DNA turnover and thymidine re-utilization in mouse tissues.

Mice were injected intravenously with tritiated thymidine (TdR) and 125I-labeled iododeoxyuridine (IUdR) in low doses to provide a simultaneous labeling of tissue DNA with non-toxic amounts of these two precursors. The total activity per organ and the ratio of the two isotopes was measured in the DNA at various times between 1 and 15 days after the injection. Since TdR from dying cells is re-utilized more effeciently than IUdR from the same cells, more labeled TdR than IUdR was retained in the tissue DNA in these experiments. From the slopes of the regression lines, the true rated of turnover of replicating tissue DNA and the per cent re-utilization of TdR were calculated. Re-utilization of TdR varied from 37 to 60% in the six tissues examined.     

Double labeling with [3H]thymidine and [125I]iododeoxyuridine as a method for determining the fate of injected DNA and cells in vivo

Mice were injected intravenously and intraperitoneally with preparations of intestinal nucleoprotein, spleen nuclei, mouse thymus cells, or human kidney T cells whose DNA had been labeled with both [3H]thymidine (TdR) and [125I]-iododeoxyuridine (IUdR). Since free TdR is reutilized more efficiently than free IUdR produced by enzymic hydrolysis of the exogenous DNA, the ratio of [3H]TdR/[125I]IUdR in the DNA fraction of the tissues of the recipient mice provides a measure of the amount of intact exogenous DNA in the tissue. In most instances, the doubly labeled exogenous DNA was almost completely hydrolyzed within 1 day injection, but survival of the DNA from whole cells could be demonstrated in some cases.     

DIFFERENCES IN REUTILIZATION OF THYMIDINE IN HEMOPOIETIC AND LYMPHOPOIETIC TISSUES OF THE NORMAL MOUSE

Swiss Albino mice received a single i.v. injection of ³H-thymidine (TdR) or of ¹²⁵I-deoxyuridine (IUdR). Bone marrow, thymus, spleen and mesenteric lymph node were examined for the efficiency of precursor incorporation into DNA, and for DNA renewal from day 1 to day 8.
TdR is 5–8 times more efficiently incorporated by the different organs in vivo and in vitro than is IUdR. This indicates that the discrimination against IUdR occurs at the level of DNA synthesizing cells.
A diminished DNA turnover rate measured with ³H-TdR in comparison with ¹²⁵I-UdR is interpreted to indicate reutilization of TdR.
TdR reutilization was observed in bone marrow and spleen from at least day 1 on, and in the thymus from day 3 on, following pulse labeling of DNA synthesizing cells. The degree of TdR reutilization appears higher in the thymus (67%) than the bone marrow (43%) and spleen (38%). The mesenteric lymph node indicates either no, or a very low efficiency of TdR reutilization. The data are also consistent with a reutilization equally efficient for TdR and IUdR.
It is suggested that the TdR salvage pathway in hemopoietic tissues is largely localized to single organs which have immediate access to TdR made available by catabolism of DNA. The contribution of TdR from systemic reutilization to the organs studied falls within the limits of error of measurements. Moreover, the TdR salvage pathway especially in the lymph node may involve other DNA breakdown products than nucleosides.     

Incorporation of thymidine and iododeoxyuridine into the DNA of mouse tissues.

Mice were injected intravenously with a solution containing tritiated thymidine (TdR) and iodine-labelled iododeoxyuridine (IUdR). The ratio of 3H/125I activities was measured in the acid-soluble fraction and in the DNA of several tissues at various times from 0.08 to 24 h after injection. There did not appear to be any discrimination in favor of TdR in the acid-soluble fraction of the tissues. The amount of TdR incorporated into the DNA was four to five times greater than the amount of IUdR incorporated; moreover, this value remained relatively constant throughout the period of DNA synthesis under the conditions used. Although IUdR was destroyed more rapidly than TdR in the body, particularly at high concentrations of both precursors, this factor did not account for the major portion of the discrimination observed with tracer amounts of the two DNA precursors. Discrimination in favor of TdR as a precursor for DNA must, therefore, occur at some stage in the utilization of intracellular precursor.     

