[3H]thymidine labels less than half of the DNA-synthesizing cells in the mouse tumour, carcinoma NT

Abstract. We have studied carcinoma NT, a transplantable mouse adenocarcinoma of spontaneous origin. Cells labelled with [³H]thymidine ([³H]TdR) were restricted to a narrow zone around the periphery of this tumour and were also found in rings up to 50 μ m wide, around isolated blood vessels in the central necrotic area. Labelling with [³H]deoxyuridine ([³H]UdR), another DNA synthesis precursor, produced a very different pattern. The labelled zone around the periphery was much wider than with [³H]TdR, and [³H]UdR labelled cells were found up to 110 μ m from isolated vessels. [³H]iododeoxyuridine ([³H]IUdR) gave the same pattern of labelling as [³H]UdR. In the heavily labelled zone, within 1 mm of the tumour periphery, the labelling index (LI) was 51% after [³H]UdR or [³H]IUdR injection, and only 36% with [³H]TdR.
The data show that at least half of the DNA-synthesizing cells in this tumour did not incorporate [³H]TdR. Previous workers reported cell loss factors for carcinoma NT of 60% calculated from [³H]TdR labelling data and 30% from the rate of loss of [¹²⁵I]UdR. The present work suggests that calculations based on [¹²⁵I]UdR data are more likely to be accurate for carcinoma NT than those using [³H]TdR data.     

Double labelling of tissue combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine: the autoradiographic significance of inhibition of thymidine incorporation into DNA by bromodeoxyuridine given simultaneously

Abstract We describe a reproducible method for combining tritiated thymidine ([³H]TdR) autoradiography with immunoperoxidase detection of bromodeoxyuridine (BrdU) in paraffin-embedded tissues. The technique has been used to examine, in mouse tongue epithelium, the inhibition of incorporation into DNA of [³H]TdR by a simultaneous injection of BrdU in the doses that both compounds are likely to be used in cell proliferation studies. The significance that this inhibition has on prolongation of autoradiograph exposure times, to ensure that all cells that incorporate [³H]TdR are scored as positive, in particular the most lightly labelled cells, has been quantified.
The inhibition of uptake into DNA of [³H]TdR from 0.23 to 1.85 MBq (6.25 to 50 μCi) per animal, produced by a simultaneous injection of 2.5 mg BrdU shows a linear, dose-dependent relationship. Provided the injected dose (in μCi per animal) multiplied by the autoradiographic exposure time (in days) is greater than a value of 700, then all cells that are labelled after incorporation of [³H]TdR alone are also labelled after simultaneous double labelling, despite the latter producing a lower average grain count.     

Effect of Vinblastine On the Cell Cycle and Migration of Ameloblasts of Mouse Incisors As Shown By Autoradiography Using 3H-Thymidine

Abstract. The effects of vinblastine on the cell cycle and the migration of ameloblasts were studied in the lower incisors of mice by labelling the cells with ³H-thymidine ([³H]TdR) and radioautography. A group of mice received 2 μg/g of body weight vinblastine intraperitoneally and 6 hr after these animals and those of a control group were injected with 1μCi/g body weight of [³H]TdR, and sacrificed at time intervals from 0.75 hr to 15 days.
The generation time of ameloblasts in the progenitor compartment was 14.8 hr in animals treated with vinblastine and 17 hr in the controls, using the FLM curve method; with the grain dilution method the duration was respectively 29.25 hr and 25.96 hr. the thymidine labelling index of the treated animals was 50% higher than the controls. the velocity of ameloblast migration, determined either by the displacement of the most incisally labelled cell or by the grain dilution method, was lower in the experimental group (2.48 cell positions/hr and 9.18 μ/hr respectively) as compared with the control (3.21 cell positions/hr and 18.88 μm/hr respectively).
The results on the ameloblast production rate are contradictory but the slowing down in the velocity of cell migration is compatible with a decrease of the rate of cell production in the progenitor compartment as a vinblastine effect.     

