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.     

Down regulation of hypertrophied follicular cell volume in thyroid hyperplastic gland

In the present study, changes in thyroid follicular cell volume and its regulation have been investigated during the early involution of a hyperplastic goitre. Male Wistar rats were administered an iodine deficient diet for 6 months with propylthiouracil (PTU, 0.15%) during the last two months. At the end of iodine deficiency (day 0), some rats were killed and the others received a normal iodine diet. These rats were killed after different periods of iodine refeeding. Thyroid follicular cell volume was very high in hyperplastic gland whereas thyroid protein concentration was low. Thyroid follicular cell volume quickly decreased when rats were normally iodine refed, whereas thyroid protein concentration increased. Electron microscopal observations showed that thyroid follicular cells retained their endocrine aspect in hyperplastic state and throughout the iodine refeeding period. Using concomitant stereological and biochemical techniques, it is shown that the amount of cellular iodide and an unknown iodinated compound strongly increased during the early iodine refeeding. Plasma TSH was high on day 0 and remained at this level until day 8 whereas plasma T3 and T4 were low on day 0 and remained at this low level until day 4. The present data show that the involution of thyroid follicular cell volume is induced by iodide and mediated by an iodinated compound at least in the initial phase, and is independent of plasma TSH, T3, T4, so indicating the involvement of a thyroid autoregulatory mechanism. These changes in cell volume may be of importance in ion transport, i.e. in the metabolism of thyroid follicular cell during the early involution of the hyperplastic goitre.     

Involution of hyperplastic goitre in the adult male rat. Tissue compartment process with early iodide effect: a stereological and biochemical study

The morphological and functional changes during involution of hyperplastic goitre have been investigated in the adult male rat. Male wistar rats received an iodine-deficient diet for 6 months and during the last 2 months received propylthiouracil (PTU, 0.15%). By the end of this treatment (day 0), a hyperplastic goitre was obtained. A normal iodine supply was then given and PTU withdrawn. During the first 8 days of iodine refeeding, the plasma thyrotropin (TSH) remained at a high level (ten times the control value), whereas the thyroid iodide content was low on day 0, markedly increased on day 1 and decreased on day 4. Plasma T3 and T4 levels remained unchanged for 4 days and only increased on day 8. The total thyroid protein concentration was low on day 0 and then increased rapidly on day 8 (by 34%). The volume density of colloid remained low and unchanged until day 8, when it started to increase. However, the thyroid epithelial cell volume and the volume density of capillaries were raised on day 0, decreased rapidly in the next 8 days and more slowly later on. The total number of thyroid epithelial cells was considerably raised in the hyperplastic gland. It did not vary until day 16, when it decreased slowly, reaching a plateau on day 45 above the control value. The present data show that involution of hyperplastic goitre in the rat is due essentially to a decrease in thyroid epithelial cell volume and to a reduction of the increased number of capillary blood vessels present. The decrease in the number of epithelial cells is only 16.5%, suggesting that the death of thyroid epithelial cells contributes little. Half the process of involution, which occurs from days 0 to 8, is controlled by the thyroid iodide concentration rather than TSH, indicating the involvement of a thyroid autoregulatory mechanism. It must be emphasized, however, that the discontinuous pattern of epithelial cell number during involution may indicate that some cells with larger nuclei and more rapid turnover disappear more quickly after iodine refeeding.     

Clinical study on early changes in thyroid function of hyperthyroidism treated with propylthiouracil and a relatively small dose of iodide.

