David Plotnik - Academia.edu (original) (raw)

Papers by David Plotnik

A phase 1 study in healthy volunteers to investigate the safety, tolerability, and pharmacokinetics of VIR-2482: a monoclonal antibody for the prevention of severe influenza A illness

Antimicrobial agents and chemotherapy, Feb 20, 2024

medRxiv (Cold Spring Harbor Laboratory), Apr 4, 2024

Background: Influenza A results in significant morbidity and mortality. VIR-2482, an engineered h... more Background: Influenza A results in significant morbidity and mortality. VIR-2482, an engineered human monoclonal antibody with extended half-life, targets a highly conserved epitope on the stem region of influenza A hemagglutinin, and may protect against seasonal and pandemic influenza. : This double-blind, randomized, placebo-controlled, phase 2 study examined the safety and efficacy of VIR-2482 for seasonal influenza A illness prevention in unvaccinated healthy adults. Participants (N = 2977) were randomized 1:1:1 to receive VIR-2482 450 mg, VIR-2482 1200 mg, or placebo via intramuscular (IM) injection. Primary and secondary efficacy endpoints were the proportions of participants with reverse transcriptase-polymerase chain reaction (RT-PCR)-confirmed influenza A infection and either protocol-defined influenza-like illness (ILI) and Centers for Disease Control and Prevention (CDC)-defined ILI or World Health Organization (WHO)-defined ILI, respectively. Results: VIR-2482 450 mg and 1200 mg prophylaxis did not reduce the risk of protocol-defined ILI with RT-PCR-confirmed influenza A versus placebo (relative risk reduction [RRR], 3.8% [95% CI : -67.3, 44.6] and 15.9% [95% CI: -49.3, 52.3], respectively). At the 1200 mg dose, the RRRs in influenza A illness were 57.2% [95% CI: -2.5, 82.2] using CDC-ILI and 44.1% [95% CI: -50.5, 79.3] using WHO-ILI definitions, respectively. Serum VIR-2482 levels were similar regardless of influenza status; variants with reduced VIR-2482 susceptibility were not detected. Local injection-site reactions were mild and similar across groups. VIR-2482 1200 mg IM was well tolerated but did not significantly prevent protocol-defined ILI. Secondary endpoint analyses suggest this dose may have reduced influenza A illness. .

Aging Cell, 2012

Increasing age is the most robust predictor of greater malignancy and treatment resistance in hum... more Increasing age is the most robust predictor of greater malignancy and treatment resistance in human gliomas. However, the adverse association of clinical course with aging is rarely considered in animal glioma models, impeding delineation of the relative importance of organismal versus progenitor cell aging in the genesis of glioma malignancy. To address this limitation, we implanted transformed neural stem/progenitor cells (NSPCs), the presumed cells of glioma origin, from 3 and 18month old mice into 3 and 20-month host animals. Transplantation with progenitors from older animals resulted in significantly shorter (p ≤ 0.0001) median survival in both 3month (37.5 vs 83 days) and 20-month (38 vs 67 days) hosts, indicating that agedependent changes intrinsic to NSPCs rather than host animal age accounted for greater malignancy. Subsequent analyses revealed that increased invasiveness, genomic instability, resistance to therapeutic agents and tolerance to hypoxic stress accompanied aging in transformed NSPCs. Greater tolerance to hypoxia in older progenitor cells, as evidenced by elevated HIF-1 promoter reporter activity and hypoxia response gene (HRG) expression, mirror the upregulation of HRGs in cohorts of older vs younger glioma patients revealed by analysis of gene expression databases, suggesting that differential response to hypoxic stress may underlie age-dependent differences in invasion, genomic instability and treatment resistance. Our study provides strong evidence that progenitor cell aging is responsible for promoting the hallmarks of age-dependent glioma malignancy and that consideration of progenitor aging will facilitate development of physiologically and clinically relevant animal models of human gliomas.

