Unlabelled iododeoxyuridine increases the cytotoxicity and incorporation of [1251]-iododeoxyuridine in two human glioblastoma cell lines

Iododeoxyuridine (IUdR), labelled with radioiodines emitting Auger, alpha or beta- radiation, has been proposed as a therapeutic tool in the treatment of cancer. However, the low per cent incorporation in tumour cells and limited cytotoxicity are major obstacles for such an application. Using unlabelled IUdR as a modulator, we have studied the in vitro cytotoxicity of [125I]-IUdR in two human glioblastoma cell lines. Surprisingly, an enhanced cytotoxicity of [125I]-IUdR was observed in the presence of 0.3-10 microM concentrations of unlabelled IUdR in U251 glioblastoma cells and to a lesser extent in LN229 cells. The presence of unlabelled IUdR unexpectedly increased the incorporation of [125I]-IUdR in both cell lines. Thymidine competitively blocked the cytotoxic effects of combined unlabelled and [125I]-labelled IUdR in these cells and DNA-incorporation of radiolabelled IUdR.     

Site-specific incorporation of [125I]iododeoxyuridine into DNA

A procedure for the incorporation of [¹²⁵I]IdU into specific sites in DNA is described. The approach depends upon attachment of radioiododeoxyuridine to a controlled pore glass support which is then used for automated synthesis of an oligomer. The resulting oligomer, containing a terminal 3′[¹²⁵I]iododeoxyuridine, is used as a primer during DNA synthesis catalyzed by the Taq polymerase employing thermal cycling. The product formed includes the radioiodonucleotide at a single internal site determined by the length of the oligomer.     

Iodine heterocycles: [125I] and [131I] ortho-iodosophenylphosphoric acid

Since organic molecules tagged with radioiodine are often subject to dehalogenation, techniques are needed for "protecting" the iodine. A suggested approach was the incorporation of iodine directly into a heterocyclic compound as one of the ring's heteroatoms. Such a compound, orthoiodosophenylphosphoric acid, was synthesized with I-125 and I-131. Upon i.v. administration to dogs and rabbits, most of the radiolabel was excreted in the urine. There was no evidence of the appearance of free iodide. The renal elimination of orthoiodosophenylphosphoric acid was contrasted with the biliary excretion of another iodine heterocycle, diphenyleneiodonium. Iodine heterocycles, with appropriate substituents, may represent a useful class of compounds for biologic studies.     

Absence of delayed chromosomal instability in a normal human fibroblast cell line after 125I iododeoxyuridine

PURPOSE: To seek the delayed appearance of chromosomal abnormalities in human fibroblasts exposed to the Auger electron emitter 125I. MATERIALS AND METHODS: Normal untransformed human fibroblasts, HF19, were exposed to a concentration of [I125]IUdR, which allowed the survival of 37% of clonogens. Chromosomal analysis using both conventional Giemsa and fluorescence in situ hybridization (FISH) was undertaken on non-clonal bulk cultures from 2 to 39 days after treatment. RESULTS: The data show a declining level of unstable aberrations in the progeny of HF19 fibroblasts exposed to [I125]IUdR, eventually reaching control levels. CONCLUSIONS: The results provide evidence that [125I]IUdR does not induce ongoing chromosomal instability in long-term culture, and gives further support to the use of Auger-electron emitting radionuclides in the treatment and diagnosis of tumours.     

Absence of delayed chromosomal instability in a normal human fibroblast cell line after 125 I iododeoxyuridine

Purpose : To seek the delayed appearance of chromosomal abnormalities in human fibroblasts exposed to the Auger electron emitter 125 I. Materials and methods : Normal untransformed human fibroblasts, HF19, were exposed to a concentration of [I 125 ]IUdR, which allowed the survival of 37% of clonogens. Chromosomal analysis using both conventional Giemsa and fluorescence in situ hybridization (FISH) was undertaken on non-clonal bulk cultures from 2 to 39 days after treatment. Results : The data show a declining level of unstable aberrations in the progeny of HF19 fibroblasts exposed to [I 125 ]IUdR, eventually reaching control levels. Conclusions : The results provide evidence that [ 125 I]IUdR does not induce ongoing chromosomal instability in long-term culture, and gives further support to the use of Auger-electron emitting radionuclides in the treatment and diagnosis of tumours.     