In order to compare the acute effects of three kinds of antithyroid agents of iodide (I-), propylthiouracil (PTU) and PTU combined with iodide (PTU+I-) on thyroid function in hyperthyroid patients with diffuse goiter, serum concentrations of thyroxine (T4), triiodothyronine (T3), T3-resin sponge uptake (T3-RU) and free thyroxine index (FT4I) were employed as thyroid function parameters. In the group given iodine (1 mg/day) as iodinated-lecithine, the initial values of T4, T3, T3-RU and FT4I were 20.9 +/- 1.6 microng/100 ml (T4), greater than 740 ng/100 ml (T3), 49.5 +/- 2.3% (T3-RU) and 14.7 +/- 1.8 (FT4I). At the end of one week of therapy, they decreased clearly to 15.6 +/- 2.2 microng/100 ml, 457 +/- 87 ng/100 ml, 42.2 +/- 4.0% and 9.7 +/- 2.4. The so-called "escape phenomenon" from iodide inhibition was observed in serum T4, T3-RU and FT4I values at the end of two weeks of iodide therapy, while serum T3 continued to decrease but the value of T3 was far outside of the normal range. In the PTU group (300 mg/day), thyroid function parameters were 22.5 +/- 0.8 microng/100 ml (T4), greater than 592 ng/100 ml (T3), 54.9 +/- 1.0% (T3-RU) and 18.7 +/- 1.0 (FT4I) before treatment. They decreased continually week by week. At the end of four-week treatment with PTU, the value of each thyroid function parameter was 11.1 +/- 1.9 microng/100 ml, 229 +/- 56 ng/100 ml, 36.6 +/- 4.4% and 5.7 +/- 1.7. In the group of hyperthyroidism simultaneously given both PTU and iodide (300 mg/PTU and 1 mg/iodine), these thyroid function parameters decreased as well as in the group treated with PTU alone for more than two weeks. More rapid or significant decrease of T4, T3, T3-RU and ft4i in PTU+I- group than in PTU group was observed in the present study. These results suggested strongly that iodide alone was not an adequate therapy for hyperthyroidism as well known and they were also compatible with the idea that the concomitant administration of PTU and iodide was more effective in the early phase of therapy of hyperthyroidism than PTU alone.     

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

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.     

Spermatogonial multiplication in the Chinese hamster. IV. Search for long cycling stem cells

In the Chinese hamster, 17 days, i.e. one cycle of the seminiferous epithelium, after two injections of [3H]TdR given 24 hr apart, labelled cells were found among all types of spermatogonia, including stem cells (As). These labelled As spermatogonia derive from one or more self-renewing divisions of the stem cells that originally incorporated [3H]TdR. In the steady state, half of the divisions of the As will be self-renewing and the other half will give rise to Apr spermatogonia that will ultimately become spermatozoa. Theoretically, the labelling index (LI) after 17 days will be similar to that after 1 hr, and in this study twice as high as for the 1-hr interval since only one injection was given. However, experimental values only half that of the theoretical LI were found after 17 days. The following causes for the loss of labelled stem cells are discussed: (1) dilution of label because of division; (2) influx of unlabelled components of false pairs (i.e. newborn stem cells that still have to migrate away, mostly during G1, from their sister cells and are scored as Apr spermatogonia) between 1 hr and 17 days; (3) the existence of long- and short-cycling stem cells, probably combined with preferential differentiation of the short-cycling elements; (4) selective segregation of DNA at stem cell mitosis; and (5) irradiation death of radiosensitive labelled stem cells. As it is not impossible that factors 1, 2, 4 and 5 together account for the total loss of labelled stem cells, LI results do not provide evidence for the existence of separate classes of short- and long-cycling stem cells. The distributions of the LIs of the As, Apr and Aal spermatogonia over the stages of the epithelial cycle at 17 days are similar to those at 1 hr after injection. Hence the regulatory mechanisms that govern the stimulation and inhibition of proliferation of As that give rise to new As for the next epithelial cycle are similar to those of the As that will divide into Apr spermatogonia during the same epithelial cycle. Grain counts revealed that more [3H]TdR is incorporated into As, Apr and Aal spermatogonia that are in S phase during epithelial stages X-IV than in stages V-IX.     

Possible regulation of the growth of thyroid tissue by plasma iodide

Rats on iodine deficient diet for 6 months received propylthiouracil (PTU) (0.15%) during the last 2 months. At the end of this treatment, PTU was withdrawn and the rats were iodine refed. 48 hrs. before the iodine refeeding all rats were injected with 3H thymidine. The results showed that some prelabelled cells in the hyperplastic goitre preferentially disappeared during its involution and therefore are more sensitive to iodine.     

