PROLIFERATION OF LIVER EPITHELIA IN THREE-WEEK-OLD RATS

The proliferative activity of liver epithelia in 3-week-old rats was studied auto-radiographically using ³H-thymidine. Only a single peak of labelled mitoses (late pro- to anaphases) at 7 hr was found in the per cent labelled mitoses curve after injection of ³H-thymidine. A second peak at about 32 hr described by Post, Huang & Hoffman (1963) and Post & Hoffman (1964, 1965) as well as Grisham (1969) and a cycle time of about 22 hr derived from the distance between the two peaks could not be confirmed by the present work.
According to the present experiments the cycle time of parenchymal liver cells in 3-week-old rats must range between 50 hr (with a growth fraction of 0·25) and 7·1 days (with a growth fraction of 1·0). The present results do not support the existence of a growth fraction of only 0·1 as assumed by Post et al. (1963).     

RECYCLING OF RESTING CELLS IN THE JB-1 ASCITES TUMOUR AFTER TREATMENT WITH 1-β-D-ARABINOFURANOSYLCYTOSINE (Ara-C)

Resting cells in tumours present a major problem in cancer chemotherapy. In the plateau phase of growth of the murine JB-1 ascites tumour (i.e. 10 days after 2–5 × 10⁶ cells i.p.) large fractions of non-cycling cells with G₁ and G₂ DNA content (Q₁ and Q₂ cells) are present, and the fate of these resting cells was investigated after treatment with l-β-d-arabinofuranosylcytosine (Ara-C). The experimental work consisted of growth curves, percentage of labelled mitoses curves after continuous labelling with ³H-TdR, and cytophotometric determination of single-cell DNA content in unlabelled tumour cells. Treatment with an i.p. single injection of Ara-C 200 mg/kg in the plateau JB-1 tumour resulted in a significant reduction in the number of tumour cells 1 and 2 days later as compared with untreated controls, while no difference in the number of tumour cells was observed after 3 days. In tumours prelabelled with ³H-TdR 24 hr before Ara-C treatment, a significant decrease in the percentage of labelled mitoses was observed 6–8 hr later followed by a return to the initial value after 12 hr, and a new pronounced fall from 20 hr after Ara-C. The second fall in the percentage of labelled mitoses disappeared when the labelling with ³H-TdR was continued also after Ara-C treatment. Cytophotometry of unlabelled tumour cells prelabelled for 24 hr with ³H-TdR before Ara-C treatment showed 20 hr after Ara-C a pronounced decrease in the fraction of Q_t cells paralleled by an increase in the fraction of unlabelled cells with S DNA content. These results indicate recycling of resting cells first with G₂ and later with G_x DNA content, which contribute to the regrowth of the tumours.     

CIRCADIAN INFLUENCE ON THE WAVE FORM OF THE FREQUENCY OF LABELED MITOSES IN MOUSE CORNEAL EPITHELIUM

The FLM was studied in mouse corneal epithelium at two different phases of the mouse circadian system. The following results were obtained: (1) a circadian rhythm was found in the corneal mitotic index of both experimental groups, with the peak near 09.00 hours, the trough near 21.00 hours and a 10-fold difference in values between the peak and the trough; (2) when ³H-TdR was injected at 09.00 hours, the first wave of labelled mitoses rose earlier, attained a broader peak and fell later when compared to the first wave of labeled mitoses obtained when ³H-TdR was injected at 21.00 hours; and (3) in both experimental groups there was a suggestion of a second wave of labeled mitoses, especially in the group injected with ³H-TdR at 21.00 hours, where a flat second wave appeared in the region of 60–90 hr. Transit times derived from FLM curves obtained from such synchronous populations of cells are of questionable validity. Circadian rhythmicity and other causes of synchronization must be ruled out before accepting a single FLM curve as a valid indication of transit times.     