3H-5-IODO-2'-DEOXYURIDINE TOXICITY PROBLEMS IN CELL PROLIFERATION STUDIES

The toxicity of ³H-5-iodo-2′-deoxyuridine (³H-IUdR) was evaluated by injecting tumor-bearing C3H mice with different concentrations of ethanol (the solvent), different doses of tritium tagged onto either IUdR or thymidine and different chemical doses of IUdR, and then measuring the ³H-IUdR incorporation into duodenal and mammary tumor DNA as well as the cellular kinetics of duodenal crypt cells. Ethanol (37% or less, 0.2 ml/mouse) does not significantly inhibit IUdR incorporation into DNA, and the incorporation after a tritium dose of 75 μCi ³H-IUdR/mouse (about 3 μCi/g body weight) is not less than the incorporation following an injection of 25 μCi ³H-IUdR/mouse when the IUdR dose is below 0.005 μmole per mouse. The toxic effects are primarily due to chemical toxicity from IUdR per se. IUdR, at doses of 0.2 μmoles per mouse does inhibit IUdR incorporation into duodenal and tumor DNA, and the duodenal labeling index and the fraction of labeled mitoses are significantly reduced when 0.013 μmole IUdR per mouse is injected. Also some of the duodenal cells containing IUdR apparently undergo only one post-labeling division and the generation time (T_c) of the cells containing IUdR (25 μCi ³H-IUdR/mouse) is 15.3 hr as compared to 13.3 hr for cells labeled with ³H-T (75 μCi/mouse). This increase in T_c is probably not statistically significant; nevertheless, these results do indicate that one must be exceedingly cautious when using ³H-IUdR as a radiotracer for studies concerned with in vivo cellular kinetics and, at least for C3H mice, the dose should be less than 0.01 μmole per 25 g mouse.     

Effects of thymidine analogues on murine and human cells.

Three mouse tumour cell lines grew continuously in 3 micro M 5-bromodeoxyuridine (BUdR). One line (MC-2) produced a retrovirus and altered in morphology in the presence of BUdR or 5-iododeoxyuridine (IUdR). These effects, which could be reversed by growth in normal medium were similar to those reported for the B-16 mouse melanoma line. The B-16 line used in this study, however, as well as a variety of human cells (six melanoma lines and three fibroblast strains), were much more sensitive to BUdR, 0.03-0.1 micro M being the maximum tolerated levels for continuous growth. No virus production or changes in morphology were induced in these cells by BUdR, deoxyuridine (UdR), 5-fluorodeoxyuridine (FUdR) or thymidine (TdR). The results of cell labelling and growth studies showed a correlation of incorporation of BUdR into DNA with toxicity. Compared on a competitive basis with 1 micro M TdR, the order of incorporation of 1 micro M nucleosides by two human cell lines was TdR = BUdR = IUdR greater than UdR greater than FUdR. In contrast to previous reports that FUdR is incorporated into RNA but not into DNA, half of the FUdR label was found in alkalistable, DNase-sensitive material. Over 90% of the other compounds was incorporated into DNA. All of the UdR and 60% of the IUdR label was incorporated as thymidine; this conversion could be inhibited by labelling in the presence of FUdR.     

Characteristics of spontaneous and induced thymidine and 5-iodo-2-deoxyuridine resistant clones of mouse lymphoma cells

