Evaluation of vertebral bone strength with a finite element method using low dose computed tomography imaging.

BACKGROUND: Focusing on compression fractures of bone by finite elements, we evaluated bone strength based on the computed tomography-based finite element method. However, the exposure dose is an issue. We aimed to investigate the quantity of reduction of the radiation dose with respect to the reference dose by comparing the calculation results of compression fractures of the vertebral body using experimental data obtained from the spine of a pig. METHODS: Computed tomography images of a self-made phantom that enclosed the lower lumbar vertebra of edible wild pigs were obtained under baseline-dose conditions using various lower tube currents. Images obtained under reference-dose conditions were reconstructed using the filtered back-projection method, whereas images obtained under low-dose conditions were reconstructed using both the filtered back-projection method and the iterative reconstruction method. Computer simulations involving the creation of finite element models using all images were implemented for the compression load calculation for vertebral body parts. Based on the calculated results, images of the low-dose and reference-dose conditions were compared. RESULTS: Using pigs' lower lumbar vertebrae, finite element model analysis of low-dose X-ray computed tomography images showed that equivalent results can be obtained with a dose of approximately 40% of the standard radiographic reference doses. As for the compression stress intensity, the same results as those under reference-dose conditions were obtained using the iterative reconstruction method in combination with computed tomography-based finite element method. CONCLUSIONS: The combination of the iterative reconstruction method with the computed tomography-based finite element method is an effective image reconstruction method for achieving dose reduction.

Development of a Knee Joint CT-FEM Model in Load Response of the Stance Phase During Walking Using Muscle Exertion, Motion Analysis, and Ground Reaction Force Data.

Background and objectives: There are no reports on articular stress distribution during walking based on any computed tomography (CT)-finite element model (CT-FEM). This study aimed to develop a calculation model of the load response (LR) phase, the most burdensome phase on the knee, during walking using the finite element method of quantitative CT images. Materials and Methods: The right knee of a 43-year-old man who had no history of osteoarthritis or surgeries of the knee was examined. An image of the knee was obtained using CT and the extension position image was converted to the flexion angle image in the LR phase. The bone was composed of heterogeneous materials. The ligaments were made of truss elements; therefore, they do not generate strain during expansion or contraction and do not affect the reaction force or pressure. The construction of the knee joint included material properties of the ligament, cartilage, and meniscus. The extensor and flexor muscles were calculated and set as the muscle exercise tension around the knee joint. Ground reaction force was vertically applied to suppress the rotation of the knee, and the thigh was restrained. Results: An FEM was constructed using a motion analyzer, floor reaction force meter, and muscle tractive force calculation. In a normal knee, the equivalent stress and joint contact reaction force in the LR phase were distributed over a wide area on the inner upper surface of the femur and tibia. Conclusions: We developed a calculation model in the LR phase of the knee joint during walking using a CT-FEM. Methods to evaluate the heteromorphic risk, mechanisms of transformation, prevention of knee osteoarthritis, and treatment may be developed using this model.

Simulating Knee-Stress Distribution Using a Computed Tomography-Based Finite Element Model: A Case Study.

This study aimed to evaluate the mechanism of progression involved in knee osteoarthritis (OA). We used the computed tomography-based finite element method (CT-FEM) of quantitative X-ray CT imaging to calculate and create a model of the load response phase, wherein the greatest burden is placed on the knee joint while walking. Weight gain was simulated by asking a male individual with a normal gait to carry sandbags on both shoulders. We developed a CT-FEM model that incorporated walking characteristics of individuals. Upon simulating changes owing to a weight gain of approximately 20%, the equivalent stress increased extensively in both medial and lower leg aspects of the femur and increased medio-posteriorly by approximately 230%. As the varus angle increased, stress on the surface of the femoral cartilage did not change significantly. However, the equivalent stress on the surface of the subchondral femur was distributed over a wider area, increasing by approximately 170% in the medio-posterior direction. The range of equivalent stress affecting the lower-leg end of the knee joint widened, and stress on the posterior medial side also increased significantly. It was reconfirmed that weight gain and varus enhancement increase knee-joint stress and cause the progression of OA.

Effectiveness of a case-based digital learning interprofessional workshop involving undergraduates in medical technology, radiological science, and physical therapy: A pre-post intervention study.

