Inhibition of Macrophage DNA Synthesis by Immunomodulators

Oil-induced guinea pig peritoneal exudate macrophages were found to incorporate actively [³H]thymidine without any tissue fluids such as conditioned medium, lymphokines or inflammatory tissue exudates. The [³H]thymidine incorporation was markedly suppressed by macrophage stimulants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS), while glucosamine incorporation was simultaneously increased by these stimulants. The degree of suppression of thymidine incorporation depended on the cell density, the concentrations of the stimulants, and sera or culture media used. The exposure of macrophages to MDP for 30 min was sufficient to cause significant suppression.     

Effects of 9-ene-tetrahydrocannabinol on uterine estrogenicity in the mouse.

Several early (Phase I) and late (Phase II) estrogenic effects of 9-ene-tetrahydrocannabinol (THC) were examined in the adult mouse uterus. An injection of THC (2.5 or 10 mg/kg body wt) in ovariectomized mice neither stimulated uterine water imbibition or accumulation of [125I]bovine serum albumin (Phase I responses) at 6 h, nor antagonized these Phase I responses elicited by estradiol-17 beta (E2). With respect to Phase II responses, although single injections of THC (2.5, 5.0 and 10 mg/kg body wt) alone were ineffective in influencing uterine weight at 24 h or incorporation of [3H]thymidine at 18 h, this drug interfered with these responses elicited by E2 in a dose-dependent manner. In contrast, an injection of THC in progesterone (P4)-primed ovariectomized mice modestly enhanced (61%) uterine incorporation of [3H]thymidine. However, E2-stimulated uterine thymidine incorporation in P4-primed ovariectomized mice was antagonized by THC treatment. Effects of THC on blastocyst implantation were examined. Single or multiple injections of various doses of THC neither induced implantation in P4-primed delayed implanting mice, nor interfered with E2-induced implantation. Furthermore, daily injections of THC (10 mg/kg body wt) during the peri-implantation period had no apparent adverse effects on implantation, or on experimentally induced decidualization (deciduomata). The data suggest that THC is neither pro- nor antiestrogenic with respect to Phase I responses. However as regards Phase II responses, THC is modestly pro-estrogenic in the P4-treated uterus, but is anti-estrogenic in the presence of E2. These estrogen agonistic/antagonistic effects of THC on uterine Phase II responses do not adversely affect the process of implantation and decidualization.     

Organ culture of adult rat colonic mucosa on fibrin foam

A system for maintaining adult rat colonic mucosa in organ culture for up to 28 days is described. Distal colonic mucosa physically separated from the muscle layers was cultured at 37 degrees C on a substrate of human fibrin foam in HEPES- and bicarbonate-buffered Waymouth's MB 752/1 medium supplemented with 10% fetal bovine serum, L-glutamine, bovine albumin, ascorbic acid, hydrocortisone, insulin, and ferrous sulfate; the optimal atmostphere for culture was 95% O2 and 5% CO2. Viability of explants was demonstrated by tissue morphology with light microscopy, incorporation of [3H]thymidine and [3H]leucine into DNA and protein, [14C]glucosamine and [3H]fucose incorporation, and glycoprotein synthesis. Two days after initiation of culture, degeneration of surface and crypt cells was observed. Secreted mucosubstances covered the explants. Explants maintained in 95% O2 retained a variable number of glandular crypts with normal columnar epithelium for 14 to 21 days in culture. At 28 days, explants contained a single layer of cuboidal surface epithelium and a rare cryptlike gland.     

3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver. STUDY DESIGN: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery. RESULTS: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery. CONCLUSION: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.     