Tracer dose and availability time of thymidine and bromodeoxyuridine: application of bromodeoxyuridine in cell kinetic studies

Abstract. The present experiments with [¹⁴C]-thymidine (TdR) and [³H]-bromo-deoxyuridine (BrdU) using mouse jejunal crypt cells show that the upper limit of the tracer dose of TdR is about 0.5 µg g body weight^-1 and that of BrdU is about 5·0 µg g body weight^-1. Applying these doses, the proportions of the endogenous DNA synthesis attributed to the exogenous DNA precursor are 2% and 9% respectively. For [³H]-TdR doses commonly used in cell kinetic studies this proportion is only 0-1-1.0%, a negligible quantity that does not influence the endogenous DNA synthesis. The maximum availability time of tracer doses of TdR as well as BrdU is 40 to 60 min, the majority of the precursors being incorporated after 20 min. The availability time is the same for TdR doses exceeding the tracer dose by a factor of 80, whereas it is prolonged in the case of BrdU doses exceeding the tracer dose by a factor of 50. BrdU is suitable to replace radioactively labelled TdR in short term cell kinetic studies, i.e. determination of the labelling index or of the S phase duration by double labelling. However, more studies are needed to elucidate how far BrdU can replace TdR in long term studies as shown by differences between the fraction of labelled mitoses (FLM) curves of a human renal cell carcinoma measured with BrdU and [³H]-TdR.     

The Synthesis and Release of [3H-Tyrosine1]Methionine5-Enkephalin from Guinea Pig Brain Slices

Abstract: Stores of methionine-enkephalin were labelled on the N -terminal by incubation of whole brain slices with [³H]tyrosine (10 °Ci/ml). The ³H radioactivity corresponding to the position of authentic Met-enkephalin after extraction on Amberlite XAD₂ and separation by thin-layer chromatography was taken as an index of synthesis. Maximal incorporation of the labelled tyrosine into Met-enkephalin was attained after 4 h of incubation at 37°C and was inhibited in the presence of 10 μ M cycloheximide. Isolated nerve terminals failed to incorporate any [³H]tyrosine. The labelled compound had opiatelike activity and consisted of the same five amino acids as an authentic standard. Incubations with leucine aminopeptidase indicated that the labelled tyrosine was on the N -terminus and removal of this tyrosine resulted in loss of opiate-like activity. The incorporation of [¹⁴C]glycine, selected as an alternative precursor, was consistent with de novo synthesis and not N -terminal exchange. A radioimmunoassay was also used to quantify the amount of labelled Met-enkephalin. KCl (50 m M ) elicited a Ca²⁺-dependent release of the synthesised [³H]Met-enkephalin from whole brain slices and also from isolated nerve terminals. The release of Met-enkephalin radioimmunoactivity paralleled that of [³H]met-enkephalin. Preliminary investigations have suggested that carbamyl choline inhibited this release and its effect was partially reversed by atropine.     

Turnover and Maturation Kinetics In the Hairless Mouse Epidermis. Continuous [3H]Tdr Labelling and Mathematical Model Analyses

Abstract. Hairless mice were continuously labelled with 10μCi of tritiated thymidine ([³H]TdR) every 4 h for 8 d, and the proportions of labelled basal and differentiating cells were recorded separately. the mitotic rate was measured by the stathmokinetic method and the cell cycle distributions were measured by flow cytometry of isolated basal cells at intervals during the labelling period. the mitotic rate of the [³H]TdR-injected animals did not deviate from control values during the first 5 d. Computer simulations of the data based on various mathematical models were made, and three main conclusions were obtained: (1) a large spread in transit times through the G₁ phase was found, together with a very narrow distribution in maturation time of differentiating cells; (2) about 20% of the differentiating cells were estimated to leave the basal cell layer directly after mitosis. This is consistent with results obtained from different sets of data; and (3) during continuous labelling more than 90% of the cells are labelled during each passage through the S phase.     