All healthcare professionals must understand information on a patient's biophysical functions, and it is important to educate professionals on how to use this information in an interprofessional team for diagnosis. However, there is little interprofessional education for students of medical technology and radiological science involved in biophysical function diagnosis. In the present study, we developed a case-based interprofessional learning tool for using biophysical information for diagnosis. The study examined the effects of a collaborative exercise workshop for healthcare professional students using the tool. Participants were 234 students from three healthcare professions (medical technology, radiological science, and physical therapy). They completed the Japanese version of the Readiness for Interprofessional Learning Scale before and after the workshops. The workshops incorporated digital materials that allowed students to examine the test results of a virtual patient, answer questions, and discuss their diagnoses and prognoses. For analysis, a two-way analysis of variance was performed on the total score on the Readiness for Interprofessional Learning Scale of the three departments, and the effectiveness of the workshop for the three departments was compared. Statistical analyses showed no interaction between time and department (p = 0.283). After the workshop, students from all three departments showed significant improvements in total scores on the Readiness for Interprofessional Learning Scale (p < 0.01) with medium to large effect sizes (r = 0.33-0.52). In the comparison between departments, there was a significant difference in the awareness levels of only medical technology and radiological science students before the workshop (p = 0.015). This study conducted case-based learning workshops with students from three departments, in which a patient's biophysical information was conveyed between occupational practices. The workshops improved the awareness of interprofessional education in students from all departments and revealed that interprofessional education is important for healthcare professions involved in biophysical function diagnosis.

Pharmacokinetics of 3-[125I]iodo-alpha-methyl-L-tyrosine, a tumor imaging agent, after probenecid loading in mice implanted with colon cancer DLD-1 cells.

INTRODUCTION: In order to improve tumor imaging, changes in the pharmacokinetics of 3-[123I]iodo-alpha-methyl-l-tyrosine ([123I]IMT), an artificial amino acid that exhibits high tumor accumulation, after probenecid (PBC) loading was studied in mice implanted with colon cancer DLD-1 cells using 125I-labeled IMT ([125I]IMT). METHODS: DLD-1-implanted KSN-slc nude male mice received 740 kBq of [125I]IMT via the tail vein at 5 min after 50 mg/kg body weight PBC loading, and autoradiography was performed at 5, 15 and 30 min after injection. Male ddY mice then received 670 kBq of [125I]IMT and 50 mg/kg 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH) or p-aminohippurate (PAH) via the tail vein, and kidney autoradiography was performed at 5 min after injection. In vitro inhibition study was then performed based on the accumulation mechanisms of [125I]IMT in DLD-1, using 1 mM l-tyrosine, BCH, alpha-(methylamino)-isobutyric acid, N-benzoyl-beta-alanine, PBC, PAH, 2,4-dinitrophenol and sodium azide. Both Na+-dependent and Na+-independent uptake were investigated. RESULTS: Higher tumor accumulation in PBC-loaded DLD-1-implanted mice was seen when compared to control mice. PAH and BCH, respectively, reduced renal accumulation in the tubule segment-2 (S2)-like and S1-like regions. We confirmed that [125I]IMT transport is predominantly mediated by l-type amino acid transporter-1 in DLD-1 cells. CONCLUSIONS: [125I]IMT uptake is mediated by organic anion and amino acid transporters in the kidney. Organic anion transporter inhibitors may yield better tumor images with good tumor/normal tissue radioactivity ratios if adequate administration plans are developed.

Transport of D-[1-14C]-amino acids into Chinese hamster ovary (CHO-K1) cells: implications for use of labeled d-amino acids as molecular imaging agents.