Effect of N-trifluoroacetyladriamycin-14-valerate on [3H]thymidine uptake and DNA synthesis of human lymphoma cells

The effects of N-trifluoroacetyladriamycin-14-valerate on the uptake of [3H]thymidine and its incorporation into DNA of human P3HR-1 lymphoma cells were studied. In the absence of the drug, at 0 degrees C, [3H]thymidine was transported into the cells but not incorporated into DNA, as determined by both the trichloroacetic acid-soluble and -precipitable counts obtained with the cells. At 37 degrees C, [3H]thymidine was readily transported into the cells and incorporated into DNA. In the presence of the drug, both [3H]thymidine uptake (as shown by acid-soluble counts) and the amount of its incorporation into acid-precipitable materials were markedly reduced. However, the uptake of [3H]thymidine at 0 degrees C was found to be equally sensitive to drug inhibition as at 37 degrees C. The incorporation at 37 degrees C of [3H]thymidine into acid-precipitable materials of the cells, which had been prelabeled at 0 degrees C with [3H]thymidine, was found to be insensitive to inhibition by the drug. The in vitro activities of DNA polymerases alpha and beta purified from human P3HR-1 cells were also found not to be susceptible to inhibition. Nuclei purified from cells pretreated with the drug continued to synthesize DNA. The cytofluorograms of the cells treated with the drug indicated that the treated cells accumulated at the G2/M phase, whereas the S phase of the cells was not arrested. These results suggest that N-trifluoroacetyladriamycin-14-valerate inhibits [3H]thymidine uptake but not cellular DNA synthesis in human P3HR-1 lymphoma cells.     

Biochemical aspects of cardiac muscle differentiation. Possible control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3':5'-monophosphate.

A single injection of either isoproternol or N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) results in an inhibition in the rate of [3H]thymidine incorporation into DNA of differentiating cardiac muscle of the neonatal rat. This inhibition is not due to substantially altered cellular uptake or catabolism of [3H]thymidine. Inhibition of [3H]thymidine incorporation by isoproterenol or dibutyryl cyclic AMP is potentiated by theophylline. Maximal inhibition (95%) is observed 24 h after administration of isoproterenol, and the rate of incorporation returns to a value 80% of control by 72 h. Norepinephrine also inhibits [3H]thymidine incorporation whereas cyclic GMP, N2, 02-Dibutyryl guanosine 3':5'-monophosphate (dibutyryl cyclic GMP), and phenylephrine have little effect. Equilibrium sedimentation analysis of cardiac muscle DNA in neutral and alkaline cesium chloride gradients using bromodeoxyuridine as a density label indicate that isoproterenol and dibutyryl cyclic AMP inhibit [3H]thymidine incorporation into DNA that is replicating semiconservatively. Administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats inhibits by approximately 60% the incorporation of [3H]thymidine into DNA of tissue slices of cardiac muscle prepared 16 h later. [3H]Thymidine incorporation into DNA of tissue slices is into chains that were growing in vivo. This incorporation is linear for at least 4 h of incubation and is inhibited by isoproterenol and dibutyryl cyclic AMP. Inhibition is not due to altered cellular uptake of [3H]thymidine nor is it due to a cytotoxic action. Several other compounds which elevate intracellular levels of cyclic AMP (epinephrine, norepinephrine, glucagon, and prostaglandin E1) also inhibit [3H]thymidine incorporation into DNA or cardiac muscle tissue slices. Cyclic GMP, dibutyryl cyclic GMP, sodium butyrate, and phenylephrine have little effect. Isoproterenol administered together with theophylline to neonatal rats signficantly stimulates the in corporation of [3H]phenylalanine into total cardiac muscle protein and into myosin. This enhanced incorporation may be due in part to an increase in the cellular uptake of [3H]phenylalanine. DNA synthesis decreases progressively in differentiating cardiac muscle of the rat during postnatal development and essentially ceases by the middle of the third week (Claycomb, W. C. (1975) J. Biol. Chem. 250, 3229-3235). In reviewing the literature it was found that this decline in synthetic activity correlates temporally with a progressive increase in tissue concentrations of norepinephrine and cyclic AMP and with the anatomical and physiological development of the adrenergic nerves in this tissue. Because of these facts and data presented in this report it is proposed that cell proliferation and cell differentiation in cardiac muscle may be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators.     