Preparation of 1-[125I]iodo-[114C]dodecane

1-[¹²⁵I]Iodo-[1¹⁴C]dodecane was prepared using the phosphorus halide/alcohol-reaction. Since the conventional synthesis of iodoalkanes requires apparatus and procedures which do not conform to radioprotection standards, a new apparatus was also constructed. Apparatus and method can also be used for the synthesis of other halogen-alkanes.     

[99mTc]pyrophosphate and [125I]phenylphosphonate behavior in the infarcted rat myocardium

The concentration of [¹²⁵I]phenylphosphonate ([¹²⁵I]φPA), was compared to that of [^99mTc]pyrophosphate ([^99mTc]Pyro) in a rat infarct model. Twenty-three hours after ligation of the anterior descending coronary artery, [¹²⁵I]φPA was given intravenously (i.v.) to 30 adult male rats. Five minutes later, 5 of these animals received [^99mTc]Pyro IV and the content of ¹²⁵I and ^99mTc was then determined at 2 h for normal (N), and infarcted (INF) heart segments. A similar investigation was completed in 5 of the animals at each of the following times post MI; 2, 3, 4, 7 and 75 days. Data were expressed as the mean (± SEM) of (% uptake/g infarct)/(% uptake/g normal) for both tracers at each study time. These data suggest that the myocardial cells which exhibit constant uptake of [^99mTc]Pyro are similar to those exhibiting chronic retention of [¹²⁵I]φPA. The myocardial infarct to normal ratio of [^99mTc]Pyro on [¹²⁵I]φPA showed excellent correlation (r = 0.928) and suggests that persistent positive pyrophosphate scans are likely due to residual damaged tissue from the acute episode of necrosis, rather than the involvement of newly injured tissue.     

Accumulation and metabolism of [125I] HIPDM in the rat pancreas

Our previous studies have shown a high accumulation of [125I]N,N,N'-trimethyl-N'-(2-hydroxy-3-methyl-5-iodobenzyl)-1,3- propanediamine (HIPDM) in the human pancreas. In this study, the pancreatic accumulation and metabolism of [125I]HIPDM were studied in rats to determine the factors influencing its uptake by this organ. In biodistribution studies, [125I]HIPDM showed a high uptake by the pancreas similar to that by the brain and lungs, both organs with a low tissue pH. TLC analysis of pancreatic homogenate after the injection of [125I]HIPDM showed that it was metabolically stable in this organ. Moreover, in the pancreatic homogenate, the bulk of the radioactivity was recovered from the microsomal fraction, and the radioactivity bound to microsomal particles showed release that was dependent on the Ca2+ or Mg2+ concentration in the incubation medium. These results suggest that the initial pancreatic uptake of [125I]HIPDM may be a function of blood flow and governed by the pH gradient hypothesis, while subsequent retention may occur secondary to ionic binding within the pancreas.     

In vitro characterization of the influx of 3-[125I]iodo-L-alpha-methyltyrosine and 2-[125I]iodo-L-tyrosine into U266 human myeloma cells: evidence for system T transport

