Basis of FLT as a cell proliferation marker: comparative uptake studies with [3H]thymidine and [3H]arabinothymidine, and cell-analysis in 22 asynchronously growing tumor cell lines

The usefulness of radiolabeled 3'-fluoro-3'-deoxythymidine (FLT), a thymidine derivative with affinity to cytoplasmic thymidine kinase 1 (TK1), as a tumor proliferation marker was evaluated using [3H]FLT and 22 cultured tumor cell lines. Asynchronously growing tumor cells were used for studies to mimic in vivo status of tumors. FLT uptake in each cell line was compared with [3H]thymidine ([3H]Thd) uptake and %S-phase fraction, both known as acceptable markers of proliferation. Uptake of the mitochondrial TK2 specific substrate [3H]arabinothymidine ([3H]AraT) was studied as a reference. Metabolic fate of FLT in tumor cells was also analyzed to elucidate the retention mechanism of FLT. [3H]FLT uptake was mildly correlated with the %S-phase fraction (r=0.76, p<0.0001) and correlated better with [3H]Thd uptake (r=0.88, p<0.0001). In contrast, the TK(2) specific substrate, [3H]AraT, was not significantly correlated with the %S-phase fraction (r=0.19, p=0.39), although it showed some correlation with the [3H]Thd uptake (r=0.47, p<0.05). Over 90% of radioactivity of [3H]Thd was found in the DNA fraction after 60 minutes incubation. In contrast, most of the radioactivity of [3H]FLT was found in the acid-soluble fraction (95%). [3H]FLT incorporation into the DNA fraction was negligible (0.2%). The [3H]AraT was mainly distributed in the acid-soluble fraction (70%) and the DNA fraction (20%). From our results, we concluded that FLT uptake in tumor cells reflects tumor cell proliferation. However, much more convincing validation is needed to clarify the difference between FLT and true substrates for DNA synthesis, like thymidine.     

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.     

[methyl-3H]Choline incorporation into MCF7 tumour cells: correlation with proliferation

Purpose The aim of the study was to investigate the intracellular location of [methyl-³H]choline in MCF7 tumour cells and to determine the relationship between [methyl-³H]choline incorporation and proliferation.Methods Tumour cells were incubated with [methyl-³H]choline for 10 min, and then in cold medium to simulate the rapid blood clearance of [methyl-¹¹C]choline. Labelled metabolites were then extracted from cells by treating them with organic and aqueous solvents to determine the distribution of tracer between phospholipid and water-soluble metabolite pools. Aqueous extracts were subjected to thin-layer chromatography, ion exchange chromatography and a choline extraction procedure to identify ³H-containing metabolites. Procedures were carried out on fast- and slow-growing populations of MCF7 cells to determine the relationship between choline incorporation and proliferation.Results Only about 5% of [methyl-³H]choline was present as phospholipid. [methyl-³H]choline incorporation was found to be related to S-phase fraction. In another experiment, [methyl-¹⁴C]choline incorporation was found to be correlated with [methyl-³H]thymidine incorporation. The V_max of choline uptake was found to be increased whilst K_m was decreased in populations of MCF7 cells with higher proliferative fractions, compared with populations having lower proliferative fractions.Conclusion Choline incorporation into tumour cells under conditions that simulate rapid blood clearance of [methyl-¹¹C]choline is correlated with proliferation. Most of the activity (about 95%) was in the non-lipid fraction of the cell.     

Lymphocyte transformation (by [3H]thymidine) in mice infected with trichinella spiralis

The blastogenic response of infected and normal mice (to stimulation by specific antigen and non specific antigens) was followed by the uptake of [³H]thymidine.It was established that there was a minimum number of cells (1 × 10⁶) per culture for these responses to be observed and that for specific stimulation by T. spiralis soluble antigen 10 μg per culture gave optimal results.     