Inhibition of macrophage DNA synthesis by immunomodulators. II. Characterization of the suppression by muramyl dipeptide or lipopolysaccharide [3H]thymidine incorporation into macrophages

Guinea pig peritoneal exudate macrophages actively incorporated [3H]thymidine into trichloroacetic acid-insoluble fraction in vitro. The incorporation of [3H]thymidine was almost completely inhibited by aphidicolin, an inhibitor of DNA polymerase alpha and an autoradiograph showed heavy labeling in nuclei of 15% of macrophage populations. These results indicate that the observed thymidine incorporation was due to a nuclear DNA synthesis. The [3H]thymidine incorporation was markedly suppressed when macrophages were activated by immunoadjuvants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS). The suppression of [3H]thymidine incorporation by MDP was neither due to the decrease in thymidine transport through the cell membrane, nor due to dilution by newly synthesized "cold" thymidine. An autoradiograph revealed that MDP markedly decreased the number of macrophages the nuclei of which were labeled by [3H]thymidine. These results suggest that the suppression of [3H]thymidine incorporation by the immunoadjuvants reflects a true inhibition of DNA synthesis. The inhibition of DNA synthesis by MDP was also observed in vivo. Further, it was strongly suggested that the inhibition was not caused by some mediators, such as prostaglandin E2, released from macrophages stimulated by the immunoadjuvants but caused by a direct triggering of the adjuvants at least at the early stage of activation. Cyclic AMP appears to be involved in the inhibitory reaction.     

The incorporation of [3H]glucosamine, [3H]fucose and [14C]mannose into protein in the rat anterior pituitary incubated in vitro

Rat anterior hemipituitaries incubated in vitro rapidly take up and incorporate into protein D-[6-³H]-glucosamine · HCl, D-[1-¹⁴C]mannose and L-[G-³H]fucose. The newly labeled protein was only slowly released into a Krebs-Ringer bicarbonate incubation medium. Glucosamine- or mannose-labeled protein was barely detectable in the medium after a 30–60 min incubation whereas about 4% of all fucose-labeled protein had already been released into the incubation medium by 30 min. Puromycin · 2HCl (1 mM) inhibited incorporation of glucosamine or mannose into protein to 40% or less of control values within 30 min; fucose incorporation was not significantly inhibited before 45 min. Acid hydrolysis followed by amino acid analysis of glucosamine-labeled protein yielded significant amounts of label in glucosamine, galactosamine and apparent glucosamine-degradation products but no significant amount of label in any amino acid.     

Nerve growth factor-induced increase in [3H]thymidine incorporation into pancreas of young rats and its partial blockade by propranolol or partial sialoadenectomy

Administration of nerve growth factor (NGF) twice daily for 2 days to rats 11 days at the time of the initial injection resulted in a 6.6-fold increase in [3H]thymidine levels of pancreas, when comparison was made to levels of untreated controls. Isoproterenol (ISO), a beta-adrenergic agonist known to produce marked increase in [3H]thymidine incorporation into DNA of salivary glands, caused increases in levels of [3H]thymidine in pancreas that were similar in magnitude to those induced by NGF. The combined administration of ISO and NGF did not cause any increase above those observed with either agent alone. Administration of propranolol (3 mg/kg body wt) prior to administration of ISO prevented the usual ISO-induced increase in DNA synthesis, but propranolol in either a 3- or 9-mg/kg body wt dose, caused only a 50% inhibition of NGF-induced thymidine incorporation. In the absence of the submandibular-sublingual glands, the ISO failed to induce the usual high levels of thymidine incorporation, whereas NGF induced the same high levels observed in rats with submandibular glands intact. NGF did not alter the distribution of beta 1- and beta 2-adrenoceptors in the pancreas but did increase norepinephrine when the initial administration was at age 5 days, but not when it was given at age 10 days. Since NGF increased DNA synthesis in the absence of submandibular-sublingual glands, whereas ISO did not, this suggests that ISO requires NGF to induce beta 1-activation and subsequent synthesis and that NGF is a direct activator.     