INTRODUCTION: The fact that d-amino acids have been found in various tissues and are involved in various functions is a clue to how to develop new imaging agents. We examined d-amino acid transport mechanisms in Chinese hamster ovary (CHO-K1) cells because CHO-K1 cells are widely used in biomedical studies and are thought to be useful for expression of genes involved in metabolism of D-amino acids. METHODS: Uptake experiments were performed. CHO-K1 cells cultured in 60-mm plastic culture dishes under ordinary culture conditions were incubated with 18.5 kBq of radiolabeled amino acid in 2 ml of phosphate-buffered-saline-based uptake solution at 37 degrees C. The following radiolabeled amino acid tracers were used: D-[1-(14)C]-alanine, L-[1-(14)C]-alanine, D-[1-(14)C]-serine, L-[1-(14)C]-serine, D-[1-(14)C]-methionine, L-[1-(14)C]-methionine, D-[1-(14)C]-phenylalanine, L-[1-(14)C]-phenylalanine, D-[1-(14)C]-leucine, L-[1-(14)C]-leucine, D-[1-(14)C]-valine, L-[1-(14)C]-valine, D-[1-(14)C]-tyrosine, L-[1-(14)C]-tyrosine, D-[1-(14)C]-glutamic acid, L-[1-(14)C]-glutamic acid, D-[1-(14)C]-lysine, L-[1-(14)C]-lysine, D-[1-(14)C]-arginine and L-[L-(14)C]-arginine. We tested the inhibitory effects of the following compounds (1.0 mM) on transport: 2-(methylamino)isobutyric acid (a specific inhibitor of system A, in Na(+)-containing uptake solution) and 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L, in Na(+)-free uptake solution). RESULTS: D-[1-(14)C]-methionine, D-[1-(14)C]-phenylalanine and D-[1-(14)C]-tyrosine accumulated mainly via system L. D-[1-(14)C]-alanine and D-[1-(14)C]-serine accumulated primarily via system ASC. High uptake of D-[1-(14)C]-alanine, D-[1-(14)C]-methionine, D-[1-(14)C]-phenylalanine and D-[1-(14)C]-leucine was observed. The uptake of radiolabeled serine, valine, tyrosine, glutamic acid and arginine into CHO-K1 was highly stereoselective for l-isomers. CONCLUSIONS: We observed high uptake of D-[1-(14)C]-alanine via system ASC (most likely alanine-serine-cysteine-selective amino acid transporter-1) and high uptake of D-[1-(14)C]-methionine and D-[1-(14)C]-phenylalanine via system L (most likely L-type amino acid transporter-1).

Differences in accumulation and the transport mechanism of l- and d-methionine in high- and low-grade human glioma cells.

INTRODUCTION: Although [S-methyl-11C]-labeled L-methionine and D-methionine (11C-L-MET and 11C-D-MET) are useful radiotracers for positron emission tomography imaging of brain tumors, it is not known whether the accumulation and transport mechanisms underlying uptake of 11C-D-MET and 11C-L-MET are the same. 11C-L-MET is mainly taken up by the amino acid transport system L. We evaluated accumulation and the transport mechanism of D-MET in high- and low-grade human glioma cells in vitro. METHODS: The expression of transport system genes in high- (A172 and T98G) and low-grade (SW1088 and Hs683) glioma cells was quantitatively analyzed. Accumulation of [S-methyl-3H]-L-MET (3H-L-MET) and [S-methyl-3H]-D-MET (3H-D-MET) in these cells was compared during 60min of incubation. The transport mechanism of 3H-L-MET and 3H-D-MET was investigated by incubating the cells with these compounds and examining the effect of the inhibitors 2-amino-2-norbornane-carboxylic acid or α-(methylamino) isobutyric acid. RESULTS: Absolute expression levels of system L and system alanine-serine-cysteine (ASC) in high-grade glioma cells were higher than in low-grade cells. In high-grade glioma cells, expression of system ASC genes was higher than that of system L genes. 3H-D-MET, which is transported by systems L and ASC, accumulated at higher levels than 3H-L-MET at all incubation times because 3H-D-MET is more sensitive to system ASC than 3H-L-MET. Conversely, in low-grade glioma cells with lower expression of system L and ASC, 3H-D-MET accumulated at higher levels than 3H-L-MET in early incubation times because 3H-D-MET may be more sensitive to system ASC than system L. CONCLUSION: 3H-D-MET was mainly transported by systems L and ASC and sensitive to system ASC, whereas 3H-L-MET was transported by system L in human glioma cells. In vitro, the accumulation of 3H-D-MET was significantly higher than that of 3H-L-MET during the entire incubation time in high-grade glioma cells, and in early incubation times in low-grade glioma cells.

Detection of maleate-induced Fanconi syndrome by decreasing accumulation of 125I-3-iodo-alpha-methyl-L-tyrosine in the proximal tubule segment-1 region of renal cortex in mice: a trial of separate evaluation of reabsorption.