SUBCELLULAR DISTRIBUTION OF ENDOGENOUS AND [3H] γ-AMINOBUTYRIC ACID IN RAT CEREBRAL CORTEX

Abstract— Slices of rat cerebral cortex were labelled by incubation with [³H]γ-aminobutyric acid (GABA) and homogenized in isotonic sucrose. The subcellular distributions of endogenous GAB A, [³H]GABA and glutamate decarboxylase (GAD) were studied by density gradient centrifugation. The subcellular distributions of the labelled and endogenous amino acid were remarkably similar, indicating that [³H]GABA is taken up into the endogenous GABA pool. About 40 per cent of both endogenous and [³H]GABA were recovered in particles which were tentatively identified as synaptosomes from their equilibrium density and sensitivity to osmotic shock. In slices labelled with [³H]GABA and [¹⁴C]α-aminoisobutyric (AIB) acid, significantly more [³H]GABA was recovered in paniculate fractions than [¹⁴C]AIB. About 80 per cent of the enzyme GAD was also recovered in the same particle fractions which contained [³H]GABA and endogenous GABA. Evidence is presented which suggests that a loss of particle-bound GABA occurs during subcellular fractionation procedures.     

Development and Localization of the Thymidine Phosphorylating Systems in Brain

Abstract: The development of the thymidine phosphorylating systems was studied in various regions of brain. Brain slices from cerebellum, brain stem, and forebrain of rabbits 2, 7, 14, 30, 90, 500, and 2500 days of age were incubated for various times in artificial CSF containing 3 nM-[³H]thymidine at 37°C under 95% O₂-5% CO₂. When slices from all brain regions of 2-day-old rabbits were incubated in [³H]thymidine for 30 min, tissue-to-medium ratios of ³H were between 2 and 4 and declined with age, and the percentages of the total ³H in perchloric acid homogenates of brain slices as [³H]DNA were 26–29%, declining to low levels with age. However, at all ages and in all regions studied, 41 -88% of the ³H within the slices was phosphorylated. After homogenization and subcellular fractionation of the brain slices incubated in [³H]thymidine for 30 min, the highest percentage of [³H]thymidine phosphates plus [³H]DNA was present in the nuclear (crude and purified) and mitochondrial fractions of all brain regions. The [³H]DNA content in the nuclear and mitochondrial fractions declined with age, but the percentage of [³H]thymidine phosphates did not. Thymidine phosphates were synthesized from thymidine in all brain regions tested throughout the entire life span.     

The proliferative response of rat liver parenchymal cells after partial hepatectomy A methodological study comparing flow cytometry of nuclear DNA content and in vivo and in vitro uptake of thymidine

Abstract. DNA synthesis in rat hepatocytes, from livers regenerating after 70% hepatectomy, was assessed by flow cytometric determination of nuclear DNA content and by incorporation of [³H]thymidine. Parenchymal liver cells were isolated by collagenase perfusion and low-speed centrifugation. Nuclei from the isolated cells were prepared for flow cytometry by a treatment with detergent, pepsin and RNase, and stained with ethidium bromide. Parallel samples of cells were incubated with [³H]thymidine and analysed for rate of incorporation of radioactivity into DNA and for labelling index determination.
The flow cytometric measure of the replicative response, i.e. the presence of cells with S-phase DNA content within the diploid and tetraploid cell populations, was compared with the incorporation of [³H]thymidine. For each of fourteen animals, including two control rats and twelve partially hepatectomized animals killed either before (at 13 hr after hepatectomy), at the onset (16 and 18 hr) or at the peak (24 hr) of regenerating activity, a fairly good correlation was found between the different methods. Satisfactory resolution of the flow cytometric detection of S-phase cells was indicated by a sorting experiment using an Ortho (system 50-H) cell sorter which demonstrated that after [³H]thymidine injection in vivo 88% of the diploid and 84% of the tetraploid S-phase nuclei were labelled, while labelling in the G¹-fractions was only 2 and 7%, respectively.     