Levels of human equilibrative nucleoside transporter-1 are higher in proliferating regions of A549 tumor cells grown as tumor xenografts in vivo

Nuclear Medicine and Biology, 2012

3&amp... more 3'-Fluoro-3'-deoxythymidine (FLT) has been proposed for positron emission tomography (PET)-based identification of tumor chemosensitivity that is mediated by the human equilibrative nucleoside transporter-1 (ENT1). ENT1 facilitates transport of FLT into cells and elevated levels of FLT are associated with both larger FLT-PET signals and increased response to nucleoside-based chemotherapies. FLT-PET is also used as a measure of tumor proliferation. The present study examined the extent to which ENT1 levels vary in a proliferation-dependent manner in tumor cells in vivo. The human adenocarcinoma cell line A549 was used to establish tumor xenografts in nude mice. FLT uptake was measured in vivo using PET, and further examined ex vivo using autoradiography. FLT uptake patterns were compared to immunohistochemical (IHC) analysis of ENT1 and the proliferation markers Ki67 and BrdU. Regional differences in FLT uptake matched differences in IHC proliferation markers. All cells stained for ENT1, but the staining intensity was twice as high for Ki67(+) cells than for Ki67(-) cells. Under in vivo conditions, proliferating regions of tumors show increased FLT uptake and higher ENT1 levels than nonproliferating tumor regions.

Transport of 3'-fluoro-3'-deoxythymidine (FLT) and its metabolites in the human lymphoblast cell line TK6

The Journal of Nuclear Medicine, 2011

The Role of CS1 Fibronectin in HIV-1 Infection of α4β7 + T Lymphocytes

Journal of Virology

Gut-homing α 4 β 7 high CD4 + T lymphocytes have been shown to be preferentially targeted by Huma... more Gut-homing α 4 β 7 high CD4 + T lymphocytes have been shown to be preferentially targeted by Human Immunodeficiency Virus-1 (HIV), and are implicated in HIV pathogenesis. Previous studies demonstrated that HIV envelope protein gp120 binds and signals through α 4 β 7 , and that this likely contributes to the infection of α 4 β 7 high T cells and promotes cell-to-cell virus transmission. Structures within the second variable loop (V2) of gp120, including the tripeptide motif LDV/I, are thought to mediate gp120-α 4 β 7 binding. However, lack of α 4 β 7 binding has been reported in gp120 proteins containing LDV/I, and the precise determinants of gp120-α 4 β 7 binding are not fully defined. In this work, we report the novel finding that fibronectins mediate indirect gp120-α 4 β 7 interactions. We show that Chinese Hamster Ovary (CHO) cells used to express recombinant gp120 produced fibronectins and other extracellular matrix proteins that co-purified with gp120. CHO fibronectins were abl...

Transport of 3'-fluoro-3'-deoxythymidine (FLT) and its metabolites in the human lymphoblast cell line TK6

Society of Nuclear Medicine Annual Meeting Abstracts, May 1, 2011

The effects of 5-fluorouracil treatment on 3'-fluoro-3'-deoxythymidine (FLT) transport and metabolism in proliferating and non-proliferating cultures of human tumor cells

Society of Nuclear Medicine Annual Meeting Abstracts, May 1, 2012

Nuclear medicine and biology, Jan 6, 2014

The use of thymidine (TdR) and thymidine analogs such as 3'-fluoro-3'-deoxythymidine (FLT... more The use of thymidine (TdR) and thymidine analogs such as 3'-fluoro-3'-deoxythymidine (FLT) as positron emission tomography (PET)-based proliferation markers can provide information on tumor response to treatment. Studies on another TdR analog, 4'-thiothymidine (4DST), suggest that it might be a better PET-based proliferation tracer than either TdR or FLT. 4DST is resistant to the catabolism that complicates analysis of TdR in PET studies, but unlike FLT, 4DST is incorporated into DNA. To further evaluate 4DST, the kinetics of 4DST transport and metabolism were determined and compared to FLT and TdR. Transport and metabolism of FLT, TdR and 4DST were examined in the human adenocarcinoma cell line A549 under exponential-growth conditions. Single cell suspensions were incubated in buffer supplemented with radiolabeled tracer in the presence or absence of nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of equilibrative nucleoside transporters (ENT). Kinetics o...