Digestive tract cell proliferation and food consumption patterns of Ha/ICR mice

The relationship between the daily pattern of food consumption and the proliferation rate of the oesophagus, stomach, forestomach, small intestine and colon of Ha/ICR mice was examined. Proliferative activity was determined by [3H]TdR incorporation on a wet weight tissue basis, along with selective counting of labelled nuclei. Under conditions of ad libitum feeding with a 12 hr light cycle (lights on at 0600) mice eat most of their food during the dark period. A distinct circadian rhythm was observed in the oesophagus, stomach, forestomach and colon with the peak of [3H]TdR incorporation between 0400 and 0600 and the nadir between 1600 and 1800. Although a circadian fluctuation was observed in the small intestine, its amplitude was much less than in other areas. This rhythmic change in proliferation rate could be phase shifted by allowing the mice to feed only between 0800 and 1600 for 14 days. Under these conditions the peak in proliferative activity occurred between 1800 and 2000. Fasting reduced the daily level of proliferative activity in all of the digestive tract sites studied, and for all areas except the oesophagus greatly reduced or eliminated the circadian fluctuation. The forestomach and colon were the most influenced by fasting with 24 hr [3H]TdR incorporation reduced to 30-40% of the control value. Refeeding following a 48 hr fast produced a rapid increase in proliferative activity peaking at levels well above the control value at 16 hr after the onset of refeeding. The major exception to this was the small intestine which slowly returned to the control value during the first 24 hr. Partial refeeding produced a diminished refeeding response. Once the normal pattern of food consumption was re-established following refeeding the normal proliferative fluctuations were again observed.     

Cell population kinetics in pig epidermis: further studies

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 tS varied from 8 to 10 hr. The cell turnover times (tT) 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 tS for cells in L1 and L2 were 9.8 hr and 11.9 hr, respectively. tG2 + 1/2tM was 7.2 hr in L1 and 9.1 hr in L2.     

Digestive Tract Cell Proliferation and Food Consumption Patterns of Ha/Icr Mice

The relationship between the daily pattern of food consumption and the proliferation rate of the qesophagus, stomach, forestomach, small intestine and colon of Ha/ICR mice was examined. Proliferative activity was determined by [³H]TdR incorporation on a wet weight tissue basis, along with selective counting of labelled nuclei. Under conditions of ad libitum feeding with a 12 hr light cycle (lights on at 0600) mice eat most of their food during the dark period. A distinct circadian rhythm was observed in the oesophagus, stomach, forestomach and colon with the peak of [³H]TdR incorporation between 0400 and 0600 and the nadir between 1600 and 1800. Although a circadian fluctuation was observed in the small intestine, its amplitude was much less than in other areas. This rhythmic change in proliferation rate could be phase shifted by allowing the mice to feed only between 0800 and 1600 for 14 days. Under these conditions the peak in proliferative activity occurred between 1800 and 2000. Fasting reduced the daily level of proliferative activity in all of the digestive tract sites studied, and for all areas except the oesophagus greatly reduced or eliminated the circadian fluctuation. the forestomach and colon were the most influenced by fasting with 24 hr [³H]TdR incorporation reduced to 30–40% of the control value. Refeeding following a 48 hr fast produced a rapid increase in proliferative activity peaking at levels well above the control value at 16 hr after the onset of refeeding. the major exception to this was the small intestine which slowly returned to the control value during the first 24 hr. Partial refeeding produced a diminished refeeding response. Once the normal pattern of food consumption was re-established following refeeding the normal proliferative fluctuations were again observed.     