DNA Strand Breakage in Chinese Hamster V79 Cells Caused by Low Levels of Incorporated [3H] and [14C]thymidine

Summary

A sensitive alkali-unwinding assay was used to measure DNA strand breakage in Chinese hamster V79 cells caused by low-level incorporation of methyl-labelled [³H] and [¹⁴C] thymidine, and to estimate the effective dose per disintegration relative to low doses of gamma-irradiation. Damage equivalent to 0·0035 ± 0·0006 and 0·0014 ± 0·0005 Gy was observed for each ³H and ¹⁴C disintegration respectively. These values agree well with those expected from the estimated nuclear radiation dose delivered by the beta particles if a relative biological effect (r.b.e.) of 1·0 is assumed, and suggest that strand-breakage produced by these isotopes is determined by the nuclear radiation dose delivered by the beta particles.     

Evaluation of Radiation Dose Resulting from the Ingestion of [3H]- and [14C]thymidine in the Rat

Summary

Average doses to rat tissues from the ingestion of 2-[¹⁴C]thymidine were compared with those from methyl-[³H]thymidine or 6-[³H]thymidine. Among the three precursors, [¹⁴C]thymidine gave the highest dose to spleen and small intestine. The doses to other tissues from [¹⁴C]thymidine were almost the same or lower as compared with those from [³H]thymidine, irrespective of the 9 times higher β-ray energy of ¹⁴C than that of ³H. In the case of [¹⁴C]thymidine, most of the dose was given by radioactivity incorporated into the organic tissue constituents (non-volatile radioactivity). In the case of [³H]thymidine, however, the dose contributions by non-volatile radioactivity were very small and the major contributions were rather from volatile radioactivity (³HHO), formed by degradation of [³H]thymidine. No significant difference in their total doses was found between the two [³H]precursors, but the dose from non-volatile radioactivity alone was 2–3 times higher with methyl-[³H]thymidine than with 6-[³H]thymidine. Estimates of the dose to cell nuclei in various tissues after the ingestion of [³H]thymidine were also made in order to predict more precisely possible radiation hazards.     

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.     

Transport mechanisms of 3-[123I]iodo-alpha-methyl-L-tyrosine in a human glioma cell line: comparison with [3H]methyl]-L-methionine.

The amino acid analog 3-[(123)I]iodo-alpha-methyl-L-tyrosine (IMT) is under clinical evaluation as a SPECT tracer of amino acid transport in brain tumors. This study investigated the carrier systems involved in IMT transport in human glioma cells in comparison with [3H-methyl]-L-methionine (3H-MET). METHODS: Human glioma cells, type 86HG-39, were cultured and incubated for 1 min at 37 degrees C with IMT and 3H-MET in the lag phase (1.2 d after seeding), exponential growth phase (3 d after seeding), and plateau phase (8 d after seeding). Experiments were performed in the presence and absence of Na+, during inhibition of system L amino acid transport by 2-aminobicyclo[2.2.1 ]heptane-2-carboxylic acid (BCH), and during inhibition of system A amino acid transport by 2-(methylamino)-isobutyric acid (MeAIB). RESULTS: IMT and 3H-MET uptake decreased by 55%-73% when the cells entered from the exponential growth phase into the plateau phase (P< 0.05; n = 3-11). Inhibition by BCH reduced uptake of IMT in the lag phase, exponential growth phase, and plateau phase by 90%-98% (P < 0.001; n = 3-6) and the uptake of 3H-MET by 73%-83% (P < 0.001; n = 3-11). In a Na+-free medium 3H-MET uptake was reduced by 23%-33% (P < 0.05; n = 3-11), whereas IMT uptake was not significantly different. MeAIB showed no significant effect on IMT or 3H-MET uptake in either phase. CONCLUSION: Transport of both IMT and 3H-MET depends on the proliferation rate of human glioma cells in vitro and is dominated by BCH-sensitive transport. These data indicate that system L is induced in rapidly proliferating glioma cells and is the main contributor to the uptake of both tracers. 3H-MET transport showed a minor Na+ dependency that was not attributable to system A. The similarity of transport mechanisms of both tracers emphasizes the clinical equivalence of IMT SPECT and (11)C-MET PET for the diagnostic evaluation of gliomas.     