The effects of 5-fluoruracil treatment on 3′-fluoro-3′-deoxythymidine (FLT) transport and metabolism in proliferating and non-proliferating cultures of human tumor cells

Nuclear Medicine and Biology, 2012

3&amp... more 3'-Fluoro-3'-deoxythymidine (FLT) positron emission tomography (PET) has been proposed for imaging thymidylate synthase (TS) inhibition. Agents that target TS and shut down de novo synthesis of thymidine monophosphate increase the uptake and retention of FLT in vitro and in vivo because of a compensating increase in the salvage pathway. Increases in both thymidine kinase-1 (TK1) and the equilibrative nucleoside transporter hENT1 have been reported to underlie this effect. We examined whether the effects of one TS inhibitor, 5-fluorouracil (5FU), on FLT uptake require proliferating cells and whether the effects are limited to increasing TK1 activity. The effects of 5FU on FLT transport and metabolism, TK1 activity, and cell cycle progression were evaluated in the human tumor cell line, A549, maintained as either a proliferating or non-proliferating culture. There were dose-dependent increases in FLT uptake that peaked after a 10 μM 5FU exposure and then declined to baseline levels or below at higher doses in both proliferating and non-proliferating cultures. The dose-dependence for FLT uptake was mirrored by changes in TK1 activity. S phase fraction did not correlate with FLT uptake in proliferating cultures. Chemical inhibition of hENT1 reduced overall levels of FLT uptake but did not affect the low dose increase in FLT uptake. 5FU only affects FLT uptake in proliferating A549 cells and increases in FLT uptake are directly related to increased TK1 activity. Our studies did not support a role for hENT1 in the increased uptake of FLT after exposure to 5FU. Our studies with A549 cells support the suggestion that FLT-PET could provide a measure of TS inhibition in vivo.

Nuclear Medicine and Biology, 2011

Introduction: Recent studies in the human adenocarcinoma cell line A549 have identified cell grow... more Introduction: Recent studies in the human adenocarcinoma cell line A549 have identified cell growth-dependent equilibrative nucleoside transporter-1 (hENT1) as a modifier of 3′-fluoro-3′-deoxythymidine (FLT) uptake and retention. In the present study, we used the ability to isolate human lymphoblastoid clones deficient in thymidine kinase 1 (TK1) to study how metabolism and nucleoside transport influence FLT uptake and retention. Methods: Transport and metabolism of FLT were measured in the human lymphoblastoid cell line TK6 and in eight clones isolated from TK6. Four clones were TK1-proficient, while four were TK1-deficient. Both influx and efflux of FLT were measured under conditions where concentrative and equilibrative transport could be distinguished. Results: Sodium-dependent concentrative FLT transport dominated over equilibrative transport mechanisms and while inhibition of hENT1 reduced FLT uptake, there were no correlations between clonal variations in hENT1 levels and FLT uptake. There was an absolute requirement of TK1 for concentration of FLT in TK6 cells. FLT uptake reached a peak after 60 min of incubation with FLT after which intracellular levels of FLT and FLT metabolites declined. Efflux was rapid and was associated with reductions in FLT and each of its metabolites. Both FLT and FLT-monophosphate were found in the efflux buffer. Conclusions: Initial rates of FLT uptake were a function of both concentrative and equilibrative transporters. TK1 activity was an absolute requirement for the accumulation of FLT. Retention was dependent on nucleoside/nucleotide efflux and retrograde metabolism of FLT nucleotides.