Spermatogonial Multiplication In the Chinese Hamster. Iv. Search For Long Cycling Stem Cells

ABSTRACT In the Chinese hamster, 17 days, i. e. one cycle of the seminiferous epithelium, after two injections of [³H]TdR given 24 hr apart, labelled cells were found among all types of spermatogonia, including stem cells (A_s). These labelled A_s spermato-gonia derive from one or more self-renewing divisions of the stem cells that originally incorporated [³H]TdR. In the steady state, half of the divisions of the A_s will be self-renewing and the other half will give rise to A_pr spermatogonia that will ultimately become spermatozoa. Theoretically, the labelling index (LI) after 17 days will be similar to that after 1 hr, and in this study twice as high as for the 1-hr interval since only one injection was given. However, experimental values only half that of the theoretical LI were found after 17 days. the following causes for the loss of labelled stem cells are discussed: (1) dilution of label because of division; (2) influx of unlabelled components of false pairs (i. e. newborn stem cells that still have to migrate away. mostly during G₁, from their sister cells and are scored as A_pr spermatogonia) between 1 hr and 17 days; (3) the existence of long- and short-cycling stem cells, probably combined with preferential differentiation of the short-cycling elements; (4) selective segregation of DNA at stem cell mitosis; and (5) irradiation death of radiosensitive labelled stem cells. As it is not impossible that factors 1, 2, 4 and 5 together account for the total loss of labelled stem cells, LI results do not provide evidence for the existence of separate classes of short- and long-cycling stem cells. The distributions of the LIs of the A_s, A_pr and A_al spermatogonia over the stages of the epithelial cycle at 17 days are similar to those at 1 hr after injection. Hence the regulatory mechanisms that govern the stimulation and inhibition of proliferation of A_s that give rise to new A_s for the next epithelial cycle are similar to those of the A_s that will divide into A_pr spermatogonia during the same epithelial cycle. Grain counts revealed that more [³H]TdR is incorporated into A_s, A_pr and A_al spermatogonia that are in S phase during epithelial stages X-IV than in stages V-IX.     

Identification of a major iodolipid from the horse thyroid gland as 2-iodohexadecanal

The incorporation of iodide into proteins (PBI) and lipids (LBI) of horse thyroid slices was measured in various conditions. Their dependency on the concentration of extracellular iodide was strikingly different. For PBI the relationship was biphasic with a decrease above 10 microM, likely to correspond to the Wolff-Chaikoff effect. On the contrary, LBI increased as a function of iodide concentration up to 100 microM. Methimazole (MMI) inhibited the incorporation of iodide into both LBI and PBI, but higher concentrations of MMI were required to depress LBI as compared to PBI. The inhibition of active iodide transport by NaCIO4 reduced both PBI and LBI. Chromatography on silica gel resolved almost equal amounts of low and high polarity iodolipids. The main unpolar iodolipid was identified as 2-iodohexadecanal (2-IHDA), on the basis of proton nuclear magnetic resonance spectroscopy, mass spectrometry, and co-elution with authentic 2-IHDA obtained by chemical synthesis in reversed-phase high performance liquid chromatography and gas chromatography. The presence of 2-IHDA was also detected in dog thyroid slices, following incubation with KI (50 microM) and in the rat thyroid, 4 hours after intraperitoneal injection of KI (650 micrograms). An incubation of bovine brain plasmalogens with lactoperoxidase, iodide, and H2O2 generated 2-IHDA. In conclusion, we have identified a major thyroid iodolipid as 2-iodohexadecanal. The biosynthesis of this compound is likely to involve the addition of iodine to the vinyl ether group of plasmalogens.     

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.     

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 microCi tritiated thymidine ( [3H]TdR) (2 microCi/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 microCi [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.     