Quiescence in S-phase and G 1 arrest induced by irradiation and/or hyperthermia in six human tumour cell lines of different p53 status

Purpose : Quiescent S-phase cells, i.e. cells with a DNA content intermediate between G1 and G2 that nevertherless do not synthesize DNA have been previously observed in human melanoma cells exposed to radiation and/or hyperthermia. This phenomenon has now been studied in more detail comparing six human tumour cell lines of different p53 status and thus different cell-cycle checkpoint control. Materials and methods : Two melanoma (Be11, MeWo), two squamous carcinoma (4197, 4451) and two glioma (EA14, U87) cell lines were used. Changes in the cell-cycle distribution after treatment were studied using two-parameter flow cytometry in order to measure DNA content and BrdU incorporation simultaneously. Results : The fraction of unlabelled cells in the S-phase compartment was determined at daily intervals after treatment. Only background levels of such cells were seen in three of the cell lines (Be11, 4197, EA14). With the other three cell lines (MeWo, 4451, U87) we observed a time- and dose-dependent increase: a few days after treatment up to 20% of all cells did not incorporate BrdU. It is interesting to note that Be11, 4197 and EA14 are p53 wild-types and show a G 1 block of several hours after irradiation and/or hyperthermia, while MeWo and 4451 are p53 mutants unable to exhibit such a delay, and U87 in spite of being a p53 wild-type has a reduced ability to do so. Conclusions : The MeWo, 4451 and U87 cell lines have less time available for the repair of DNA damage before entering into the S-phase, which leads to problems during replication and causes some kind of interphase death. Radiation-induced apoptosis does not seem to be involved here, as it is not unequivocally correlated with the induction of a G 1 block or with p53 status.     

Quiescence in S-phase and G1 arrest induced by irradiation and/or hyperthermia in six human tumour cell lines of different p53 status

PURPOSE: Quiescent S-phase cells, i.e. cells with a DNA content intermediate between G1 and G2 that nevertheless do not synthesize DNA have been previously observed in human melanoma cells exposed to radiation and/or hyperthermia. This phenomenon has now been studied in more detail comparing six human tumour cell lines of different p53 status and thus different cell-cycle checkpoint control. MATERIALS AND METHODS: Two melanoma (Be11, MeWo), two squamous carcinoma (4197, 4451) and two glioma (EA14, U87) cell lines were used. Changes in the cell-cycle distribution after treatment were studied using two-parameter flow cytometry in order to measure DNA content and BrdU incorporation simultaneously. RESULTS: The fraction of unlabelled cells in the S-phase compartment was determined at daily intervals after treatment. Only background levels of such cells were seen in three of the cell lines (Be11, 4197, EA14). With the other three cell lines (MeWo, 4451, U87) we observed a time- and dose-dependent increase: a few days after treatment up to 20% of all cells did not incorporate BrdU. It is interesting to note that Bell, 4197 and EA14 are p53 wild-types and show a G1 block of several hours after irradiation and/or hyperthermia, while MeWo and 4451 are p53 mutants unable to exhibit such a delay, and U87 in spite of being a p53 wild-type has a reduced ability to do so. CONCLUSIONS: The MeWo, 4451 and U87 cell lines have less time available for the repair of DNA damage before entering into the S-phase, which leads to problems during replication and causes some kind of interphase death. Radiation-induced apoptosis does not seem to be involved here, as it is not unequivocally correlated with the induction of a G1 block or with p53 status.     