Journal of Nuclear Medicine, 2010

The basis for the use of nucleoside tracers in PET is that activity of the cell-growth-dependent ... more The basis for the use of nucleoside tracers in PET is that activity of the cell-growth-dependent enzyme thymidine kinase 1 is the rate-limiting factor driving tracer retention in tumors. Recent publications suggest that nucleoside transporters might influence uptake and thereby affect the tracer signal in vivo. Understanding transport mechanisms for different nucleoside PET tracers is important for evaluating clinical results. This study examined the relative role of different nucleoside transport mechanisms in uptake and retention of [methyl-3 H]-3′-deoxy-3′fluorothymidine (3 H-FLT), [methyl-3 H]-thymidine (3 H-thymidine), and 3 H-1-(2-deoxy-2-fluoroβ-D-arabinofuranosyl)-5-methyluracil (3 H-FMAU). Methods-Transport of 3 H-FLT, 3 H-thymidine, and 3 H-FMAU was examined in a single human adenocarcinoma cell line, A549, under both nongrowth and exponential-growth conditions. Results-3 H-Thymidine transport was dominated by human equilibrative nucleoside transporter 1 (hENT1) under both growth conditions. 3 H-FLT was also transported by hENT1, but passive diffusion dominated its transport. 3 H-FMAU transport was dominated by human equilibrative nucleoside transporter 2. Cell membrane levels of hENT1 increased in cells under exponential growth, and this increase was associated with a more rapid rate of uptake for both 3 H-thymidine and 3 H-FLT. 3 H-FMAU transport was not affected by changes in growth conditions. All 3 tracers concentrated in the plateau phase, nonproliferating cells at levels many-fold greater than their concentration in buffer, in part because of low levels of nucleoside metabolism, which inhibited tracer efflux. Conclusion-Transport mechanisms are not the same for 3 H-thymidine, 3 H-FLT, and 3 H-FMAU. Levels of hENT1, an important transporter of 3 H-FLT and 3 H-thymidine, increase as proliferating cells enter the cell cycle. Keywords proliferation; nucleoside transport; nucleoside metabolism; 3 H-FLT; 3 H-FMAU Pet provides a noninvasive approach to measuring tumor growth and response to therapy (1,2). Labeled thymidine and thymidine analogs such as [methyl-3 H]-3′-deoxy-3′fluorothymidine (3 H-FLT) and 3 H-1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5

Tp53 codon-72 polymorphisms identify different radiation sensitivities to g2-chromosome breakage in human lymphoblast cells

Environmental and Molecular Mutagenesis, 2011

Both the G2 chromosomal radiosensitivity assay and allelic differences in TP53 codon-72 have been... more Both the G2 chromosomal radiosensitivity assay and allelic differences in TP53 codon-72 have been associated with cancer predisposition. The relationship between the two endpoints was determined in 56 human EBV-transformed lymphoblastoid cell lines. Although there were overlapping distributions of sensitivity for the different genotypes, cell lines that were homozygous for the proline coding allele were more likely to be resistant to chromatid break formation than those containing two arginine coding alleles, whereas cell lines expressing both the proline and arginine codon were either resistant like proline-proline lines or sensitive like arginine-arginine lines. The results support an important role of the TP53 codon-72 polymorphism in modifying G2-chromosome radiosensitivity. Distinguishing the effect of TP53 codon-72 variations from other modifiers of G2-chromosome radiosensitivity might aid in identifying new markers of cancer risk.

Levels of human equilibrative nucleoside transporter-1 are higher in proliferating regions of A549 tumor cells grown as tumor xenografts in vivo

Nuclear Medicine and Biology, 2012

3&amp... more 3'-Fluoro-3'-deoxythymidine (FLT) has been proposed for positron emission tomography (PET)-based identification of tumor chemosensitivity that is mediated by the human equilibrative nucleoside transporter-1 (ENT1). ENT1 facilitates transport of FLT into cells and elevated levels of FLT are associated with both larger FLT-PET signals and increased response to nucleoside-based chemotherapies. FLT-PET is also used as a measure of tumor proliferation. The present study examined the extent to which ENT1 levels vary in a proliferation-dependent manner in tumor cells in vivo. The human adenocarcinoma cell line A549 was used to establish tumor xenografts in nude mice. FLT uptake was measured in vivo using PET, and further examined ex vivo using autoradiography. FLT uptake patterns were compared to immunohistochemical (IHC) analysis of ENT1 and the proliferation markers Ki67 and BrdU. Regional differences in FLT uptake matched differences in IHC proliferation markers. All cells stained for ENT1, but the staining intensity was twice as high for Ki67(+) cells than for Ki67(-) cells. Under in vivo conditions, proliferating regions of tumors show increased FLT uptake and higher ENT1 levels than nonproliferating tumor regions.