“Suppressor factor” of neutrophils: A short story of a long-term misconception

A large body of evidence obtained during the last decade has demonstrated that neutrophils suppress T cell proliferation in different models of inflammation and cell interaction. The commonly used method for assessing cell proliferation and proliferation inhibition is measuring [³H]thymidine incorporation into cells. Earlier, we observed inhibition of [³H]thymidine uptake in experiments on neutrophil-mediated regulation of T cell response in tuberculosis immunity. Here, we used different types of proliferating cells to analyze the nature of the soluble “neutrophil factor” by a variety of methods (dialysis, HPLC, mass spectrometry, and NMR) and unambiguously demonstrated that neutrophils do not synthesize a specific factor inhibiting cell proliferation, but secrete high concentrations of extracellular thymidine that competitively inhibit [³H]thymidine incorporation. Although the physiological significance of thymidine secretion by neutrophils remains unknown, this phenomenon should be carefully considered when designing test systems for studying cell–cell interactions.     

Sertoli cell-secreted protein(s) stimulates DNA synthesis in purified rat Leydig cells in vitro

We have examined the effects of Sertoli cell-secreted proteins (SCSP) on [3H]thymidine incorporation by purified preparations (greater than 96%) of rat Leydig cells to determine whether Sertoli cells influence DNA synthesis in these cells in vitro. Incubation of Leydig cells isolated from testes of rats of ages 16 to 90 days with SCSP (Mr greater than 10,000) induced significant dose-, time- and age-related increases in [3H]thymidine incorporation by the cells. A dose-response curve to SCSP showed that as little as 0.2 micrograms SCSP/ml consistently induced a small but significant increase (31% and 10% above control; P less than 0.001) in [3H]thymidine incorporation by Leydig cells isolated from immature (26 days) and mature (70 days) rats, respectively. The maximum response (230% and 48% above control) was obtained with a concentration of 18 micrograms SCSP/ml in cells isolated from immature and mature rats, respectively. Hydroxyurea, a specific inhibitor of replicative DNA synthesis, significantly (P less than 0.001) inhibited both basal and SCSP-induced [3H]thymidine incorporation in Leydig cells from immature and adult rats without affecting the viability of the cells. Incubation of immature rat Leydig cells in SCSP for 48 h also stimulated a 3-fold increase in cell number. The component of the crude SCSP which stimulated Leydig cell [3H]thymidine incorporation is trypsin-sensitive, heat-stable, and adsorbs to a heparin-agarose affinity column but not to concanavalin A-Sepharose. The secretion of this factor(s) by Sertoli cells is stimulated independently by FSH and testosterone. These results demonstrate for the first time that cultured Sertoli cells secrete a protein(s) which, in vitro, stimulates rat Leydig cell replicative DNA synthesis.     

[3H]Thymidine Incorporation To Estimate Growth Rates of Anaerobic Bacterial Strains

The incorporation of [³H]thymidine by axenic cultures of anaerobic bacteria was investigated as a means to measure growth. The three fermentative strains and one of the methanogenic strains tested incorporated [³H]thymidine, whereas the sulfate-reducing bacterium and two of the methanogenic bacteria were unable to incorporate [³H]thymidine during growth. It is concluded that the [³H]thymidine incorporation method underestimates bacterial growth in anaerobic environments.     