[3H]西达本胺在大鼠体内的排泄研究

目的:运用氧化燃烧炉预处理样品技术对[3H]西达本胺在大鼠体内的排泄进行研究;并运用同位素标记技术初步推断西达本胺在大鼠体内的代谢产物。方法:采用氧化燃烧炉处理[3H]西达本胺粪、尿、胆汁样品,用液闪计数仪测定其放射性水平。结果:方法学确证研究结果显示粪、尿、胆汁样品3H回收率基本为100%,精密度和准确度均小于10%。大鼠单次灌胃给[3H]西达本胺(1.2 mg/kg,1.24 mC i/kg),336 h后从粪中收集到给药剂量的(63.28±12.40)%,尿中收集到(18.77±3.21)%,粪尿合计(82.25±13.15)%;给药后48 h胆汁排泄出给药剂量的(4.28±2.72)%。另外,经HPLC仪收集纯化样品后测定放射性得到的色谱图发现在大鼠血浆、粪、尿和胆汁中均有西达本胺代谢物峰,原形药物在图谱上的放射性约占50%。结论:氧化燃烧法用于处理[3H]西达本胺的大鼠粪、尿、胆汁样品简便、准确。大鼠灌服给药后,在血浆、粪、尿和胆汁中均发现有西达本胺原形药物和代谢产物,约有一半以原形排出体外。     

Enhanced binding of the hypoxic cell marker [3H]fluoromisonidazole in ischemic myocardium

The 2-nitroimidazole fluoromisonidazole is metabolically trapped in viable hypoxic cells in inverse proportion to PO2. This attribute suggests that [18F]fluoromisonidazole may be useful for imaging hypoxic tissue using positron emission tomography. To examine this potential, we studied the pharmacokinetics and biodistribution of [3H]fluoromisonidazole in six open chest dogs. In two normal dogs, plasma and urine samples were collected over a 4-hr period following i.v. injection of the drug. In four animals, regional myocardial ischemia was produced 2 hr prior to drug injection by occlusion of the circumflex coronary artery and maintained during the 4-hr sampling period. In all animals, postmortem samples of myocardium and other organs were obtained and tissue, plasma, and urine tritium activity were determined by liquid scintillation counting. In areas of reduced flow, [3H]fluoromisonidazole accumulated in myocardium in inverse proportion to myocardial blood flow measured by microspheres, indicating enhanced binding in hypoxic tissues. Maximum tissue concentrations in ischemic myocardium were two- to three-fold greater than in normal myocardium and plasma. Plasma clearance data indicate the drug is rapidly distributed into the total-body water, clears from the body with a half-life of 275 +/- 50 min, and undergoes minimal metabolism by 4 hr. We conclude [18F]fluoromisonidazole may be a suitable agent for radionuclide imaging of hypoxic myocardium.     