A phase 1 study in healthy volunteers to investigate the safety, tolerability, and pharmacokinetics of VIR-2482: a monoclonal antibody for the prevention of severe influenza A illness

Antimicrobial agents and chemotherapy, Feb 20, 2024

medRxiv (Cold Spring Harbor Laboratory), Apr 4, 2024

Background: Influenza A results in significant morbidity and mortality. VIR-2482, an engineered h... more Background: Influenza A results in significant morbidity and mortality. VIR-2482, an engineered human monoclonal antibody with extended half-life, targets a highly conserved epitope on the stem region of influenza A hemagglutinin, and may protect against seasonal and pandemic influenza. : This double-blind, randomized, placebo-controlled, phase 2 study examined the safety and efficacy of VIR-2482 for seasonal influenza A illness prevention in unvaccinated healthy adults. Participants (N = 2977) were randomized 1:1:1 to receive VIR-2482 450 mg, VIR-2482 1200 mg, or placebo via intramuscular (IM) injection. Primary and secondary efficacy endpoints were the proportions of participants with reverse transcriptase-polymerase chain reaction (RT-PCR)-confirmed influenza A infection and either protocol-defined influenza-like illness (ILI) and Centers for Disease Control and Prevention (CDC)-defined ILI or World Health Organization (WHO)-defined ILI, respectively. Results: VIR-2482 450 mg and 1200 mg prophylaxis did not reduce the risk of protocol-defined ILI with RT-PCR-confirmed influenza A versus placebo (relative risk reduction [RRR], 3.8% [95% CI : -67.3, 44.6] and 15.9% [95% CI: -49.3, 52.3], respectively). At the 1200 mg dose, the RRRs in influenza A illness were 57.2% [95% CI: -2.5, 82.2] using CDC-ILI and 44.1% [95% CI: -50.5, 79.3] using WHO-ILI definitions, respectively. Serum VIR-2482 levels were similar regardless of influenza status; variants with reduced VIR-2482 susceptibility were not detected. Local injection-site reactions were mild and similar across groups. VIR-2482 1200 mg IM was well tolerated but did not significantly prevent protocol-defined ILI. Secondary endpoint analyses suggest this dose may have reduced influenza A illness. .

Aging Cell, 2012

Increasing age is the most robust predictor of greater malignancy and treatment resistance in hum... more Increasing age is the most robust predictor of greater malignancy and treatment resistance in human gliomas. However, the adverse association of clinical course with aging is rarely considered in animal glioma models, impeding delineation of the relative importance of organismal versus progenitor cell aging in the genesis of glioma malignancy. To address this limitation, we implanted transformed neural stem/progenitor cells (NSPCs), the presumed cells of glioma origin, from 3 and 18month old mice into 3 and 20-month host animals. Transplantation with progenitors from older animals resulted in significantly shorter (p ≤ 0.0001) median survival in both 3month (37.5 vs 83 days) and 20-month (38 vs 67 days) hosts, indicating that agedependent changes intrinsic to NSPCs rather than host animal age accounted for greater malignancy. Subsequent analyses revealed that increased invasiveness, genomic instability, resistance to therapeutic agents and tolerance to hypoxic stress accompanied aging in transformed NSPCs. Greater tolerance to hypoxia in older progenitor cells, as evidenced by elevated HIF-1 promoter reporter activity and hypoxia response gene (HRG) expression, mirror the upregulation of HRGs in cohorts of older vs younger glioma patients revealed by analysis of gene expression databases, suggesting that differential response to hypoxic stress may underlie age-dependent differences in invasion, genomic instability and treatment resistance. Our study provides strong evidence that progenitor cell aging is responsible for promoting the hallmarks of age-dependent glioma malignancy and that consideration of progenitor aging will facilitate development of physiologically and clinically relevant animal models of human gliomas.