The aim of this study was to investigate the cellular uptake mechanisms responsible for the accumulation of 3-[(125)I]iodo-L-alpha-methyltyrosine ((125)I-3-IMT) and 2-[(125)I]iodo-L-tyrosine ((125)I-2-IT), two radiotracers for metabolic tumor imaging, using single-photon emission tomography, into U266 human myeloma cancer cells. Time course and concentration dependency of (125)I-3-IMT uptake was assessed. Kinetic parameters were calculated using an Eadie Hofstee plot. A set of competitive inhibitors of the main amino acid transport systems was used for the discrimination of the transporters responsible for the uptake of (125)I-3-IMT and (125)I-2-IT. Protein incorporation of both tracers was determined using acid precipitation. The measured maximum velocity for (125)I-3-IMT transport was 4.199 nmol per mg protein 20 s(-1), and the Michaelis constant was 107.9 microM. Addition of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH), a competitive inhibitor of System L, reduced the influx by 39.0+/-3.3% for (125)I-3-IMT and 66.3+/-0.9% for (125)I-2-IT. The BCH-insensitive influx was further reduced by Tryptophan (Trp) by 43.8+/-3.5% for (125)I-3-IMT and 15.3+/-1.3% for (125)I-2-IT. This suggests involvement of System T transport. We measured <2% of radioactivity in the acid precipitable fractions of both tracers with no increase in time. We conclude that the influx of (125)I-3-IMT and (125)I-2-IT into U266 human myeloma cells is mediated by both System L and System T amino acid transporters. The kinetic parameters suggest that elevated plasma levels of aromatic amino acids will reduce (123)I-3-IMT uptake in myeloma patients. Both tracers do not enter protein synthesis significantly.     

Subcellular distribution of [125I]iodoaryl beta-methyl fatty acids

Subcellular distribution studies of two aryl branched-chain and one aryl straight-chain iodinated fatty acids were carried out as part of a continuing effort to determine if such acids are metabolized by beta-oxidation. For the omega-iodoaryl fatty acids, a change in subcellular radioactivity location was observed which was chain-length dependent. Chain lengths of 15 carbons, straight and branched, were largely found in the nuclear-membrane fraction, whereas a chain length of 8 carbons was largely located in the cytosol. No unequivocal evidence for metabolic trapping in the mitochondria was observed for omega-iodoaryl branched-chain fatty acids using the centrifugation technique employed in this study.     

Characterization of 3-[125I]iodo-alpha-methyl-L-tyrosine transport via human L-type amino acid transporter 1

We examined transport of 3-[(125)I]iodo-alpha-methyl-L-tyrosine ([(125)I]IMT) in Xenopus laevis oocytes co-expressing human L-type amino acid transporter 1 (a component of system L) and human 4F2hc. Human LAT1 mediated transport of [(125)I]IMT. [(125)I]IMT uptake was decreased by the presence of L-isomers of Cys, Leu, Ileu, Phe, Met, Tyr, His, Trp and Val and D-isomers of Leu, Phe and Met. Human LAT1-mediated [(125)I]IMT uptake was highly stereoselective for the L-isomers of Tyr, His, Trp, Val and Ileu. To examine the effects of 3-iodination and alpha-methylation on IMT transport, kinetic parameters of IMT were compared with those of mother Tyr and 3-[(125)I]iodo-L-tyrosine (3-I-Tyr). Uptake of Tyr, 3-I-Tyr and [(125)I]IMT followed Michaelis-Menten kinetics, with K(m) values of 29.0 +/- 5.1, 12.6 +/- 6.1 and 22.6 +/- 4.1 microM, respectively. Neither the alpha-methyl group nor the size of the 3-iodinated Tyr residue was an obstacle to transport via hLAT1. Furthermore, affinity of IMT for hLAT1 is higher than that of the natural parent tyrosine. The level of efflux mediated by hLAT1 was highly stimulated by extracellularly applied L-Leu, suggesting exchange of [(125)I]IMT and L-Leu via hLAT1.     