Differential uptake of [18F]FET and [3H]l-methionine in focal cortical ischemia

Amino acids such as [(11)C-methyl]l-methionine are particularly useful in brain tumor diagnosis, but unspecific uptake (e.g., in cerebral ischemia) has been reported. O-(2-[(18)F]fluoroethyl)-l-tyrosine ([(18)F]FET) shows a clinical potential similar to that of l-methionine (MET) in brain tumor diagnosis but is applicable on a wider clinical scale. The aim of this study was to evaluate the uptake of [(18)F]FET and [(3)H]MET in focal cortical ischemia in rats by dual-tracer autoradiography. METHODS: Focal cortical ischemia was induced in 25 CDF rats using the photothrombosis (PT) model. At different time points up to 6 weeks after the induction of PT, [(18)F]FET and [(3)H]MET were injected intravenously. Additionally, contrast-enhanced magnetic resonance imaging (MRI) was performed in 10 animals. One hour after tracer injection, brains were cut in coronal sections and evaluated by dual-tracer autoradiography. Lesion-to-brain (L/B) ratios were calculated by dividing the maximal uptake in the lesion by the mean uptake in the brain. An L/B ratio of >2.0 was considered indicative of pathological uptake. Histological slices were stained by cresyl violet and supplemented by immunostainings for glial fibrillary acidic protein (GFAP) and CD68 in selected cases. RESULTS: A variably increased uptake of both tracers was observed in the PT lesion and its demarcation zone up to 7 days after PT for [(18)F]FET and up to 6 weeks for [(3)H]MET. The cutoff level of 2.0 was exceeded in 12/25 animals for [(18)F]FET and in 18/25 animals for [(3)H]MET. Focally increased tracer uptake matched contrast enhancement in MRI in 3/10 cases for [(18)F]FET and in 5/10 cases for [(3)H]MET. Immunohistochemical staining in lesions with differential uptake of [(18)F]FET and [(3)H]MET revealed that selective uptake of [(18)F]FET was associated with GFAP-positive astrogliosis while selective [(3)H]MET uptake correlated with CD68-positive macrophage infiltration. CONCLUSIONS: [(18)F]FET, like [(3)H]MET, may exhibit significant uptake in the periphery of cortical infarctions, which has to be considered in the differential diagnosis of unknown brain lesions. There are discrepancies between [(18)F]FET and [(3)H]MET uptake in the area of infarctions that appear to be caused by the preferential uptake of [(18)F]FET in reactive astrocytes versus the preferential uptake of [(3)H]MET in macrophages.     

电离辐射诱导的细胞G2期阻滞

哺乳动物受X射线照射后,可以使细胞周期延迟或阻滞,包括G1期阻滞、S期延迟和G2期阻滞。G1期阻滞仅在野生型p53基因存在时出现,在清除DNA损伤的细胞中具有重要作用;而G2期阻滞更有利于损伤后DNA的修复和细胞存活,并且与p53基因存在状态无关。因此,对电离辐射诱导细胞G2期阻滞机制的探讨成为近年来国内外放射生物学领域的研究热点。     

Effects of Irradiation on the [Methyl-3H]Choline Uptake in the Human Prostate Cancer Cell Lines LNCaP and PC3

BACKGROUND AND PURPOSE: Choline positron emission tomography (PET) can help to optimize radiation treatment strategy of prostate cancer. Therefore, the aim of this study was to elucidate the effects of ionizing radiation on the choline uptake in an androgen-dependent (LNCaP) and an androgen-independent (PC3) prostate cancer cell line. MATERIAL AND METHODS: Uptake of [methyl-(3)H]choline chloride was investigated between 4 and 96 h after irradiation with 6 Gy. Dose dependence of choline uptake was examined following irradiation with 2-12 Gy, and cell survival was analyzed via the clonogenic assay. Michaelis-Menten kinetics was determined 24 h (PC3) and 48 h (LNCaP) after irradiation with 6 Gy. RESULTS: PC3 cells showed a significant transitory increase of [methyl-(3)H]choline uptake with a maximum at 24 h after irradiation. In LNCaP cells irradiation induced a significant decrease with a minimum at 48 h. Changes in choline uptake in both cell lines were almost dose-independent up to 12 Gy. Following irradiation with 6 Gy, transport capacity (v(max)) increased and Michaelis-Menten constant (K(M)) decreased in PC3 cells, while in LNCaP cells the two parameters behaved vice versa. CONCLUSION: Changes in choline uptake following irradiation might be due to metabolic changes associated with initiation of processes that finally cause cell death. Thus, changes in tumor choline uptake monitored by PET after radiotherapy might not exclusively reflect therapeutic success but also altered tracer uptake as a consequence of irradiation.     