Clones resistant to 5-iodo-2-deoxyuridine (IUdR) were isolated from P388 cells and cultured in the absence of selective medium. Thymidine kinase assays were performed on 8 clones which had arisen spontaneously and 19 isolated after exposure to X-rays or alkylating agents. All the clones tested showed significantly reduced thymidine kinase activity relative to wild-type cultures, but none showed zero levels. 14 of these clones were tested for thymidine (TdR) uptake and all showed a marked reduction in the rate of [³H]TdR incorporation into acid soluble fractions and into DNA. 7 IUdR-resistant (IUdR^r) clones were tested for revertibility as measured by growth of colonies in HAT medium. 5 of the 7 were found to revert at measurable rates either spontaneously or after a low dose of mutagen.Thymidine kinase activity was also measured in 8 thymidine resistant P388 clones (TdR^r). Initial rates of thymidine phosphorylation were not significantly altered in 5 of the 8 clones tested but significantly lower amounts of phosphorylated products were observed in 6 of the 8 clones. [³H]TdR uptake was reduced in 9 of 12 clones tested, and 2 of them showed no corresponding reduction in the thymidine kinase activity, suggesting the occurence of mutants with altered permeability for thymidine.IUdR resistant L5178Y clones could not be isolated. Thymidine resistant L5178Y clones were similar to TdR^r P388 clones, i.e. they showed changes in initial rates of thymidine kinase activity and reduced accumulation of phosphorylated products. Only one clone could be shown to be a membrane mutant. These results are discussed in relation to the genetic nature of the thymidine kinase locus in the two cell lines.     

SYNCHRONIZATION OF MITOCHONDRIAL DNA SYNTHESIS IN CHINESE HAMSTER CELLS (LINE CHO) DEPRIVED OF ISOLEUCINE

Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([³H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([³H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9–12 h after addition of isoleucine and virtually all [³H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G₁ because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.     

Toxicity and mutagenicity of X-rays and [I]dUrd or [H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Diesterified Derivatives of 5-Iodo-2′-Deoxyuridine as Cerebral Tumor Tracers

With the aim to develop beneficial tracers for cerebral tumors, we tested two novel 5-iodo-2′-deoxyuridine (IUdR) derivatives, diesterified at the deoxyribose residue. The substances were designed to enhance the uptake into brain tumor tissue and to prolong the availability in the organism. We synthesized carrier added 5-[¹²⁵I]iodo-3′,5′-di-O-acetyl-2′-deoxyuridine (Ac₂[¹²⁵I]IUdR), 5-[¹²⁵I]iodo-3′,5′-di-O-pivaloyl-2′-deoxyuridine (Piv₂[¹²⁵I]IUdR) and their respective precursor molecules for the first time. HPLC was used for purification and to determine the specific activities. The iodonucleoside tracer were tested for their stability against human thymidine phosphorylase. DNA integration of each tracer was determined in 2 glioma cell lines (Gl261, CRL2397) and in PC12 cells in vitro. In mice, we measured the relative biodistribution and the tracer uptake in grafted brain tumors. Ac₂[¹²⁵I]IUdR, Piv₂[¹²⁵I]IUdR and [¹²⁵I]IUdR (control) were prepared with labeling yields of 31–47% and radiochemical purities of >99% (HPLC). Both diesterified iodonucleoside tracers showed a nearly 100% resistance against degradation by thymidine phosphorylase. Ac₂[¹²⁵I]IUdR and Piv₂[¹²⁵I]IUdR were specifically integrated into the DNA of all tested tumor cell lines but to a less extend than the control [¹²⁵I]IUdR. In mice, 24 h after i.p. injection, brain radioactivity uptakes were in the following order Piv₂[¹²⁵I]IUdR>Ac₂[¹²⁵I]IUdR>[¹²⁵I]IUdR. For Ac₂[¹²⁵I]IUdR we detected lower amounts of radioactivities in the thyroid and stomach, suggesting a higher stability toward deiodination. In mice bearing unilateral graft-induced brain tumors, the uptake ratios of tumor-bearing to healthy hemisphere were 51, 68 and 6 for [¹²⁵I]IUdR, Ac₂[¹²⁵I]IUdR and Piv₂[¹²⁵I]IUdR, respectively. Esterifications of both deoxyribosyl hydroxyl groups of the tumor tracer IUdR lead to advantageous properties regarding uptake into brain tumor tissue and metabolic stability.     