OBJECTIVE: Fanconi syndrome is a renal dysfunction characterized by various combinations of renal tubular transport dysfunction involving amino acids, glucose, protein and other substances. Most reabsorption of amino acids occurs in proximal renal tubule segment 1 (S1). The present study evaluated the possibility of early detection of drug-induced Fanconi syndrome, based on decreased renal accumulation of 125I-3-iodo-alpha-methyl-L-tyrosine (125I-IMT), an amino acid transport marker, in the S1 region of renal cortex. The present experimental model used maleate (MAL)-induced Fanconi syndrome in mice. Results were compared between 125I-IMT and 3 other clinical renal radiopharmaceuticals: 99mTc-2,3-dimercaptosuccinic acid (99mTc-DMSA); 99mTc-mercaptoacetylglycylglycylglycine (99mTc-MAG3); and 99mTc-diethylenetriaminepentaacetic acid (99mTc-DTPA). METHODS: Male ddY mice (age, 6 weeks; body weight, 25 g) were used to create a Fanconi model of renal dysfunction. A single dose of maleate disodium salt was administered by intraperitoneal injection (6 mmol/kg). Hematoxylin and eosin (HE) staining of the renal cortex, renal autoradiography and measurement of renal radioactivity of labeled compounds were performed at 30, 60, 90 and 120 min after MAL injection. At 5 min after injection of labeled compounds (18.5 kBq for accumulation experiment, 670 kBq for autoradiography), animals were sacrificed by ether overdose and kidneys were removed. For the accumulation experiment, radioactivity was measured using a well-type scintillation counter. For autoradiography, 20-microm sections of frozen kidney were used with Bio-Imaging Analyzer. RESULTS: At 30 min after MAL injection, HE staining showed pyknosis in some proximal tubule cells. At that time, accumulations of 125I-IMT and 99mTc-DMSA in the S1 region were approximately 67% and 55% of control levels (p < 0.005). MAL increased accumulation of 99mTc-DTPA in the S1 region, but had no effect on accumulation of 99mTc-MAG3 in the S 1 region. CONCLUSIONS: Decreased accumulation of 123I-IMT in the S1 region appears to represent a useful marker for detection of MAL-induced Fanconi syndrome. In future, we plan to assess the efficacy of using 125I-IMT to monitor renal dysfunction induced by nephrotoxic clinical drugs.

A strategy for improving FDG accumulation for early detection of metastasis from primary pancreatic cancer: stimulation of the Warburg effect in AsPC-1 cells.

INTRODUCTION: Early detection and/or prediction of metastasis provide more prognostic relevance than local recurrence. Direct spread into the peritoneum is frequently found in pancreatic cancer patients, but positron emission tomography (PET) with 2-deoxy-2-fluoro-d-glucose (FDG) is not useful for identifying such metastasis. We investigated a method to enhance FDG accumulation using AsPC-1 human ascites tumor cells. METHODS: (14)C-FDG accumulation was assessed under the following conditions: 1) characteristics of (14)C-FDG transport were examined using phloridzin, a Na(+)-free buffer, and various hexoses, and 2) accumulation of (14)C-FDG was measured in cells that were pretreated with hexose for various time periods, and activity of 6-phosphofructo-1-kinase (PFK-1) was assayed. RESULTS: (14)C-FDG transport into AsPC-1 cells was mediated primarily by a Na(+)-independent transport mechanism. Aldohexoses such as d-glucose, D-mannose, and D-galactose inhibited (14)C-FDG transport. Cells pretreated with d-glucose, D-mannose, or D-fructose exhibited augmented (14)C-FDG accumulation. Pretreatment with higher concentrations of D-glucose or D-fructose tended to increase PFK-1 activity. CONCLUSIONS: Very little information has been published about the association between PFK-1 and FDG accumulation, and we confirmed the impacts of various hexoses on the activity of PFK-1 and FDG accumulation in AsPC-1 cells. Clarifying the relevance of PFK-1 in FDG accumulation will contribute to developing new features of FDG-PET, because PFK-1 is the main regulator of glycolysis.

Transcellular transport of 4-iodo-L-meta-tyrosine via system L across monolayers of kidney epithelial cell line LLC-PK1.

The substance 4-[(125)I]iodo-L-meta-tyrosin (4-[(125)I]mTyr) is a radioiodinated amino acid that exhibits high in vivo stability and rapid renal elimination in vivo. We investigated transport of 4-[(125)I]mTyr in LLC-PK(1) (porcine kidney epithelial cell line) monolayers grown on collagen-coated, micro-porous membrane filters. We found that 4-[(125)I]mTyr transport in LLC-PK(1) cells was carrier-mediated and sodium-independent, and that 4-[(125)I]mTyr transport was similar to that of L-Tyr and 3-iodo-alpha-methyl-L-tyrosine. The results of the inhibition experiments suggest that 4-[(125)I]mTyr transport is predominantly mediated by a L-type amino acid transporter 1-like porcine homologue (a component of system L) in both basolateral and apical membrane.