THE KINETICS OF CELL PROLIFERATION IN THE TRACHEOBRONCHIAL EPITHELIA OF RATS WITH AND WITHOUT CHRONIC RESPIRATORY DISEASE

Labelling indices of the tracheobronchial epithelia of conventionally-derived rats with chronic respiratory disease (CRD) and minimal-disease rats without CRD have been determined. The duration of the DNA synthesis phase (t_s) computed from the percentage of mitoses labelled at various intervals of time after injection of tritiated thymidine was 7 hr: t_G2 was 3.5 hr. Using the measured value of t_s and the labelling indices, the mean turnover times of the tracheobronchial epithelia in three groups of six 5-week-old conventionally-derived rats were calculated to be 11.2, 14.6 and 22.4 days, while in similar groups of 5-week-old minimal-disease rats the turnover times were found to be 24.3, 36.5 and 41.6 days. The majority of cell divisions in the tracheobronchial epithelium of these minimal disease rats were probably required for growth rather than renewal. The mean turnover time of this tissue in 5-week-old Syrian hamsters was 73 days. The cells of the rat tracheobronchial epithelium have been classed as basal or superficial, depending on their shape and proximity to the basement membrane. The mean turnover time of the basal cells in 5-week-old minimal-disease rats was 11.7 days calculated from labelling indices. The migration method of Brown & Oliver (1968) gave a similar value for the basal cells in minimal-disease rats, and a value of 9.5 days for the basal cells in a group of conventionally-derived rats. The mean turnover time in the latter was only 5.4 days if two rats with tracheobronchitis were included. Consideration of the slow rate of fall in mean grain count over labelled cells at intervals of time after labelling and the calculated turnover times suggests that the proliferative fraction of the basal cell population is close to unity. Well-labelled cells were still present in both basal and superficial populations in the minimal-disease rats at 10 days after labelling. The marked effects of CRD on cell proliferation in this epithelium are emphasized and the significance of this in relation to published work is discussed.     

Recycling of resting cells in the JB-1 ascites tumour after treatment with 1-beta-D-arabinofuranosylcytosine (ara-C).

Resting cells in tumours present a major problem in cancer chemotherapy. In the plateau phase of grwoth of the murine JB-1 ascites tumour (i.e. 10 days after 2-5 X 10(6) cells i.p.) large fractions of non-cycling cells with G1 and G2 DNA content (Q1 and Q2 cells) are present, and the fate of these resting cells was investigated after treatment with 1-beta-D-arabinofuranosylcytosine (Ara-C).The experimental work of growth curves, percentage of labelled mitoses curves after continuous labelling with 3H-TdR, and cytophotometric determination of single-cell DNA content in unlabelled tumour cells. Treatment with an i.p. single injection of Ara-C 200 mg/kg in the plateau JB-1 tumour resulted in a significant reduction in the number of tumour cells 1 and 2 days later as compared with untreated controls, while no difference in the number of tumour cells was observed after 3 days. In tumours prelabelled with 3H-TdR 24 hr before Ara-C treatment, a significant decrease in the percentage of labelled mitoses was observed 6-8 hr later followed by a return to the initial value after 12 hr, and a new pronounced fall from 20 hr after Ara-C. The second fall in the percentage of labelled mitoses disappeared when the labelling with 3H-TdR was continued also after Ara-C treatment. Cytophotometry of unlabelled tumour cells prelabelled for 24 hr with 3H-TdR before Ara-C treatment showed 20 hr after Ara-C a pronounced decrease in the fraction of Q1 cells paralleled by an increase in the fraction of unlabelled cells with S DNA content. The results indicate recycling of resting cells first with G2 and later with G1 DNA content, which contribute to the regrowth of the tumours.     

EFFECTS OF UTERINE DISTENSION AND OESTRADIOL ON CELL KINETICS IN THE ENDOMETRIAL EPITHELIUM OF OVARIECTOMIZED RATS

Cell kinetics in the uterine epithelium of ovariectomized rats were studied after uterine distension and/or an oestradiol injection, by cumulative ³H-TdR labelling and percentage of labelled mitoses (PLM). With both methods it was found that distension shortens the total cell cycle at the expense of G₁ more than does oestradiol. Both treatments act in a cumulative manner since the greatest reduction in T_c is observed after distension plus oestradiol. PLM curves showed that distension and/or oestradiol induce a 30% reduction in S phase duration. The evolution of percentages of labelled cells and colchicine-blocked mitoses after these treatments confirms their additive effects and indicates that the mitogenic action of oestradiol is delayed compared to that of distension. It is suggested that these factors stimulate epithelial cell division in the uterus through partly different metabolic channels.     