The induction of thymidine- and IUdR-resistant variants in P388 mouse lymphoma cells by x-rays, UV and mono- and bi-functional alkylating agents

Dose-response curves for “mutation” to resistance to 5-iodo-2-deoxyuridine (IUdR) and excess thymidine (TdR) in P388 mouse lymphoma cells have been established after exposure of these cells to six chemical mutagens, UV |and| ionising radiations. The dose-response curves for all mutagens in both selective system show considerable similarities when induced mutation frequencies are plotted against survival. Expression time for both types of variants, IUdR^r and TdR^r, are similar, i.e. maximum frequencies are reached by 48 h and there is no fall in variant frequency at late expression times up to 144 h. Over the range of survival levels studied there appears to be little or no dependence of expression time on dose of mutagen. Some loss of mutants after high doses (i.e. at low survival levels) was observed due to the fact that a significant proportion of both TdR^r and IUdR^r clones were more sensitive to the mutagens than the wild-type population. The similarities in induced dose-response curves for different mutagens suggest that the mutants have a common origin, probably an error in repair, but it seems unlikely that errors in “cut and patch” repair are responsible. A comparison of spontaneous frequencies of IUdR^r and TdR^r variants suggests that IUdR is mutagenic in P388 cells.     

Uptake of 125I-iododeoxyuridine in mouse organs during deuteration of body water: Evidence for a thymus-specific effect

The effects of body water deuteration on mammalian DNA synthesis $\text{in vivo}$ during the deuterium equilibration period in the body were studied. Young adult mice were given 15% or 30% D₂O in the drinking water for 4, 10 or 21 days. Control mice were given distilled water. Eighteen hours prior to sacrifice, ¹²⁵IUdR, a conveniently monitored synthetic analogue of the DNA precursor thymidine, was injected intravenously. Although neither radioiodine activity of the total body nor body weight varied significantly among the three groups, thymic radioactivity per g tissue was significantly lower in mice given 30% D₂O and, to a lesser extent, in mice given 15% D₂O than in the control group. In contrast, intestine and hemopoietic bone marrow displayed minor changes in ¹²⁵IUdR incorporation. This reduction of ¹²⁵IUdR incorporation is discussed in relation to the particular importance of thymidine reutilization in the thymus.     

[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.     

Effects of Pulse Labelling With Tritiated Thymidine On the Circadian Rhythmicity In Epidermal Cell-Cycle Distribution In Mice

The influence of pulse labelling with 50 °Ci tritiated thymidine ([³H]TdR) (2 μCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G₂ phases of the cell cycle were estimated from the obtained DNA frequency distributions. the proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [³H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [³H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G₂ and M phase during the first 32 hr after the injection. the number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 °Ci [³H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

DISTRIBUTION OF 3H-THYMIDINE IN ARABIDOPSIS VEGETATIVE MERISTEMS AFTER 5-IODODEOXYURIDINE TREATMENT

Arabidopsis thaliana vegetative meristems, growing under short photoperiods, respond to the application of IUdR by precocious floral morphogenesis. ³H-thymidine was used to label cells active in DNA synthesis and study the effect of analogue application on the amount and distribution of DNA synthesis throughout the meristem during 78 hr subsequent to IUdR treatment. Photomicrographs, autoradiographs, and composite plots of label distribution in transparent superimposed sequential sections revealed a non-uniform distribution of labelling in control vegetative meristems, which typically contained a central and axial core incorporating little ³H-thymidine and a peripheral flanking tunica region which contained densely labelled cells. The ratio of labelled cells in the peripheral region to labelled cells in the central region was about 10:1 in the controls. In meristems pretreated with IUdR there was a brief suppression of ³H-thymidine incorporation during 6–12 hr after treatment, followed by two waves of enhanced incorporation in the peripheral region, and a progressive increase in the frequency of labelled cells in the central core of the meristem. After 78 hr the frequency of labelled cells in the central core of IUdR-treated meristems was 8-fold higher than in untreated meristems, and the frequency in the peripheral regions was about the same in both IUdR and control series. The enhancement in amount and uniformity of DNA synthesis after temporary inhibition by IUdR parallels the normal enhancement which is observed when vegetative meristems are transferred to long photoperiods causing floral induction.     