Levels of human equilibrative nucleoside transporter-1 are higher in proliferating regions of A549 tumor cells grown as tumor xenografts in vivo

Nuclear Medicine and Biology, 2012

3&amp... more 3'-Fluoro-3'-deoxythymidine (FLT) has been proposed for positron emission tomography (PET)-based identification of tumor chemosensitivity that is mediated by the human equilibrative nucleoside transporter-1 (ENT1). ENT1 facilitates transport of FLT into cells and elevated levels of FLT are associated with both larger FLT-PET signals and increased response to nucleoside-based chemotherapies. FLT-PET is also used as a measure of tumor proliferation. The present study examined the extent to which ENT1 levels vary in a proliferation-dependent manner in tumor cells in vivo. The human adenocarcinoma cell line A549 was used to establish tumor xenografts in nude mice. FLT uptake was measured in vivo using PET, and further examined ex vivo using autoradiography. FLT uptake patterns were compared to immunohistochemical (IHC) analysis of ENT1 and the proliferation markers Ki67 and BrdU. Regional differences in FLT uptake matched differences in IHC proliferation markers. All cells stained for ENT1, but the staining intensity was twice as high for Ki67(+) cells than for Ki67(-) cells. Under in vivo conditions, proliferating regions of tumors show increased FLT uptake and higher ENT1 levels than nonproliferating tumor regions.

Transport of 3'-fluoro-3'-deoxythymidine (FLT) and its metabolites in the human lymphoblast cell line TK6

The Journal of Nuclear Medicine, 2011

The Role of CS1 Fibronectin in HIV-1 Infection of α4β7 + T Lymphocytes

Journal of Virology

Gut-homing α 4 β 7 high CD4 + T lymphocytes have been shown to be preferentially targeted by Huma... more Gut-homing α 4 β 7 high CD4 + T lymphocytes have been shown to be preferentially targeted by Human Immunodeficiency Virus-1 (HIV), and are implicated in HIV pathogenesis. Previous studies demonstrated that HIV envelope protein gp120 binds and signals through α 4 β 7 , and that this likely contributes to the infection of α 4 β 7 high T cells and promotes cell-to-cell virus transmission. Structures within the second variable loop (V2) of gp120, including the tripeptide motif LDV/I, are thought to mediate gp120-α 4 β 7 binding. However, lack of α 4 β 7 binding has been reported in gp120 proteins containing LDV/I, and the precise determinants of gp120-α 4 β 7 binding are not fully defined. In this work, we report the novel finding that fibronectins mediate indirect gp120-α 4 β 7 interactions. We show that Chinese Hamster Ovary (CHO) cells used to express recombinant gp120 produced fibronectins and other extracellular matrix proteins that co-purified with gp120. CHO fibronectins were abl...

Transport of 3'-fluoro-3'-deoxythymidine (FLT) and its metabolites in the human lymphoblast cell line TK6

Society of Nuclear Medicine Annual Meeting Abstracts, May 1, 2011

The effects of 5-fluorouracil treatment on 3'-fluoro-3'-deoxythymidine (FLT) transport and metabolism in proliferating and non-proliferating cultures of human tumor cells

Society of Nuclear Medicine Annual Meeting Abstracts, May 1, 2012

Nuclear medicine and biology, Jan 6, 2014

The use of thymidine (TdR) and thymidine analogs such as 3'-fluoro-3'-deoxythymidine (FLT... more The use of thymidine (TdR) and thymidine analogs such as 3'-fluoro-3'-deoxythymidine (FLT) as positron emission tomography (PET)-based proliferation markers can provide information on tumor response to treatment. Studies on another TdR analog, 4'-thiothymidine (4DST), suggest that it might be a better PET-based proliferation tracer than either TdR or FLT. 4DST is resistant to the catabolism that complicates analysis of TdR in PET studies, but unlike FLT, 4DST is incorporated into DNA. To further evaluate 4DST, the kinetics of 4DST transport and metabolism were determined and compared to FLT and TdR. Transport and metabolism of FLT, TdR and 4DST were examined in the human adenocarcinoma cell line A549 under exponential-growth conditions. Single cell suspensions were incubated in buffer supplemented with radiolabeled tracer in the presence or absence of nitrobenzylmercaptopurine ribonucleoside (NBMPR), an inhibitor of equilibrative nucleoside transporters (ENT). Kinetics o...