Transcellular transport of radioiodinated 3-iodo-alpha-methyl-L-tyrosine across monolayers of kidney epithelial cell line LLC-PK1.

OBJECTIVE: 3-[123I]iodo-alpha-methyl-L-tyrosine ([123I]IMT) is an imaging agent for amino acid transport. In order to obtain fundamental data related to tumor imaging with [123I]IMT and renal physiological accumulation of [123I]IMT, we investigated the transport characteristics of [125I]IMT in porcine kidney epithelial cell line LLC-PK1 using cell monolayers grown on microporous membrane filters. METHODS: LLC-PK1 monolayers were created on a collagen-coated microporous (3 microm) membrane (4.7 cm2). To examine transcellular transport (secretion and reabsorption) and accumulation, the monolayers were incubated for up to 90 min at 37 degrees C with 18.5 kBq [125I]IMT in Dulbecco's phosphate-buffered saline (pH 7.4) as an uptake solution. After incubation, transcellular transport was assessed by quantifying the radioactivity of the solutions on each side of the monolayer. For the accumulation experiment, the cells were solubilized in NaOH solution, and the radioactivity was quantified. For the inhibition experiment, the inhibitor was added at a final concentration of 1 mM. For the pH dependence experiment, the pH of the apical-side uptake solution was varied from pH 5 to pH 8. Transport of [14C]Tyr was examined for comparison. RESULTS: Bi-directional transcellular transport of [125I]IMT was observed, corresponding to secretion and reabsorption in proximal tubule. Accumulation of [125I]IMT from the basolateral side (1.62 +/- 0.15%) and the apical side (2.62 +/- 0.35%) was observed at 90 min. 2-Amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L), L-Tyr (mother compound of [125I]IMT) and 2-aminoisobutyric acid (an inhibitor of system L and A) inhibited both directional transport (p < 0.01) and accumulation (p < 0.01). 2-(Methylamino)isobutyric acid (a specific inhibitor of system A) appeared to inhibit transport and accumulation, but the results were not significant. Decreasing apical pH significantly enhanced accumulation of [125I]IMT from both sides (p < 0.001), whereas accumulation of mother L-Tyr was significantly suppressed. CONCLUSIONS: The inhibition experiments suggest that the main contributor to [125I]IMT transport is system L, rather than Na+ -dependent transport, in both apical and basolateral membrane. [125I]IMT was transported by the system that transported L-Tyr, but the observed pH dependence of transport suggests that different mechanisms are involved in accumulation of [125I]IMT and [14C]Tyr.

Renal accumulation and excretion of radioiodinated 3-iodo-alpha-methyl-L-tyrosine.

OBJECTIVE: We investigated mechanisms of renal accumulation of radioiodinated 3-iodo-alpha-methyl-L-tyrosine (IMT), which has been used clinically for tumor imaging and as an amino acid transport marker in studies of brain and pancreas function. METHODS: In this study, we used 125I- or 123I-labeled IMT ([125I]IMT or [123I]IMT) as the transport marker. Partition coefficients of [125I]IMT were determined for hypothetic urine at pH ranging from 5 to 8. The examination of uptake and inhibition of [125I]IMT was performed using normal human renal proximal tubule epithelial cells (RPTEC), which are characteristic of the proximal convoluted tubule. The plasma protein binding ratio of [125I]IMT was determined using rats. In the in vivo experiments using mice, we examined biodistribution and excretion inhibition, and performed whole body autoradiography. Also, renal SPECT using [123I]IMT was performed using a normal canine. RESULTS: Very low lipophilicity of [125I]IMT in hypothetic urine suggests that a carrier-mediated pathway contributes to its marked kidney accumulation. [125I]IMT uptake into RPTEC was significantly inhibited by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) in a sodium-dependent manner, suggesting reabsorption mainly via system B0 in apical membrane of proximal tubule. Plasma protein binding ratio of IMT was 45.4 +/- 5.6%. At 6 hr after administration of IMT to mice, excretion via urinary tract was 77.51% of injected dose, and excretion into feces was 0.25%. Furosemide, ethacrynic acid and probenecid inhibited tubular secretion of [125I]IMT in mice. We obtained very clear autoradiographs of mouse renal cortex and a canine renal SPECT image (S2-like region). CONCLUSIONS: We believe that [123I]IM-T is useful for kidney imaging. In future studies, we plan to examine the use of [123I]IMT in diagnosis of disease.