Food restriction induced thyroid changes and their reversal after refeeding in female rats and their pups

In the present study, two groups of pregnant female rats were submitted to food restriction (24 h fast versus 24 h diet intake) from the 14th day of pregnancy until either the 14th day (group B) or the 4th day after parturition (group C). All pups and their mothers were sacrificed on day 14 after delivery. The body weight of the 14-day-old pups (group B) was 46% less than the controls (group A). Free thyroxine and free triiodothyronine levels in the plasma were reduced by 44 and 16% in pups and by 20 and 36% in their mothers, respectively. These reductions were correlated with a decrease in thyroid iodine content of the pups (-50%) and their mothers (-24%). Radioiodine uptake (131I) by the thyroid gland of pups was significantly increased by 27%. Plasma TSH levels were decreased by 38% in pups and by 44% in dams. Morphological changes in thyroid glands were observed in energy restricted dams and in their pups. Some of follicles in pups were empty. Moroever in dams, we noted the presence of peripheral resorbed vacuoles, sign of thyroid hyperactivity. After a refeeding (group C) period of ten days, total recovery occurred in plasma thyroid hormone levels (FT4 and FT3) and in thyroid iodine contents of pups in spite of a partial recovery of body weights and plasma TSH levels. In dams, a partial recovery occurred in plasma thyroid hormone levels in spite of total recovery in thyroid iodine contents, while plasma TSH levels exceeded control values. A significant amelioration in thyroid histological aspects was observed in pups and their dams.     

Retention of labelled DNA precursor by murine cells after a single administration of tritium labelled thymidine

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.     

Quantitative study of deoxycytidine incorporation in large and small lymphocytes of the mouse

Using radioautographic smear preparations of thymocytes and mesenteric lymph node (MLN) cells labelled with three different tritiated pyrimidine deoxyribonucleosides, the incorporation of DNA precursors was studied separately on large lymphocytes and small lymphocytes. Radioautographic reaction due to generally tritiated deoxycytidine ( [G-3H]CdR) labelling in vivo in large lymphocytes was more intense than that in small lymphocytes. When mice were sacrificed 6 hr after the administration of tritiated thymidine ( [3H]TdR), small lymphocytes were labelled more heavily than large lymphocytes. However, labelling intensity with [3H]TdR in large lymphocytes was greatly enhanced by the administration of 5-fluoro-deoxyuridine, whereas in small lymphocytes labelling intensity was only fairly enhanced by the same treatment. When cells were incubated in vitro with 5-tritium labelled deoxycytidine [( 5-3H]CdR) for 10 min, there was no significant difference in labelling intensities between large and small lymphocytes. In the case of [G-3H]CdR incorporation, the labelling intensity in large lymphocytes was found to be significantly stronger than that in small lymphocytes. Large as well as small lymphocytes incorporated [3H]TdR very well in vitro. However, addition of 5 X 0 X 10(-5) M of non-radioactive CdR to the medium greatly decreased the incorporation of [3H]TdR by large lymphocytes, whereas the effect of non-radioactive CdR in small lymphocytes was not so marked as that in large lymphocytes. Furthermore, the [3H]TdR-labelling percentages were decreased at the same rate by the addition of non-radioactive CdR in both large and small lymphocytes. These results indicate that large lymphocytes and a proportion of small lymphocytes have a strong tendency to convert CdR to thymidine mono-phosphate, which is utilized for DNA synthesis, whereas this ability is relatively weak in the rest of small lymphocytes. Thus, it is probably that this metabolic ability changes during the transition of the large lymphocyte to the small lymphocyte.     

Evidence for discrete cell kinetic subpopulations in mouse epidermis based on mathematical analysis

Abstract. Continuous (repeated) labelling studies in mouse epidermis indicate that nearly all cells are labelled after about 100 hr. Percentage labelled mitoses studies ([³H]TdR at 15.00 and 03.00 hours) have a first peak that does not reach 100% and has a half-width of about 10 hr. Small second and third peaks can be detected at about 90 and 180 hr, respectively. The changes with time in the number of labelled cells show a difference dependent on the time of day of [³H]TdR administration. Both curves show an early doubling in labelled cells which then decline, forming a peak of labelled cells. A second peak occurs at about 120 hr. This is followed by a progressive decline with no further peaks until values of about 1% labelling are obtained at 340 hr.
These experiments have been investigated mathematically. A computer programme has been devized that permits all three types of experiments to be analysed simultaneously. More importantly, it can analyse situations with a heterogeneity in cell cycle parameters in all proliferative subpopulations.
Various models for epidermal cell replacement have been considered. The data as a whole can best be explained if the basal layer contains at least two distinct subpopulations of cells and an exponentially decaying post-mitotic population with a half-life of about 30 hr. The proliferative sub-populations must be characterized by near integer differences in the length of cycle, the precursor (stem) compartment having the longer cycle. An inverse relationship is required for the length of S, i.e. the shortest time for the stem cells.
A full range of cell kinetic parameters can be calculated and are tabulated for the most appropriate model system which is one involving three transit proliferating subpopulations.     

