The distribution and kinetics of nuclei in rat osteoclasts

Osteoclast development and growth were studied by determining the number of labelled nuclei in osteoclasts of different sizes (based on the number of nuclei per osteoclast, N/O) and the number of osteoclasts with labelled nuclei at various intervals after tritiated thymidine [( 3H]TdR) injection in young rats. The osteoclast smears were made from the cellular periosteum of the proximal tibia. The frequency distribution of the N/O osteoclasts types in the smears had profiles similar to that of in situ osteoclasts in whole mounts of proximal tibia, which indicates that the osteoclast population of the smears was representative of that on the bone surface. A vast majority of the osteoclasts had a 1-6 N/O, and a number of the cells had as many as 26 or more nuclei. Furthermore, profiles of N/O frequency distributions were similar over the course of the study. Nuclei with [3H]TdR label were initially observed in osteoclasts between 4 and 12 hr after isotope injection. However, fusion of labelled nuclei to osteoclasts continued for at least 150 hr. In general, the labelled osteoclasts exhibited a significantly larger number of nuclei than the unlabelled osteoclasts. The probability of an osteoclast incorporating one or more labelled nuclei increased with time after injection and with an increase in N/O. Labelling intensity decreased with time post injection and with an increase in N/O. The data suggest that turnover of nuclei is more rapid in osteoclasts with high N/O values.     

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]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.     

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.     

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.     

Non-Circadian Rhythm In Proliferation of Haematopoietic Stem Cells

Abstract. The proportion of haematopoietic stem cells (CFU-s) engaged in DNA synthesis was determined by means of the [³H]-thymidine ([³H]TdR) suicide technique during recovery of bone marrow from the damage caused by a sublethal total body irradiation. In contrast with previous reports the [³H]TdR suicide rate was not permanently increased. It was observed that CFU-s passed through S phase in synchronous waves, following a dose of irradiation of 1.5 Gy. After a dose of 2.6 Gy, there was only one initial wave of increased CFU-s sensitivity to the action of [³H]TdR. Following the depression occurring 26 hr after the irradiation with 2.6 Gy, the proportion of CFU-s killed by the [³H]TdR was permanently increased until 5-6 days after irradiation. Thereafter large differences in the [³H]TdR suicide data were observed among individual mice. Evidence was obtained that individual mice, which had been irradiated by a dose of 2.6 Gy 8-9 days before, had identical values of the CFU-s [³H]TdR suicide rate in the bone marrow from different bones of the lower extremities. the recurrence of the synchronous waves in CFU-s passage through the cell cycle was recorded when the CFU-s population regenerated to only about 10% of its normal value. These waves were obviously not related to a particular time of the day and, consequently, they did not represent the circadian rhythm. It is concluded that the synchronous waves in which CFU-s proliferation occurred reflected the action of the control mechanism on CFU-s proliferation. This mechanism should be endowed with an important systemic component besides locally operating factors.     

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).     

Autoradiographic Investigations on Cell Proliferation in the Digestive Mucosa of Fishes

The labelling index (TLI) of the digestive mucosa of some fish species was determined following a pulse labelling with tritiated thymidine ([³H]TdR) and light microscopic autoradiography. In the oesophageal epithelium, proliferation was observed to occur in non mucus-secreting cells. In the intestine, both undifferentiated and absorptive cells incorporated [³H)TdR within 1 h after injection. Statistically significant differences in [³H]TdR incorporation were observed between the upper intestine region and both the middle and lower parts on the one hand, and between the middle and lower parts on the other hand. Mucus-secreting cells seemed unable to proliferate. In the stomach, significantly fewer labelled nuclei were counted; they were located in the isthmus epithelium. No significant difference was observed between the TLI of these regions in the different species.     

Retention of Labelled Dna Precursor By Murine Cells After A Single Administration of Tritium Labelled Thymidine

Abstract. The central zone of the rat lens epithelium, extending half way from the centre to the periphery of a whole mount preparation, normally has less than 1% of the cells in the cell cycle at any given time. Mechanical wounding initiates a burst of proliferation in the central zone. DNA synthesis begins 14 hr after wounding followed by mitosis 10 hr later. When [³H]TdR was applied at 2 hr prior to S phase, some moderately heavy and some light labelling was observed after the onset of S phase. When [³H]TdR was applied 5 hr before S phase (9 hr after wounding), all the cells were lightly labelled. Only small amounts of the label were available to these cells 5 hr after application. It is significant that there was labelling in this group because it indicates the persistence of relatively small intracellular pools of [³H]TdR for several hours after the initial 'pulse' labelling of cells. Determinations of the duration of S phase were based on the assumption that pulse labelling may be affected by the persistence of the pools of [³H]TdR and consequent light labelling of the cells.     