The effects of 5-fluoruracil treatment on 3′-fluoro-3′-deoxythymidine (FLT) transport and metabolism in proliferating and non-proliferating cultures of human tumor cells

Nuclear Medicine and Biology, 2012

3&amp... more 3'-Fluoro-3'-deoxythymidine (FLT) positron emission tomography (PET) has been proposed for imaging thymidylate synthase (TS) inhibition. Agents that target TS and shut down de novo synthesis of thymidine monophosphate increase the uptake and retention of FLT in vitro and in vivo because of a compensating increase in the salvage pathway. Increases in both thymidine kinase-1 (TK1) and the equilibrative nucleoside transporter hENT1 have been reported to underlie this effect. We examined whether the effects of one TS inhibitor, 5-fluorouracil (5FU), on FLT uptake require proliferating cells and whether the effects are limited to increasing TK1 activity. The effects of 5FU on FLT transport and metabolism, TK1 activity, and cell cycle progression were evaluated in the human tumor cell line, A549, maintained as either a proliferating or non-proliferating culture. There were dose-dependent increases in FLT uptake that peaked after a 10 μM 5FU exposure and then declined to baseline levels or below at higher doses in both proliferating and non-proliferating cultures. The dose-dependence for FLT uptake was mirrored by changes in TK1 activity. S phase fraction did not correlate with FLT uptake in proliferating cultures. Chemical inhibition of hENT1 reduced overall levels of FLT uptake but did not affect the low dose increase in FLT uptake. 5FU only affects FLT uptake in proliferating A549 cells and increases in FLT uptake are directly related to increased TK1 activity. Our studies did not support a role for hENT1 in the increased uptake of FLT after exposure to 5FU. Our studies with A549 cells support the suggestion that FLT-PET could provide a measure of TS inhibition in vivo.

Nuclear Medicine and Biology, 2011

Introduction: Recent studies in the human adenocarcinoma cell line A549 have identified cell grow... more Introduction: Recent studies in the human adenocarcinoma cell line A549 have identified cell growth-dependent equilibrative nucleoside transporter-1 (hENT1) as a modifier of 3′-fluoro-3′-deoxythymidine (FLT) uptake and retention. In the present study, we used the ability to isolate human lymphoblastoid clones deficient in thymidine kinase 1 (TK1) to study how metabolism and nucleoside transport influence FLT uptake and retention. Methods: Transport and metabolism of FLT were measured in the human lymphoblastoid cell line TK6 and in eight clones isolated from TK6. Four clones were TK1-proficient, while four were TK1-deficient. Both influx and efflux of FLT were measured under conditions where concentrative and equilibrative transport could be distinguished. Results: Sodium-dependent concentrative FLT transport dominated over equilibrative transport mechanisms and while inhibition of hENT1 reduced FLT uptake, there were no correlations between clonal variations in hENT1 levels and FLT uptake. There was an absolute requirement of TK1 for concentration of FLT in TK6 cells. FLT uptake reached a peak after 60 min of incubation with FLT after which intracellular levels of FLT and FLT metabolites declined. Efflux was rapid and was associated with reductions in FLT and each of its metabolites. Both FLT and FLT-monophosphate were found in the efflux buffer. Conclusions: Initial rates of FLT uptake were a function of both concentrative and equilibrative transporters. TK1 activity was an absolute requirement for the accumulation of FLT. Retention was dependent on nucleoside/nucleotide efflux and retrograde metabolism of FLT nucleotides.

