Lys411Ala RFC, radiolabeling was abolished. When Lys411 was replaced with Ala, Arg, Gln, Glu, Leu, and Met, only Lys411Glu RFC showed substantially decreased transport. Nine classical diamino furo[2,3-d]pyrimidine antifolates with unsubstituted α- and γ-carboxylates (1), hydrogen- or methyl-substituted α- (2, 3) or γ- (4, 5) carboxylates, or substitutions of both α- and γ-carboxylates (6, 7, 8, 9) were used to inhibit 3H-MTX transport with RFC-null K562 cells expressing wt and Lys411Ala RFCs. For wt and Lys411Ala RFCs, inhibitory potencies were in the order 4>5>1>3>2; 6-9 were poor inhibitors. Inhibitions decreased in the presence of physiologic anions. When NHS esters of 1, 2, and 4 were used to covalently modify wt RFC, inhibitory potencies were in the order 2>1>4; inhibition was abolished for Lys411Ala RFC. These results suggest that Lys411 participates in substrate binding via an ionic association with the substrate γ-carboxylate, however, this is not essential for transport. An unmodified α-carboxylate is required for high affinity substrate binding to RFC, whereas the γ-carboxyl is not essential.
doi:10.1124/mol.107.043190
PMCID: PMC3806200  PMID: 18182479
24.  A Unique Role of GATA1s in Down Syndrome Acute Megakaryocytic Leukemia Biology and Therapy 
PLoS ONE  2011;6(11):e27486.
Background
Acute megakaryocytic leukemia (AMkL) in Down syndrome (DS) children is uniformly associated with somatic GATA1 mutations, which result in the synthesis of a shorter protein (GATA1s) with altered transactivation activity compared to the wild-type GATA1. It is not fully established whether leukemogenesis and therapeutic responses in DS AMkL patients are due to loss of the wild-type GATA1 or due to a unique function of GATA1s.
Methodology
Stable clones of CMK cells with decreased GATA1s or Bcl-2 levels were generated by using GATA1- or BCL-2-specific lentivirus shRNAs. In vitro ara-C, daunorubicin, and VP-16 cytotoxicities of the shRNA stable clones were determined by using the Cell Titer-blue reagent. Apoptosis and cell cycle distribution were determined by flow cytometry analysis. Changes in gene transcript levels were determined by gene expression microarray and/or real-time RT-PCR. Changes in protein levels were measured by Western blotting. In vivo binding of GATA1s to IL1A promoter was determined by chromatin immunoprecipitation assays.
Results
Lentivirus shRNA knockdown of the GATA1 gene in the DS AMkL cell line, CMK (harbors a mutated GATA1 gene and only expresses GATA1s), resulting in lower GATA1s protein levels, promoted cell differentiation towards the megakaryocytic lineage and repressed cell proliferation. Increased basal apoptosis and sensitivities to ara-C, daunorubicin, and VP-16 accompanied by down-regulated Bcl-2 were also detected in the CMK GATA1 shRNA knockdown clones. Essentially the same results were obtained when Bcl-2 was knocked down with lentivirus shRNA in CMK cells. Besides Bcl-2, down-regulation of GATA1s also resulted in altered expression of genes (e.g., IL1A, PF4, and TUBB1) related to cell death, proliferation, and differentiation.
Conclusion
Our results suggest that GATA1s may facilitate leukemogenesis and potentially impact therapeutic responses in DS AMkL by promoting proliferation and survival, and by repressing megakaryocytic lineage differentiation, potentially by regulating expression of Bcl-2 protein and other relevant genes.
doi:10.1371/journal.pone.0027486
PMCID: PMC3217966  PMID: 22110660

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