Modulation of in vitro thymidine incorporation into crypt cells from the murine small intestine

Abstract. A method is described for the isolation of enriched populations of crypt cells from the murine small intestine. The method was developed to study the response of cells to various stimuli in vitro . The properties of the isolated cell preparations varied with the state of the intestinal mucosa of the mice from which they were isolated. Thus we could distinguish between cells from lactating and non-lactating mice. Polyamines, which are putative modulators of crypt cell division, failed to stimulate [³H]TdR incorporation in vitro . Lymphocyte culture supernatants suppressed [³H]TdR incorporation at dilutions of 1:4 to 1:64. Supernatants of 12- O -tetradecanoylphorbol-13-acetate-stimulated EL-4 cells and of mixed lymphocyte cultures failed to stimulate [³H]TdR incorporation of any dilution. Supernatants of concanavalin A-stimulated spleen cells gave less suppression of [³H]TdR incorporation than those of unstimulated spleen cells and stimulated incorporation at dilutions of 1:64 and 1:128. Phytohaemagglutinin stimulated [³H]TdR incorporation at high concentrations, whereas concanavalin A (con A) had no effect. This study shows that the isolated murine crypt cells may have the potential to provide a useful in vitro model for crypt cell responses to stimuli.     

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.     

Proliferation of cells undergoing chondrogenesis in vitro

Abstract. Continuous exposure of chicken embryo limb bud mesenchyme cells undergoing chondrogenesis in vitro to [³H] thymidine ([³H]TdR) revealed that more than 90% of the cells synthesized DNA at least once during 120 h of culture. When cells were exposed to [³H]TdR for 24 h beginning at various times throughout the culture period, the percentage of cells which incorporated [³H]TdR during each period was approximately 92%. However, when the period for incorporation of radioisotope was limited to two hours, the number of cells which incorporated [³H]TdR was found to decline during chondrogenesis in vitro. This decline was coincident with the appearance of extracellular matrix material and occurred in those cells which had, and had not, expressed the cartilage phenotype.
We conclude from these studies that (1) practically all of the cells continue to proliferate while chondrogenesis is occurring in vitro, (2) there is an increase in the length of the cell cycle during chondrogenesis in vitro, and (3) withdrawal from the cell cycle is not required for differentiation of mesenchyme into cartilage.     

Cell population kinetics in pig epidermis: further studies

Abstract. The cell population kinetics of the epidermis were studied in 4-month-old pigs. Mitotic figures were confined to the basal cell (L1) and the first suprabasal cell layer (L2). The mitotic index (MI) was 0.17 ± 0.04% for L1 and 0.08 ± 0.03% for L2. Labelled nuclei were distributed throughout the viable epidermis, the majority (79.1 ± 1.1%) were in L1 with 19.5 ± 1.2% in L2. The labelling indices (LI) in layers L1 and L2 were 7.1 ± 0.4% and 3.4 ± 0.1%, respectively. After labelling with two injections of tritiated thymidine [³H]TdR separated by 90 min, the LI increased to 8.2 ± 0.3% in L1 and to 4.0 ± 0.2% in L2. This increased labelling confirmed that cell proliferation occurs in both layers, L1 and L2, of the epidermis.
The cell production rate ( K ) in L1 and L2 had an upper limit of 10.7 ± 1.0 and 6.2 + 1.8 cells per 1000 cells per hour respectively. The cell flow rate per hour (cell flux), into and out of the DNA synthesis phase (S), and the duration of DNA synthesis were determined from double-labelling studies with [³H]TdR and [¹⁴C]TdR. The cell flux into and out of S was identical and was calculated as 0.6 ± 0.1%/hr (L1) and 0.5 ± 0.1%/hr (L2). Values for t _S varied from 8 to 10 hr. The cell turnover times ( t _T) were in the range 89–129 hr and 180–261 hr for L1 and L2, respectively.
Log normal curves were fitted to the fraction labelled mitoses data for L1 and L2. Values for t _S for cells in L1 and L2 were 9.8 hr and 11.9 hr, respectively. t _G2+ 1/2 t _M was 7.2 hr in L1 and 9.1 hr in L2.     

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.     

DNA synthesis in Langerhans'cells of mouse ear epithelium revealed by tritiated thymidine autoradiography and histochemical staining for non-specific esterase and beta-glucuronidase activity

Abstract The proportion of Langerhans'cells in DNA synthesis in normal mouse skin was assessed by combining tritiated thymidine [³H]TdR autoradiography with enzyme histochemistry. After injection of [³H]TdR, ear skin was treated in two ways. Epithelial sheet preparations were stained for the presence of non-specific esterase and cytospin preparations of epithelial cell suspensions were stained for beta-glucuronidase activity. The labelling index (p± SE mean) for cytospins, 40 min after injecting [³H]TdR, was 1.6 ± 0.15%, doubling to 3–4% from 7–17 days after injection. The sheet preparations showed the proportion of label attributable to paired Langerhans'cells rising from 18% at 40 min after injection, to approximately 45%, on days 1–4 after injection. These results suggest that the proliferation of Langerhans'cells in normal mouse skin might be higher than was previously thought to be the case.     