Local Re-Utilization of Thymidine In Normal Mouse Tissues As Measured With Iododeoxyuridine

Abstract. Thymidine (TdR) and its analogue, iododeoxyuridine (IdUdr), were used to quantitate nucleoside re-utilisation in vivo. Significantly different results are obtained, however, depending upon what form of isotopically labelled iododeoxyuridine is used. No measurable local thymidine re-utilization was found in mouse thymus, spleen or bone marrow when the retention of [³H]IdUdR was compared with [¹⁴C]TdR. On the other hand, significant differences were found between the retention of [¹²⁵I]IdUdR and [³H]IdUdR, which is attributed to de-iodination of iododeoxyuridine. Some thymidine re-utilization was found in duodenum using both [³H]IdUdR and [¹²⁵I]IdUdR. Information on the in vivo distribution of TdR and the contention that a large degree of TdR re-utilization in the thymus is evidence of extensive cell death must be re-interpreted in the light of these results. In addition, evidence for little or no local re-utilization in some tissues will greatly simplify the use of [¹¹C]TdR as an imaging agent for measuring tissue proliferation in vivo with positron emission tomography (PET).     

DNA-synthesizing cells in oral epithelium have a range of cell cycle durations: evidence from double-labelling studies using tritiated thymidine and bromodeoxyuridine

Abstract Mouse tongue epithelium is characterized by a circadian variation in the number of DNA-synthesizing cells (labelling index, LI). Cells undergoing DNA synthesis were labelled with tritiated thymidine ([³H]TdR) at 0300 (peak LI) or 1200 h (low LI). The fate of these cells was assessed by injecting animals with bromodeoxyuridine (BrdU) at intervals from 12–48 h after [³H]TdR, to follow them from one cell cycle to the next. Labelling was revealed by combining [³H]TdR autoradiography with immunoperoxidase detection of BrdU in the same sections.
A single peak in the appearance of double-labelled cells was seen at 44 h, if [³H]TdR was given at 1200 h; following [3H]TdR at 0300 h, a peak of double labelling was seen at 48 h with the possibility of smaller peaks at 24 h and 36 h.
These results show that the 24 h periodicity in LI in this tissue is associated with a predominant cell cycle duration of 44–48 h, but that a few cells cycle more quickly. Double labelling with [³H]TdR and BrdU provides a useful method for establishing cell cycle duration by labelling S-phase cells in successive cell cycles.     

Influence of Growth Hormone and Thyroxine On Cell Kinetics In the Proximal Tibial Growth Plate of Snell Dwarf Mice

Abstract. In Snell dwarf mice, the influence of short-term treatment with human growth hormone (hGH) or thyroxine on the proliferative and sulphation activity of the proximal tibial growth plate was studied. By autoradiographic methods, the [³H]methylthymidine incorporation after a single injection was measured, after 2 hr incorporation time. the labelling index was calculated and the number of labelled mitoses was counted. In addition, the distribution of the labelled nuclei over the proliferating and degenerating zones was determined by continuous labelling for 25 and 73 hr.
In untreated dwarf mice after [³H]-methylthymidine administration, the number of labelled nuclei in the growth plate is low. Labelling occurs, as expected, mainly in the cells of the proliferative zones. the number of labelled nuclei in control dwarf mice was similar after 25 and 73 hr continuous labelling. This suggests that many cells are in a resting G_o or prolonged G₁ phase. Both hGH and T₄ treatment induce a significant increase of the number of labelled nuclei per growth plate and of the number of mitoses. Since hormonal treatment induces a small number of mitoses after 2 hr incorporation of the label, the minimal G₂ phase of the cell cycle is less than 2 hr. In addition, treatment with hGH and T₄ stimulates chondrocytes in the zone of proliferative and hypertrophic cells to actively incorporate [³⁵S]-sulphate.     

[3H]Nemonapride and [3H]Spiperone Label Equivalent Numbers of D2 and D3 Dopamine Receptors in a Range of Tissues and Under Different Conditions

Abstract: [³H]Nemonapride and [³H]spiperone are very widely used to study dopaminergic systems in vitro and in vivo, but it has been reported that [³H]nemonapride and [³H]spiperone give markedly different B _max values for preparations of D₂ dopamine receptors from recombinant cell lines or animal tissues. We have used the two radioligands in parallel to study a range of dopamine receptors [D_2(short), D_2(long), and D₃] in different buffers. B _max values derived using either radioligand differ by an average of <20%, independent of receptor type or buffer conditions. All competition experiments show that the two ligands compete at a single site. It seems that [³H]spiperone and [³H]nemonapride do not differentiate between different forms or populations of D₂-like receptors.     