The subcellular distribution of 32P-labelled phospholipids, 32P-labelled ribonucleic acid and 125I-labelled iodoprotein in pig thyroid slices. Effect in vitro of thyrotrophic hormone and dibutyryl-3′,5′-(cyclic)-adenosine monophosphate

1. The incorporation in vitro of [(32)P]phosphate into phospholipids and RNA and of [(125)I]iodide into protein-bound iodine by pig thyroid slices incubated for up to 6hr. was studied. The subcellular distribution of the labelled products formed after incubation with radioactive precursor in the nuclear, mitochondrial, smooth-microsomal, rough-microsomal and cell-sap fractions was also studied. 2. Pig thyroid slices actively took up [(32)P]phosphate from the medium during 6hr. of incubation; the rate of incorporation of (32)P into phospholipids was two to five times that into RNA. 3. The uptake of [(125)I]iodide by the slices from the medium was rapid for 4hr. of incubation, 6-10% of the label being incorporated into iodoprotein. 4. Much of the (32)P-labelled phospholipid accumulated in mitochondria and microsomes, whereas the nuclear fraction contained most of the (32)P-labelled RNA. After 2hr. of incubation most of the (32)P-labelled cytoplasmic RNA accumulated in the rough-microsomal fraction. The major site of localization of proteinbound (125)I was the smooth-microsomal fraction, and gradually increasing amounts appeared in the soluble cytoplasm fraction, suggesting a vectorial discharge of [(125)I]iodoprotein (presumably thyroglobulin) from smooth vesicles into the colloid. 5. The addition of 0.1-0.4 unit of thyrotrophic hormone/ml. of incubation medium markedly enhanced the accumulation of (32)P-labelled phospholipids in the microsomal fractions and to a much smaller extent that of (32)P-labelled RNA without any increase in the total uptake of the label. Almost simultaneously the hormone increased the uptake of [(125)I]iodide by the slices and enhanced the accumulation of protein-bound (125)I in the smooth-microsomal fraction. 6. As a function of time of incubation, thyrotrophic hormone had a biphasic effect on [(125)I]iodide uptake and protein-bound (125)I formation, the stimulatory effect being reversed after 4hr. of incubation. 7. 6-N-2'-O-Dibutyryl-3',5'-(cyclic)-AMP, but not 3',5'-(cyclic)-AMP or 5'-AMP, mimicked the action of thyrotrophic hormone on iodine uptake as well as on iodination of protein. On the other hand, the mimicry by 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP of the stimulatory effect of thyrotrophic hormone on the formation of labelled thyroid phospholipids and RNA was only an apparent one resulting from an enhanced uptake of [(32)P]phosphate. 8. It is concluded that thyrotrophic hormone causes a co-ordinated increase in the formation or accumulation of phospholipids, RNA and iodoprotein associated with the endoplasmic reticulum, and that 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP mimics the more rapid effects of thyrotrophic hormone on transport and metabolic functions of thyroid cells, but does not influence their slower biosynthetic responses to the hormone.     

Comparative analysis of different approaches to investigate cell kinetics

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 (rs = 0.90; P less than 0.01), [3H]TdR LI and S phase (rs = 0.62; P less than 0.01) and [3H]TdR LI and Ki67 (rs = 0.64; P less than 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.