[3H]thymidine incorporation in bovine lymphocytes treated with the tumor promoter, 12-O-tetradecanoyl phorobol-13-acetate

Treatment of bovine lymph node lymphocytes with the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) leads to depressed [³H]thymidine incorporation in response to phytohemagglutinin (PHA). Radioautographic and morphological analyses showed that depression was at the level of blast-cell formation. Isotope-dilution experiments, and the use of [³H]deoxycytidine to label DNA indicated that the inhibition was not due to a block in thymidine transport in the treated cells. These experiments, as well as a bioassay designed to measure thymidine in the culture medium, showed that the apparent inhibition of [³H]thymidine incorporation and DNA synthesis was not the result of production of cold thymidine in the cultures. The results taken together support the idea that most TPA-treated cells are inhibited from responding to the mitogenic lectins. Those cells which do respond appear to form blast cells and synthesize DNA at the same rate as do untreated cells.     

Growth hormone inhibits renotropic action of luteinizing hormone-isoform(s)

We recently purified luteinizing hormone (LH)-isoforms with renotropic activity from ovine pituitaries based on the stimulation of [3H] thymidine incorporation into renal DNA of castrated-hypophysectomized rats. In this study, we examined the hormonal interactions between ovine growth hormone (GH) and this LH-isoform in renal DNA synthesis. A single injection of LH-isoform (40 micrograms) significantly increased [3H] thymidine incorporation, but an injection of GH (200 micrograms) did not, during experimental periods of up to 26 hours. Repetitive ovine GH treatment (5 days) did not change basal [3H] thymidine incorporation, either, although its biological activity was evidenced by an increase in insulin-like growth factor-I (IGF-I). Stimulated [3H] thymidine incorporation by LH-isoform (100 micrograms) was significantly suppressed by an injection of GH (200 micrograms) and was, to a greater extent, by repetitive treatment with GH (200 micrograms/day, for 3 or 5 consecutive days). These results demonstrated one example of the effect of complex hormonal interactions on kidney growth.     

[H]thymidine incorporation in bovine lymphocytes treated with the tumor promoter, 12-O-tetradecanoyl phorobol-13-acetate

Treatment of bovine lymph node lymphocytes with the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) leads to depressed [³H]thymidine incorporation in response to phytohemagglutinin (PHA). Radioautographic and morphological analyses showed that depression was at the level of blast-cell formation. Isotope-dilution experiments, and the use of [³H]deoxycytidine to label DNA indicated that the inhibition was not due to a block in thymidine transport in the treated cells. These experiments, as well as a bioassay designed to measure thymidine in the culture medium, showed that the apparent inhibition of [³H]thymidine incorporation and DNA synthesis was not the result of production of cold thymidine in the cultures. The results taken together support the idea that most TPA-treated cells are inhibited from responding to the mitogenic lectins. Those cells which do respond appear to form blast cells and synthesize DNA at the same rate as do untreated cells.     

Inhibition by specific monosaccharides of interleukin 2-induced thymocyte proliferation

When rat thymocytes are cultured for 3 days in serum-free medium and are stimulated to divide by interleukin 2 (IL 2), concanavalin A, or sodium periodate oxidation, addition to the medium of 10–25 mMd-ribose, 2-deoxy-d-ribose, or N-acetyl-d-galactosamine inhibits by 40% or more the incorporation of [³H]thymidine. d-ribose and lectin-free IL 2 generated from sodium periodate oxidation of rat spleen cells were used to study the characteristics of this inhibition and to test possible mechanisms of inhibition. Viability of thymocytes cultured with d-ribose is similar to that of cells cultured without this sugar. In order to be inhibitory, d-ribose has to be added to the cultures within the first 24 hr, and the inhibition can be prevented if the sugar is removed 18–24 hr after the start of culture. d-Ribose does not block the absorption of IL 2 by unstimulated rat thymocytes or by concanavalin A-generated thymic or splenic blast cells. When thymocytes are cultured with d-ribose for 24 hr, inactivated with mitomycin C, and then cultured for 3 days with fresh mitogenically stimulated cells, [³H]thymidine incorporation into the latter is not altered. This suggests that the sugar does not generate suppressor cells or suppressor supernates. d-Ribose does not appear to be a general metabolic inhibitor since [³H]leucine incorporation into thymocyte proteins and the release of [³H]leucine into medium after a 2-hr. [³H]leucine pulse are not altered by d-ribose. Trivial or artifactual effects (nonspecific cytotoxicity, changes in thymidine transport, or changes in isotonicity of the culture medium) cannot explain the inhibition. A hypothetical mechanism of inhibition is discussed.     