Stimulation of 125I-3-iodo-alpha-methyl-L-tyrosine uptake in Chinese hamster ovary (CHO-K1) cells by tyrosine esters.

INTRODUCTION: Transport of the amino acid analog (123)I-3-iodo-alpha-methyl-L-tyrosine, which is used in clinical SPECT imaging, occurs mainly via L-type amino acid transporter type 1 (LAT1; an amino acid exchanger). As LAT1 is highly expressed in actively proliferating tumors, we made a preliminary investigation of the effects of amino acid esters on enhancement of (125)I-3-iodo-alpha-methyl-L-tyrosine (IMT) uptake via LAT1 in Chinese hamster ovary (CHO-K1) cells. METHODS: Because the sequence of the CHO-K1 LAT1 gene is not available, we confirmed LAT1 expression through IMT (18.5 kBq) uptake mechanisms using specific inhibitors. L-Gly, L-Ser, L-Leu, L-Phe, L-Met, L-Tyr, D-Tyr, L-Val and L-Lys ethyl/methyl esters were tested in combination with IMT. Time-course studies over a 3-h period were conducted, and the concentration dependence of L-Tyr ethyl and methyl esters (0.001 to 10 mM) in combination with IMT was also examined. For a proof of de-esterification of L- and D-Tyr ethyl and methyl esters in the cells (by enzymatic attack or other cause), the concentration of L- and D-Tyr was analyzed by high-performance liquid chromatography of the esters in phosphate buffer (pH 7.4) and cell homogenates at 37 degrees C or under ice-cold conditions. RESULTS: Inhibition tests suggested that LAT1 is involved in IMT uptake by CHO-K1 cells. Co-administration of 1 mM of l-Tyr ethyl or methyl ester with IMT produced the greatest enhancement. The de-esterification reaction was stereo selective and temperature dependent in the homogenate. De-esterification kinetics were very fast in the homogenate and very slow in the phosphate buffer. CONCLUSIONS: The L-Tyr ethyl or methyl esters were the most effective enhancers of IMT uptake into CHO-K1 cells and acted by trans-stimulation of the amino acid exchange function of LAT1. This result suggests that de-esterification in the cells may be caused by enzymatic attack. We will use IMT and L-Tyr ethyl or methyl esters to examine LAT1 function in tumor cells or tissues in vivo.

Uptake of 3-[125I]iodo-alpha-methyl-L-tyrosine into colon cancer DLD-1 cells: characterization and inhibitory effect of natural amino acids and amino acid-like drugs.

INTRODUCTION: We examined 3-[(123)I]iodo-alpha-methyl-L-tyrosine ([(123)I]IMT) uptake and inhibition by amino acids and amino acid-like drugs in the human DLD-1 colon cancer cell line, to discuss correlation between the inhibition effect and structure. METHODS: Expression of relevant neutral amino acid transporters was examined by real-time PCR with DLD-1 cells. The time course of [(125)I]IMT uptake, contributions of transport systems, concentration dependence and inhibition effects by amino acids and amino acid-like drugs (1 mM) on [(125)I]IMT uptake were examined. RESULTS: Expression of system L (4F2hc, LAT1 and LAT2), system A (ATA1, ATA2) and system ASC (ASCT1) was strongly detected; system L (LAT3, LAT4) and MCT8 were weakly detected; and B(0)AT was not detected. [(125)I]IMT uptake in DLD-1 cells involved Na(+)-independent system L primarily and Na(+)-dependent system(s). Uptake of [(125)I]IMT in Na(+)-free buffer followed Michaelis-Menten kinetics, with a K(m) of 78 microM and V(max) of 333 pmol/10(6) cells per minute. Neutral D- and L-amino acids with branched or aromatic large side chains inhibited [(125)I]IMT uptake. Tyrosine analogues, tryptophan analogues, L-phenylalanine and p-halogeno-L-phenylalanines, and gamma amino acids [including 3,4-dihydroxy-L-phenylalanine (L-DOPA), DL-threo-beta-(3,4-dihydroxyphenyl)serine (DOPS), 4-[bis(2-chloroethyl)amino]-L-phenylalanine and 1-(aminomethyl)-cyclohexaneacetic acid] strongly inhibited [(125)I]IMT uptake, but L-tyrosine methyl ester and R(+)/S(-)-baclofen weakly inhibited uptake. The substrates of system ASC and A did not inhibit [(125)I]IMT uptake except L-serine and D/L-cysteine. CONCLUSIONS: [(125)I]IMT uptake in DLD-1 cells involves mostly LAT1 and its substrates' (including amino acid-like drugs derived from tyrosine, tryptophan and phenylalanine) affinity to transport via LAT1. Whether transport of gamma amino acid analogues is involved in LAT1 depends on the structure of the group corresponding to the amino acid residue. Beta-hydroxylation may confer reduction of transport affinity of tyrosine analogues via LAT1.