Comparison of the uptake of [123/125I]-2-iodo-D-tyrosine and [123/125I]-2-iodo-L-tyrosine in R1M rhabdomyosarcoma cells in vitro and in R1M tumor-bearing Wag/Rij rats in vivo

INTRODUCTION: Recently, promising results concerning uptake in vivo in tumors of D-amino acids have been published. Therefore, we decided to evaluate the tumor uptake of the D-analogue of [(123)I]-2-iodo-L-tyrosine, a tracer recently introduced by our group into clinical trials. The uptake of 2-amino-3-(4-hydroxy-2-[(123/125)I]iodophenyl)-D-propanoic acid (2-iodo-D-tyrosine) was studied in vitro in LAT1-expressing R1M rat rhabdomyosarcoma cells and in vivo in R1M tumor-bearing Wag/Rij rats. METHODS: The uptake of [(125)I]-2-iodo-L-tyrosine and [(125)I]-2-iodo-D-tyrosine into R1M cells was determined in appropriate buffers, allowing the study of the involved transport systems. In vivo, the biodistribution in R1M-bearing rats of [(123)I]-2-iodo-L-tyrosine and [(123)I]-2-iodo-D-tyrosine was performed by both dynamic and static planar imaging with a gamma camera. RESULTS: In in vitro conditions, the uptake of both [(125)I]-2-iodo-L-tyrosine and [(125)I]-2-iodo-D-tyrosine in the HEPES buffer was 25% higher in the presence of Na(+) ions. In the absence of Na(+) ions, [(125)I]-2-iodo-D-tyrosine was taken up reversibly in the R1M cells, with an apparent accumulation, probably for the larger part by the LAT1 system. Dynamic planar imaging showed that the uptake in the tumors of [(123)I]-2-iodo-D-tyrosine was somewhat lower than that of [(123)I]-2-iodo-L-tyrosine. At 30 min postinjection, the mean differential uptake ratio values of the L- and D-enantiomers are 2.5+/-0.7 and 1.7+/-0.6, respectively. Although the uptake of the D-isomer is lower, probably due to a faster clearance from the blood, the tumor-background ratio is the same as that of the l-analogue. CONCLUSION: A large part (75%) of [(125)I]-2-iodo-D-tyrosine in vitro and [(123)I]-2-iodo-D-tyrosine in vivo is reversibly highly taken up in R1M tumor cells by Na(+)-independent LAT transport systems, more likely by the LAT1. The clearance from the blood of [(123)I]-2-iodo-D-tyrosine in the rats is faster than that of the L-analogue, resulting in a slightly lower tumor uptake but with the same tumor-background ratio.     

[125I] Radioiodinated metaraminol: a new platelet-specific labeling agent

In our search for a platelet-specific labeling agent, metaraminol (MA), a low-toxic pharmaceutical for the treatment of hypotension and cardiogenic shock, attracted our attention. Its active incorporation and accumulation by platelets have been recognized. At first, the preparation of ¹²⁵I radioiodinated metaraminol (¹²⁵I-MA) was carried out using the chloramine-T method. Then, upon the harvest of platelets as platelet-rich plasma (PRP), their labeling with this new radiopharmaceutical was easily performed by incubation for 10 min at 37° C. The cell-labeling efficiency was dependent on cell density, reaching 63.0%±3.1% at 2.4x10⁹ cells/ml. The specific incorporation of ¹²⁵I-MA by an active transport system similar to that of 5-hydroxytryptamine (5-HT) as well as by passive diffusion was demonstrated. In in vitro studies, the unaltered state of ¹²⁵I-MA-labeled platelets with their cellular functions fully retained was estimated. In vivo studies carried out in rabbits with induced thrombi in the femoral artery showed a rather rapid disappearance of the radioactivity from circulating blood, reaching a high thrombus-to-blood activity ratio of 19.8±4.3 within 30 min of the administration of ¹²⁵I-MA-labeled autologous platelets. Thus, with the potential availability of ¹²³I, ¹²³I-MA-labeled platelets appear to be a promising agent for thrombus imaging using single-emission computed tomography (CT) studies.     