Lack of correlation between G1 arrest and radiation age-response in three synchronized human tumour cell lines

PURPOSE: To determine radiosensitivity as a function of cell age (the age-response) in three human tumour cell lines, and investigate the dependence of the age-response on G1 arrest and on cell-age heterogeneity in synchronized cell populations. MATERIALS AND METHODS: Variation in radiosensitivity throughout the cell cycle and G1 arrest was measured in mitotically selected populations of synchronized human tumour cells. In order to examine the effects of desynchronization and cell age heterogeneity on the measured age-response, a mathematical model was developed based on an existing kinetic model of the cell cycle. The model was used to describe the age-response for mitotically selected populations of cells, which was then compared with experimentally measured age responses. RESULTS: Three different human tumour cell lines had qualitatively similar age-responses, with periods of radiosensitivity in mitosis and in late G1 phase/early S phase, and periods of radioresistance in early/mid G1 phase and late S/G2 phase. Radiosensitivity appeared to increase in G1 phase before the onset of DNA synthesis. One of the cell lines displayed a prolonged G1 arrest after irradiation in G1 phase. Model results demonstrated that the measured age-responses were consistent with a simple model in which the cell cycle was divided into four regions. Radiosensitivity was assumed to be constant within each region, and changed abruptly at the borders between regions. CONCLUSIONS: Human tumour cell lines can exhibit qualitatively similar age-responses despite having markedly different G1 checkpoint responses. This suggests that modulation of the G1 arrest response may not prove to be a useful clinical strategy because it may not lead to significant cell age specific changes in radiosensitivity. The mathematical model of the radiation response of mitotically selected synchronized cells was a useful way to quantitatively describe cell age heterogeneity in these populations, and demonstrated the important impact of this heterogeneity on measured age-responses.     

Lack of correlation between G1 arrest and radiation age-response in three synchronized human tumour cell lines

Purpose: To determine radiosensitivity as a function of cell age (the age-response) in three human tumour cell lines, and investigate the dependence of the age-response on G1 arrest and on cell-age heterogeneity in synchronized cell populations. Materials and methods: Variation in radiosensitivity throughout the cell cycle and G1 arrest was measured in mitotically selected populations of synchronized human tumour cells. In order to examine the effects of desynchronization and cell age heterogeneity on the measured age-response, a mathematical model was developed based on an existing kinetic model of the cell cycle. The model was used to describe the age-response for mitotically selected populations of cells, which was then compared with experimentally measured age-responses. Results: Three different human tumour cell lines had qualitatively similar age-responses, with periods of radiosensitivity in mitosis and in late G1 phase/early S phase, and periods of radioresistance in early/mid G1 phase and late S/G2 phase. Radiosensitivity appeared to increase in G1 phase before the onset of DNA synthesis. One of the cell lines displayed a prolonged G1 arrest after irradiation in G1 phase. Model results demonstrated that the measured age-responses were consistent with a simple model in which the cell cycle was divided into four regions. Radiosensitivity was assumed to be constant within each region, and changed abruptly at the borders between regions. Conclusions: Human tumour cell lines can exhibit qualitatively similar age-responses despite having markedly different G1 checkpoint responses. This suggests that modulation of the G1 arrest response may not prove to be a useful clinical strategy because it may not lead to significant cell age specific changes in radiosensitivity. The mathematical model of the radiation response of mitotically selected synchronized cells was a useful way to quantitatively describe cell age heterogeneity in these populations, and demonstrated the important impact of this heterogeneity on measured age-responses.     

3-[123I]Iodo-L-alpha-methyl tyrosine transport into human fibroblasts and comparison with Ewing's sarcoma cells

The cellular transport systems and the transport kinetics of [123I]IMT uptake into non-malignant extracranial cells were characterized for the first time. Human fibroblasts were chosen as non-malignant extracranial cells as they are found ubiquitous in the body. [123I]IMT is exclusively transported into fibroblasts via the sodium independent system L. An apparent Michaelis constant K(m) = 116.2 +/- 18.9 microM and a maximum transport velocity V(max) = 191.6 +/- 13.9 pmol x (10(6) cells)(-1) x min(-1) were calculated for the sodium-independent transport. These results were compared with those determined in two malignantly transformed extracranial cell lines, the human Ewing's sarcoma cell lines VH-64 and CADO-ES-1.