Radioiodinated 4-iodo-L-meta-tyrosine, a system L selective artificial amino acid: molecular design and transport characterization in Chinese hamster ovary cells (CHO-K1 cells).

INTRODUCTION: High expression of the system L amino acid transporter has been observed in clinically important tissues including tumors and the blood-brain barrier. We examined amino acid transport system L selectivity of (14)C(U)-L-tyrosine ((14)C-Tyr), (125)I-4-iodo-L-meta-tyrosine (4-(125)I-mTyr), (125)I-6-iodo-L-meta-tyrosine (6-(125)I-mTyr), (125)I-3-iodo-α-methyl-L-tyrosine ((125)I-IMT) and (125)I-3-iodo-L-tyrosine (3-(125)I-Tyr) using Chinese hamster ovary cells (CHO-K1). METHODS: Cells in the exponential growth phase were incubated with 18.5 kBq of labeled amino acid in 2 mL of phosphate-buffered saline-based uptake solution and an uptake solution with/without Na(+) at 37°C or 4°C. We examined the effects of the following compounds (1.0 mM) on transport: 2-(methylamino)isobutyric acid (a specific inhibitor of system A, in Na(+)-containing uptake solution); 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L, in Na(+)-free uptake solution); sodium azide and 2,4-dinitrophenol (NaN(3) and DNP, inhibitors of the generation of adenosine triphosphate); p-aminohippurate and tetraethylammonium (PAH and TEA, inhibitors of organic anion and cation transporters); and L- and D-isomers of natural amino acids. RESULTS: (14)C-Tyr exhibited affinity for systems L, A and ASC. 4-(125)I-mTyr and 3-(125)I-Tyr exhibited high specificity for system L, whereas 6-(125)I-mTyr and (125)I-IMT exhibited affinity for both systems L and ASC. Uptake of 4-(125)I-mTyr was markedly reduced by incubation at 4 °C, and was not significantly inhibited by NaN(3), DNP, PAH or TEA. The inhibition profiles of the L- and D-isomers of natural amino acids indicated that system L mediates the transport of 4-(125)I-mTyr. CONCLUSIONS: 4-(125)I-mTyr exhibited the greatest system L specificity (93.46 ± 0.13%) of all of the tested amino acids.

Heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin potentiates the radiation response of tumor cells grown as monolayer cultures and spheroids by inducing apoptosis.

Activation of the PI3K-Akt pathway is known to induce tumor radioresistance. In the current study, we examined the ability of 17AAG, which decreases the levels of Hsp90 client proteins including components of the PI3K-Akt pathway, to sensitize radioresistant human squamous cell carcinoma cells to X-irradiation. Human squamous cell carcinoma cell lines (SQ20B, SCC61 and SCC13) were incubated for 16 h at 37 degrees C in medium containing 17AAG. Radiation sensitivity was determined by clonogenic assays, and protein levels were examined by western blotting. Apoptosis was determined in monolayer cells by AO/EB double staining and in spheroids using the TdT-mediated dUTP nick end labeling assay. 17AAG (0.2 microM) enhanced the radiosensitivity more effectively in radioresistant SQ20B and SCC13 cells than in radiosensitive SCC61 cells. However, in all three cell lines, 17AAG increased radiation-induced apoptosis by reducing the expression of EGFR and ErbB-2 and inhibiting the phosphorylation of Akt. Furthermore, 17AAG (1 microM) sensitized SQ20B spheroids to radiation, and inhibition of Akt activation by 17AAG increased radiation-induced apoptosis in spheroids. The findings suggest that 17AAG effectively sensitizes radioresistant cells to radiation by inhibiting the PI3K-Akt pathway. Targeting the PI3K-Akt pathway with 17AAG could be a useful strategy for radiosensitization of carcinomas.