Cytotoxicity of [125I]iodoHoechst 33342: contribution of scavengeable effects

PURPOSE: The incubation of the DNA minor-groove binder [125I]iodoHoechst 33342 (125IH) with plasmid DNA leads to the production of one double-strand break (dsb) per decay, both in the presence and absence of dimethylsulfoxide (DMSO). In contrast, when 125I is incorporated into mammalian cell DNA as an iodinated pyrimidine base, DMSO decreases the dsb yield and enhances survival. Because these variations in radioprotective effects may be due either to the location of 125I vis-à-vis the DNA helix or to differences in DNA architecture, the toxicity of 125IH and its modification by DMSO were examined in mammalian cells. METHODS: Uptake and retention of 125IH in V79 cells were measured, and survival was determined after accumulation of 125I decays at 0.3 degrees C +/-10% DMSO. RESULTS: A linear-quadratic survival curve was obtained both in the absence [D37 = 114+/-36 decays/cell, alpha = (5.39 1.17) x10(-3) cell/decay] and presence [D37 = 211+/-65 decays/cell, alpha = (1.27+/-0.52) x10(-3) cell/decay] of DMSO. The dose modification factor for the linear component of the survival curve was 4.25+/-1.97, indicating the predominance of indirect mechanisms. This value is similar to that obtained with DNA-incorporated 125I (4.05+/-1.72) and for the initial slope (alpha) of 137Cs gamma-rays (4.43+/- 1.41). CONCLUSIONS: Cytotoxicity resulting from the decay of the Auger electron emitter 125I in the mammalian cell nucleus is caused mainly by indirect mechanisms.     

Selective binding of 2-[125I]iodo-nisoxetine to norepinephrine transporters in the brain

A radioiodinated ligand, (R)-N-methyl-(2-[(125)I]iodo-phenoxy)-3-phenylpropylamine, [(125)I]2-INXT, targeting norepinephrine transporters (NET), was successfully prepared. A no-carrier-added product, [(125)I]2-INXT, displayed a saturable binding with a high affinity (K(d)=0.06 nM) in the homogenates prepared from rat cortical tissues as well as from LLC-PK(1) cells expressing NET. A relatively low number of binding sties (B(max)=55 fmol/mg protein) measured with [(125)I]2-INXT in rat cortical homogenates is consistent with the value reported for a known NET ligand, [(3)H]nisoxetine. Competition studies with various compounds on [(125)I]2-INXT binding clearly confirmed the pharmacological specificity and selectivity for NET binding sites. Following a tail-vein injection of [(125)I]2-INXT in rats, a good initial brain uptake was observed (0.56% dose at 2 min) followed by a slow washout from the brain (0.2% remained at 3 hours post-injection). The hypothalamus (a NET-rich region) to striatum (a region devoid of NET) ratio was 1.5 at 3 hours post-i.v. injection. Pretreatment of rats with nisoxetine significantly inhibited the uptake of [(125)I]2-INXT (70-100% inhibition) in locus coeruleus, hypothalamus and raphe nuclei, regions known to have a high density of NET; whereas escitalopram, a serotonin transporter ligand, did not show a similar effect. Ex vivo autoradiography of rat brain sections of [(125)I]2-INXT (at 3 hours after an i.v. injection) displayed an excellent regional brain localization pattern corroborated to the specific NET distribution in the brain. The specific brain localization was significantly reduced by a dose of nisoxetine pretreatment. Taken together, the data suggest that [(123)I]2-INXT may be useful for mapping NET binding sites in the brain.     

Characteristics of the binding of N-isopropyl-p-[125I]iodoamphetamine in the rat brain synaptosomal membranes

N-isopropyl-p-[125I]iodoamphetamine (125I-IMP) binding to crude synaptosomal membranes of rat brain was saturable and pharmacologically displacable. Scatchard analysis of binding data revealed an apparent dissociation constant, KD of 56.2 +/- 5.2 microM, and the estimated maximum number of binding sites, Bmax of 7.5 +/- 1.1 nmoles mg-1 protein; and competition studies demonstrated structure activity relationships among structural analogues of arylalkylamines. These findings suggest that the retention mechanism of IMP in the brain is probably associated with saturable binding of IMP to extreme high-density, relatively low-affinity binding sites rather than any of the well-known amine receptors.     