Temperature dependent phosphorylation of [H]thymidine and its incorporation into DNA by KB cells

The incorporation of [³H]TdR into DNA by KB cells cooled to 4 °C falls rapidly to about 1–2% of that of controls held at 37 °C. The amounts of four enzymes involved in TdR metabolism: TdR kinase, thymidylate kinase, cytoplasmic DNA polymerase, and nuclear DNA polymerase, never fall below 50% of those in the control cells even after 12 h at 4 °C. The activities of these enzymes were measured in vitro at different temperatures and it was found that whereas the two kinases retained appreciable activity at low temperature, the DNA polymerase activities were severely inhibited. Cultures of cells rewarmed to 37 °C after 12 h at 4 °C immediately re-started incorporation of labelled TdR into DNA, showing that sufficient enzyme activity for normal functioning had been preserved during the cold period.     

Stimulation of adenovirus replication in simian cells in the absence of a helper virus by pretreatment of the cells with iododeoxyuridine.

Pretreatment of African green monkey kidney cells with 50 mu g of 5'-iododeoxyruidine (IUdR) per ml can modify their susceptibility to the replication of human adenovirus type 7 in the absence of simian virus 40 (SV40) although this enhancement of adenovirus replication is not as efficient as that of the helper SV40 virus. Since the number of infectious centers remains unchanged after IUdR pretreatment whereas the burst size of virus from each infected cell increases, the IUdR appears to allow each infected cell to produce more virus. Cell DNA synthesis appears to be stimulated in IUdR pretreated cells infected with adenovirus 7, but the host cell DNA synthesized is small enough to remain in the Hirt supernatant fluid. The modification of susceptibility to adenovirus replication and the changed pattern of cell DNA synthesis is stable for at least two additional cell passages of the pretreated cells.     

Detection of bacterial populations in oyster tissue homogenates using direct microscopic counts and thymidine incorporation

Acridine orange direct counts and incorporation of [³H]thymidine ([³H]TdR) were used to determine the effectiveness of an antibiotic treatment on reducing bacterial activity in oyster tissue. Cell counts, as well as total [³H]TdR incorporation into the acid insoluble pool, were significantly lower in antibiotically treated oyster tissue homogenates than in untreated controls. However, rates of [³H]TdR incorporation were not significantly different between treatments, indicating increased metabolic activity (on a per cell basis) in the antibiotically treated bacterial population versus the control population.     

Density Centrifugation of Murine Fibrosarcoma Cells Following In Situ Labelling With Tritiated Thymidine

Murine fibrosarcoma (FSa) cells form at least five unique subpopulations after centrifugation in linear Renografin density gradients. Each of these subpopulations has been characterized with respect to selected kinetic parameters using pulse-labelling techniques and flow microfluorometry (FMF) analysis. Tumour-bearing mice were first injected intraperitoneally with a pulse label of tritiated thymidine ([³H]TdR, 50 μCi). Following 15, 30, 60 min or 24 hr these animals were injected with cold thymidine. Animals were killed, their tumours removed and made into suspension, and separated by density gradient centrifugation. Each gradient was fractionated and the density, cell number, tritium activity, and labelling index (LI) per fraction were determined. These data were then compared to FMF data for selected cell density bands. the results indicated a relatively higher uptake of [³H]TdR in the cells recovered at the lighter (1.06–1.12 g/cm³) as compared to the heavier (>1.12 g/cm³) densities. Following a 30-min pulse, the LI's of light cells (<1.12 g/cm³) ranged from 25 to 30%, while the heavier cells (>1.12 g/cm³) had LI's between 10 and 15%. the unseparated control cells had an LI of 19%. Comparable results were found at the other times tested. In contrast, the FMF profiles describing the DNA contents of the cells banding in the gradient showed no difference in proportion of S-phase cells among the separated subpopulations. This lack of correlation between the FMF determination of S-phase cells and labelling index for the denser cell populations implies that DNA content alone is not an effective measurement of the functional activity of cells in solid tumours. Finally, the relatively reduced uptake of [³H]TdR by these denser cells suggests that they may have resided at relatively large distances from the functional vasculature in the tumour.