H]thymidine incorporation to estimate growth rates of anaerobic bacterial strains

The incorporation of [H]thymidine by axenic cultures of anaerobic bacteria was investigated as a means to measure growth. The three fermentative strains and one of the methanogenic strains tested incorporated [H]thymidine, whereas the sulfate-reducing bacterium and two of the methanogenic bacteria were unable to incorporate [H]thymidine during growth. It is concluded that the [H]thymidine incorporation method underestimates bacterial growth in anaerobic environments.     

Correlation between the presence of T-antigen and the reinitiation of host DNA synthesis in senescent human diploid fibroblasts after SV40 infection

Senescent human diploid fibroblasts, TIG-1, had labelling indices of about 0.5-3% when labelled with [3H]thymidine for 3 days in fresh medium containing 10% fetal bovine serum. When these cells were infected with SV40, the percentage of nuclei incorporating [3H]thymidine increased by about 10-fold. The frequency of T-antigen-positive cells and that of [3H]thymidine-incorporating cells were almost the same. About 80% of T-antigen-positive cells were also positive to incorporation of [3H]thymidine, and the same result was obtained in infected young cells. These results indicated that senescent human diploid cells which are brought to synthesize T-antigen always initiate DNA synthesis as young cells do. The characteristics of senescent cells as compared with younger cells was low incidence of T-antigen-positive cells after infection. The basis of low susceptibility of senescent cells to initiate DNA synthesis by SV40 infection thus seems to be concerned with an event after the adsorption of virus, but before the synthesis of a detectable amount of T-antigen.     

Lymphocyte surface poisons: disulfides and thiolsulfonates.

Eight disulfides (I-VIII) and a thiolsulfonate (IX) were promising blocking agents of lymphocytes in graft-versus-host reactions (GvHR) without comensurate intracellular effects. The blocking effects were assayed through inhibition of the local GvHR after parental lymphocytes had been incubated with agents at suitable concentrations and then inoculated into F1 hybrid offspring. The intracellular effects were assessed beforehand by measuring the inhibition of [6-3H]thymidine incorporation by lymphocytes in the presence of a wide range of concentrations of agents. Concentration levels which induced no greater than approx. 50% inhibition of the [6-3H] thymidine incorporation were considered to reflect sufficiently small intracellular effects and were used for the subsequent GvHR comparisons. Cellular survival always was 90% or more for the GvHR tests (unless stated otherwise), even when inhibition of thymidine incorporation was as high as 50%; hence the thymidine data are useful not only as guides for dose levels in the GvHR but also as leads to new agents that may show immunosuppressive or anti-leukemic activity through intracellular effects. Structural specificity of the active compounds as cell-surface poisons is evidenced by little or no activity (less than 30% inhibition of GvHR) of 28 other disulfides, 2 trisulfides, 2 Bunte salts, and 8 other thiolsulfonates. Active agents may owe this function to replacement of the H of SH in cell-surface thiol receptors by an SR group. Glutathione did not significantly inactivate agents, probably because the products of reaction also are active disulfides. When two agents (III, IX) were given orally or intraperitoneally to F1 hybrid recipients of untreated parental cells, doses of 10--15 mg/kg produced a GvHR inhibition of 17--53%.     

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

We describe a reproducible method for combining tritiated thymidine ([H]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 mu Ci) per animal, produced by a simultaneous injection of 2.5 mg BrdU shows a linear, dose-dependent relationship. Provided the injected dose (in mu Ci 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.