Synthesis and in vitro examination of [124I]-, [125I]- and [131I]-2-(4-iodophenylamino) pyrido[2,3-d]pyrimidin-7-one radiolabeled Abl kinase inhibitors

The pyridopyrimidinones are a potent class of inhibitors of c-Abl kinase and Bcr-Abl kinase, the causative fusion protein in chronic myelogenous leukemia and Src family kinases. A novel method for routine, high-yield no-carrier-added synthesis of [(124)I]-, [(125)I]- and [(131)I]-6-(2,6-dichlorophenyl)-2-(4-iodophenylamino)-8-methyl-8H-pyrido[2,3-d]pyrimidin-7-one has been developed. The 4'-trimethylstannyl- or 4'-tri-n-butylstannyl-pyridopyrimidinone precursors were prepared from the aryl bromide via a palladium-mediated coupling with hexaalkylditin (dioxane/microwave irradiation/10 min at 160 degrees C). The radioiodination of 4'-stannylpyridopyrimidinones was found to optimally occur via an iododestannylation with Na(124)I, Na(125)I or Na(131)I in the presence of an oxidant [30% H(2)O(2)/HOAc (1:3)/10 min] in 79-87% radiochemical yield with >99% radiochemical purity. The total radiosynthesis time was 30 min. The 4-iodophenylpyridopyrimidinone 2 inhibited recombinant Abl kinase activity with an IC(50) of 2.0 nM. Cell proliferation of K562 and A431 cells was inhibited with an IC(50) of 2.0 and 20 nM, respectively. Rapid cellular uptake and equilibrium were observed within 10-15 min using [(131)I]-4-iodophenylpyridopyrimidinone 6c in K562 and A431 cells and demonstrated a 2.8-fold uptake selectivity for the Bcr-Abl-expressing K562 cells at 60 min. These results suggest that pyridopyrimidinone radiotracers may be useful in imaging Abl-, Bcr-Abl- or Src-expressing malignancies.     

hTPO和hNIS共转染胶质瘤细胞U251介导放射性碘摄取的研究

目的 将人甲状腺过氧化物酶（hTPO）基因及人钠/碘同向转运体（hNIS）基因共转入胶质瘤细胞系U251后,研究其摄碘能力的变化.方法 克隆、重组、包装并扩增纯化得到重组腺病毒（AdTPO）,测定病毒滴度,Western-Blotting检测重组腺病毒的表达.使用脂质体转染法将hNIS基因转染入人胶质瘤细胞系U251中,经过G418硫酸盐筛选获得稳定表达hNIS的细胞系（hNIS-U251）,为hNIS-U251组;使用重组腺病毒将hTPO基因转导入hNIS-U251中,使胶质瘤细胞获得hTPO基因（AdTPO-hNIS-U251）,为AdTPO-hNIS-U251组;未转入hTPO和hNIS的细胞为对照组（U251）.然后进行3组稳定表达细胞系体外摄125I实验及体外125I外流实验.3组间两两比较用q检验（Newman-Keuls法）.结果 AdTPO-hNIS-U251细胞、hNIS-U251细胞和U251细胞所摄取125I分别为（74 647.53±3605.88）、（55 769.96±4353.26）和（507.67±57.69）计数/min,3组间差异有统计学意义（F=836.17,P＜0.01）.AdTPO-hNIS-U251组较对照组（U251）摄125I能力增高约147倍（q=55.64,P＜0.01）,hNIS-U251组较对照组（U251）摄碘能力增高约110倍（q=41.47,P＜0.01）.AdTPO-hNIS-U251组较hNIS-U251组高约1.3倍（q=14.17,P＜0.01）.在体外125I外流实验中证实AdTPO-hNIS-U251组125I外流减慢,其有效半衰期延长至13 min.结论 将hTPO和hNIS基因共转染至胶质瘤细胞系U251后,能有效提高U251的摄碘能力.