Metabolism of Deoxyuridine in Rabbit Brain

Abstract: The metabolism of [³H]deoxyuridine by rabbit brain was investigated in vitro and in vivo . In vitro , brain slices from various regions of brain and from all age groups accumulated [³H]deoxyuridine from artificial CSF. Within the slices, a portion of the accumulated [³H]deoxyuridine was metabolized to [³H]deoxyuridine phosphate, with subsequent conversion to [³H]thymidine phosphate, and ultimately [³H]DNA. The percentage of the [³H]deoxyuridine phosphorylated and subsequently converted into [³H]DNA was highest at birth and declined to adult levels in 3-month-old rabbits. Thymidine, when added to the incubation medium with the [³H]deoxyuridine, was approximately 10 times as potent as unlabeled deoxyuridine in inhibiting the intracellular phosphorylation and conversion of [³H]deoxyuridine to [³H]thymidine phosphate in brain slices. In vivo , 2.5 h after intraventricular injection of [³H]deoxyuridine, over 90% of the [³H]deoxyuridine was cleared from the central nervous system at all ages. However, in both newborn and 3-month-old rabbits, approximately 40 and 12%, respectively, of the ³H remaining in brain was phosphorylated and converted to [³H]thymidine phosphates; and 11 and 4%, respectively, of the ³H remaining in brain was converted to [³H]DNA. These results show that both immature and mature rabbit brain is able to incorporate deoxyuridine into DNA. Thus, all the enzymes involved in this conversion, including thymidylate synthetase (EC 2.1.1.45), are present and active in brain throughout life.     

Localization and Mechanism of Thymidine Transport in the Central Nervous System

Abstract: The localization and mechanism of thymidine and deoxyuridine transport in the central nervous system were studied in vivo and in vitro . Previous studies have shown that thymidine enters brain from blood in part via the CSF. In vitro , isolated adult bovine cerebral microvessels, which readily concentrated and phosphorylated deoxyglucose, were unable to concentrate thymidine and deoxyuridine. In vivo , [³H]thymidine (0.2 μ M ) and [³H]deoxyuridine(0.4 μ M ) were not extracted more readily than [¹⁴C]sucrose in a single pass through the cerebral circulation of rats. In vivo , [³H]thyrnidine retention in CSF and brain after entry from blood was increased when the efflux of [³H]thymidine from CSF and the phosphorylation of [³H]thymidine in brain were depressed by the intraventricular injection of unlabeled thymidine. These studies and previous work suggest that the transfer of thymidine (and deoxyuridine) through the blood-brain barrier in either direction must be extremely low. The present studies are consistent with the postulate that thymidine is transported by an active transport system in the choroid plexus that transfers thymidine from blood into the CSF; from the CSF, the thymidine enters brain cells and is phosphorylated.     

RADIOTOXICITY OF INCORPORATED [3H]THYMIDINE AS STUDIED BY AUTORADIOGRAPHY AND FLOW CYTOMETRY CONSEQUENCES FOR THE INTERPRETATION OF FLM DATA

The radiotoxic effects of incorporated [³H]thymidine on proliferation kinetics of flash-labelled (30 min, 0.3 μCi/ml, 40 Ci/mM) L-929 cells in vitro were studied by means of autoradiography and flow cytometry. the flow cytometric results obtained by applying the BUdR-33258 Hoechst technique, using new evaluation procedures, showed that the labelled cells are delayed in their progression through the S and G₂+ M phases, leading to mitotic delay. From autoradiographs, the fraction of labelled mitoses was determined and, in addition, the ratio of labelled and of unlabelled mitotic cells to all cells. the radiotoxic effects are not evident from the FLM curve, even if the ratio of labelled mitotic cells to all cells shows a highly distorted shape. A mathematical model has been developed that describes the perturbed cell kinetics due to radiotoxic effects of the incorporated [³H]thymidine. These findings have considerable consequences for the interpretation of autoradiograhic data, especially of labelled mitoses curves.     