Comparative Analysis of Different Approaches to Investigate Cell Kinetics

Abstract. The potential of different methods to investigate proliferative activity of cell populations was analysed for non-Hodgkin's lymphomas. Cells in S phase and all cycling cells were determined on cell suspensions obtained from fresh lymph node material by [³H]-thymidine autoradiography ([³H]TdR LI), a monoclonal antibody to bromodeoxyuridine (BrdU LI), and the monoclonal antibody Ki67. A good correlation was observed between the values of [³H]TdR LI and BrdU LI ( r _s= 0.90; P < 0.01), [³H]TdR LI and S phase ( r _s= 0.62; P < 0.01) and [³H]TdR LI and Ki67 ( r _s= 0.64; P < 0.01) in individual lymphomas. Using the median values obtained from the different approaches as cut-off points to define slowly and rapidly proliferating tumours, the best agreement was observed between [³H]TdR LI and BrdU LI (91%) and poorer agreements, even though statistically significant, were observed between [³H]TdR LI and S phase (73%) or Ki67 (76%). In conclusion, the kinetic information derived from different approaches was more or less concordant and newly proposed approaches should be directly and carefully verified for their prognostic relevance before using them as alternatives to conventional methods.     

A long-lived thymidine pool in epithelial stem cells

Abstract. The labelling index (LI) of the individual basal cell positions of the anterior column of mouse tongue filiform papillae was assessed with time after an injection of [³H]TdR at 12.00 hours (the minimum point in the circadian LI rhythm). An initial doubling of the LI in the stem cell zone due to cell division was followed by a second rise of 14–16% 16 hr after injection and this occurred even in the presence of vincristine. Although the uptake of [³H]TdR and the initial LI doubling were largely prevented by a preceding injection of hydroxyurea, the 14–16% LI rise was still observed. The possible explanations are discussed, the favoured one being that an average of one of the six or seven cells (the stem cell) in each stem cell zone can store [³H]TdR in a long-lived precursor pool for at least 16 hr before being utilized for DNA synthesis. This complements previously published work which suggested that one cell in each stem cell zone may selectively segregate DNA at mitosis.     

Negative Effect of Iodide On the Survival of Newly Divided Epithelial Cells In Chronically Stimulated Rat Thyroid

Abstract. The aim of this work was to investigate some aspects of the thyroid epithelial cell kinetics during the iodide-induced involution of a hyperplastic goitre in the rat. Rats were made iodine-deficient for 6 months, and propylthiouracil (PTU) (0.15%) was added to the diet during the last 2 months. Thereafter, rats were refed with iodide and PTU was removed (day 0).
Forty-eight hours previously, all the rats were injected with tritiated thymidine ([³H]TdR) (1 μCi/g). Some animals were killed 1 hr or 24 hr after [³H]TdR injection (i.e. on day -2 and -1, day O corresponding to the restoration of a normal iodine diet); the other animals were killed after different delay periods and following L³H]TdR injection. Autoradiography of thyroid sections, iodine determination of plasma iodide and protein-bound iodine (PBI), and RIA of plasma thyroid stimulatory hormone (TSH) were performed. Plasma TSH concentration was very high on day O of iodide refeeding (3000 ± 330 ng/ml) and remained at this level until day 8. Plasma PBI was very low on day O, remained so until day 4 and greatly increased on day 8. Plasma iodide was also very low on day O, but markedly increased on day 1, then did not vary significantly until day 43 of iodine refeeding. Thyroid weight, elevated on day O, decreased relatively quickly until day 30, then more slowly until day 73.     

Heterogeneity in the mouse epidermal cell cycle analysed by computer simulations

Abstract. Different sets of cell kinetic data obtained over many years from hairless mouse epidermis have been simulated by a mathematical model including circadian variations. Simulating several independent sets of data with the same mathematical model strengthens the validity of the results obtained. The data simulated in this investigation were all obtained with the experimental system in a state of natural synchrony. The data include cell cycle phase distributions measured by DNA flow cytometry of isolated epidermal basal cells, fractions of tritiated thymidine ([³H]TdR) labelled cells within the cell cycle phases measured by cell sorting at intervals after [³H]TdR pulse labelling, bivariate bromodeoxyuridine (BrdUrd)/DNA data from epidermal basal cells isolated at intervals after pulse labelling with BrdUrd, mitotic rate and per cent labelled mitosis (PLM) data from histologic sections. The following main new findings were made from the simulations: the second PLM peak observed at about 35 h after pulse labelling is hardly influenced by circadian variations; the peak is mainly determined by persisting synchrony of a rapidly cycling population with a G₁-duration (T_G1) of 20 h to 30 h; and there is a highly significant population of slowly cycling G₁-cells (G_1σ). However, no significant circadian variations were found in the number of these cells.     

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.