Journal of Nuclear Medicine, 2010

The basis for the use of nucleoside tracers in PET is that activity of the cell-growth-dependent ... more The basis for the use of nucleoside tracers in PET is that activity of the cell-growth-dependent enzyme thymidine kinase 1 is the rate-limiting factor driving tracer retention in tumors. Recent publications suggest that nucleoside transporters might influence uptake and thereby affect the tracer signal in vivo. Understanding transport mechanisms for different nucleoside PET tracers is important for evaluating clinical results. This study examined the relative role of different nucleoside transport mechanisms in uptake and retention of [methyl-3 H]-3′-deoxy-3′fluorothymidine (3 H-FLT), [methyl-3 H]-thymidine (3 H-thymidine), and 3 H-1-(2-deoxy-2-fluoroβ-D-arabinofuranosyl)-5-methyluracil (3 H-FMAU). Methods-Transport of 3 H-FLT, 3 H-thymidine, and 3 H-FMAU was examined in a single human adenocarcinoma cell line, A549, under both nongrowth and exponential-growth conditions. Results-3 H-Thymidine transport was dominated by human equilibrative nucleoside transporter 1 (hENT1) under both growth conditions. 3 H-FLT was also transported by hENT1, but passive diffusion dominated its transport. 3 H-FMAU transport was dominated by human equilibrative nucleoside transporter 2. Cell membrane levels of hENT1 increased in cells under exponential growth, and this increase was associated with a more rapid rate of uptake for both 3 H-thymidine and 3 H-FLT. 3 H-FMAU transport was not affected by changes in growth conditions. All 3 tracers concentrated in the plateau phase, nonproliferating cells at levels many-fold greater than their concentration in buffer, in part because of low levels of nucleoside metabolism, which inhibited tracer efflux. Conclusion-Transport mechanisms are not the same for 3 H-thymidine, 3 H-FLT, and 3 H-FMAU. Levels of hENT1, an important transporter of 3 H-FLT and 3 H-thymidine, increase as proliferating cells enter the cell cycle. Keywords proliferation; nucleoside transport; nucleoside metabolism; 3 H-FLT; 3 H-FMAU Pet provides a noninvasive approach to measuring tumor growth and response to therapy (1,2). Labeled thymidine and thymidine analogs such as [methyl-3 H]-3′-deoxy-3′fluorothymidine (3 H-FLT) and 3 H-1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5

Tp53 codon-72 polymorphisms identify different radiation sensitivities to g2-chromosome breakage in human lymphoblast cells

Environmental and Molecular Mutagenesis, 2011

Both the G2 chromosomal radiosensitivity assay and allelic differences in TP53 codon-72 have been... more Both the G2 chromosomal radiosensitivity assay and allelic differences in TP53 codon-72 have been associated with cancer predisposition. The relationship between the two endpoints was determined in 56 human EBV-transformed lymphoblastoid cell lines. Although there were overlapping distributions of sensitivity for the different genotypes, cell lines that were homozygous for the proline coding allele were more likely to be resistant to chromatid break formation than those containing two arginine coding alleles, whereas cell lines expressing both the proline and arginine codon were either resistant like proline-proline lines or sensitive like arginine-arginine lines. The results support an important role of the TP53 codon-72 polymorphism in modifying G2-chromosome radiosensitivity. Distinguishing the effect of TP53 codon-72 variations from other modifiers of G2-chromosome radiosensitivity might aid in identifying new markers of cancer risk.

Levels of human equilibrative nucleoside transporter-1 are higher in proliferating regions of A549 tumor cells grown as tumor xenografts in vivo

Nuclear Medicine and Biology, 2012

3&amp... more 3'-Fluoro-3'-deoxythymidine (FLT) has been proposed for positron emission tomography (PET)-based identification of tumor chemosensitivity that is mediated by the human equilibrative nucleoside transporter-1 (ENT1). ENT1 facilitates transport of FLT into cells and elevated levels of FLT are associated with both larger FLT-PET signals and increased response to nucleoside-based chemotherapies. FLT-PET is also used as a measure of tumor proliferation. The present study examined the extent to which ENT1 levels vary in a proliferation-dependent manner in tumor cells in vivo. The human adenocarcinoma cell line A549 was used to establish tumor xenografts in nude mice. FLT uptake was measured in vivo using PET, and further examined ex vivo using autoradiography. FLT uptake patterns were compared to immunohistochemical (IHC) analysis of ENT1 and the proliferation markers Ki67 and BrdU. Regional differences in FLT uptake matched differences in IHC proliferation markers. All cells stained for ENT1, but the staining intensity was twice as high for Ki67(+) cells than for Ki67(-) cells. Under in vivo conditions, proliferating regions of tumors show increased FLT uptake and higher ENT1 levels than nonproliferating tumor regions.