Trifluoperazine inhibits the incorporation of labelled precursors into lipids, proteins and DNA of Mycobacterium tuberculosis H37Rv

Abstract We have recently demonstrated that the calmodulin antagonist trifluoperazine has antitubercular activity in vitro against Mycobacterium tuberculosis H₃₇R_v susceptible and resistant to isoniazid. It is shown that trifluoperazine at a concentration of 50 μ g ml⁻¹ when added to the cells along with the labelled precursors inhibited the incorporation of [¹⁴C]acetate into lipids (63%) and uptake of [¹⁴C]glycine (74%) and [^3H]thymidine (52%) bu whole cells of M. tuberculosis H₃₇R_v by 6 h of exposure. After 48 h, the inhibition was 87%, 97% and 74%, respectively. However, when the drug was added to cells taking up and metabolizing the labelled precursors at a later point (3 h for [¹⁴C]acetate and [³H]thymidine and 12 h for [¹⁴C]glycine) it inhibited completely the uptake of all the precursors, at least up to 24 h. The onset of inhibitory action was very rapid, i.e. 3 h. It is suggested that trifluoperazine has multiple sites of action and acts probably by affecting the synthesis of lipids, proteins and DNA.     

The Peripheral-Type Benzodiazepine Receptor Ligands [3H]Ro 5-4864 and [3H]PK 11195 Bind to the Retina of Rabbit, but Not of Turtle

Abstract: The binding of [³H]flunitrazepam, [³H]RO 5-4864, and [³H]PK 11195 to membrane preparations of the retina was studied in the turtle and rabbit. Only a single population of [³H]flunitrazepam binding sites was detected in the turtle, whereas two populations appeared to be present in the rabbit. No specific binding for [³H]RO 5-4864 and [³H]PK 11195 could be detected in the turtle. In rabbit, both ligands bound with high affinity, revealing a significant population of binding sites (K_D values of 24 ± 2.3 and 2.2 ± 0.8 nM, and B_max values of 440 ± 35 and 1,482 ± 110 fmol/mg of protein, respectively). The binding was temperature - and protein-dependent. Displacement studies showed a similar rank order of potency of various unlabeled ligands against both [³H]RO 5-4864 and [³H]PK 11195 (PK 11195 > Ro 5-4864 > flunitrazepam > flumazenil). These results suggest that peripheral-type benzodiazepine receptors are present in the retina of the rabbit, but not of the turtle.     

Measurement of the Membrane Potential of Isolated Nerve Terminals by the Lipophilic Cation [3H]Triphenylmethylphosphonium Bromide

Abstract: The lipophilic cation [³H]triphenylrnethylphosphonium bromide ([³H]TPMP⁺) was investigated as a measure of the membrane potential of synaptosomes. Conditions under which [³H]TPMP⁺ achieved an equilibrium distribution were tested. The toxicity of TPMP has been studied relative to its inhibitory effects on [³H]y-aminobutyric acid ([³H]GABA) transport. In some experiments the distribution of ⁸⁶RbZ⁺ and [³H]TPMP⁺ was changed upon incubation in the presence of elevated levels of K⁺, ouabain, or KCN, or at 0°C in a way that would be expected from the membrane potential. In normal incubation conditions a membrane potential of ∼−60 mv was calculated.     

Uptake of [3H]Dopamine into Dopaminergic and Noradrenergic Neurones of the Isolated Neurointermediate Lobe of the Rat Hypophysis. Effects of Desipramine and Nomifensine

Abstract: The isolated neurointermediate lobe (NIL) of the rat hypophysis accumulates [³H]dopamine from the incubation medium. Column chromatographic analysis showed that 92% of the tissue radioactivity was contained in the catecholamine fraction. [³H]Dopamine represented 70% and [³H]noradrenaline 30% of the [³H]catecholamines. Desipramine (1 μM) prevented the formation of [³H]noradrenaline without affecting the storage of [³H]dopamine. Nomifensine (10 μM) blocked the storage of [³H]dopamine and [³H]noradrenaline. Thus, in the NIL, [³H]dopamine is taken up into dopaminergic and noradrenergic neurones. In the latter, [³H]dopamine is converted to [³H]noradrenaline, indicating a significant dopamine β-hydroxylase activity in the NIL tissue. A selective labeling of the dopamine stores with [³H]dopamine can be achieved in the presence of desipramine.