THYROID PROLIFERATIVE POTENTIAL AS A FUNCTION OF AGE

The effect of a goitrogenic stimulus on thyroid weight and thyroid cell ³HTdR labeling of Sprague-Dawley rats varying from 2 to 40 weeks of age was determined. Propylthiouracil ad libitum in drinking water produced a spurt in follicle cell labeling index and thyroid weight evident after 24 hr for all age groups. The increase in labeling index reached a peak at 5–7 days and then decreased to a level a few times greater than that of the normal unstimulated thyroid. The tritiated thymidine labeling index for thyroid follicle cells and the effect of PTU thereon was determined for August male rats of 3 days to 12 weeks of age. In the older rats, the follicle cell labeling index rose to 5–6% after 4–5 days of PTU treatment and then slowly fell to about 1%, For the unstimulated control rat of comparable age, the labeling index was about 0.1%. At all ages the thyroid showed a rapid response to PTU. Examination of the time sequence of mitotic labeling showed that the DNA synthesis period was 7.5 hr for normal 2-week-old rats and for 10–12-week-old rats that had received PTU for 4 days. There was no second wave of labeled mitoses in either group during the 48-hr interval studied. From the curve of thyroid weight vs time on PTU and from the labeled mitoses curve, inferences regarding the minimum fraction of proliferating follicle cells in the stimulated ‘adult’rat thyroid were made.     

CHARACTERISTICS OF THE ISOPRENALINE STIMULATED PROLIFERATIVE RESPONSE OF RAT SUBMAXILLARY GLAND

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using ³H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMW,) waves gave a mean G_is transit time of 21-2 hr, mean G_is+ S transit time of 270 hr, and mean G_is+ S + G₂ transit time of 35-8 hr for a single injection of isoprenaline, where G_is is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the G_is phase dropped from 18-1 to 1–3 while the other variances remained unchanged. The estimated proliferative fraction was 0–24 after a single injection of isoprenaline, and 0–31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated ³H-TdR injections, were 0–21 and 0–36 respectively.     

SPERMATOGONIAL CHALONE(S): EFFECT ON THE PHASES OF THE CELL CYCLE OF TYPE A SPERMATOGONIA IN THE RAT

Adult rats with X-irradiated testes were used to analyze the effect of the spermatogonial chalone(s) on the phases of the cell cycle of type A spermatogonia. Twelve days after irradiation, the animals were used in two experiments designed to test the existence of hypothetical G₂ and S phase chalones. For the G₂ assay, rats injected twice with testicular extract (Group I), liver extract (Group II) or physiological saline (Group III) were killed 10 hr after the initial injection. Mitoses of type A, Intermediate and type B spermatogonia were counted in whole mounts of dissected seminiferous tubules. To test for an S phase inhibitor, two groups of rats were given multiple injections of either testicular extract (Group IV) or saline solution (Group V). Twenty-two hr after the first injection they were injected with [³H]thymidine and killed 2 hr later. Silver grains over labelled type A nuclei were counted in radioautographed sections of testes from these animals. The average grain counts were identical in Groups IV and V, indicating that the testicular extract did not affect type A spermatogonia during the S phase. Counts of type A mitoses in Groups I, II and III revealed that in the animals injected with the testicular extract (Group I) the number of divisions was 50% lower than in the control groups (Groups II and III). In contrast, mitotic activity of differentiating spermatogonia (In + B) was similar in all three groups of animals. This result is attributed to a testicular chalone which specifically inhibits type A spermatogonia during the G₂ phase of the cell cycle. Indirect evidence for a G₁ spermatogonial chalone is also presented, as a result of an analysis of published data (Clermont & Mauger, 1974).     

KINETICS OF ERYTHROPOIETIN STIMULATED PROLIFERATION OF ERYTHROID PROGENITORS IN THE SPLEEN

The effect of RBC transfusion and erythropoietin (EPO) on the proliferation of immature erythrocyte progenitors was studied in the spleens of RBC transfused, lethally irradiated mice injected with bone marrow. Transfusion decreased expansion of the progenitors and slowed their proliferation: the mean cycle time as measured by per cent labelled mitosis (PLM) on the third day after injection of bone marrow was 10.7 hr in transfused as compared to 5.6 hr in non-transfused mice. One injection of five units of erythropoietin on day 2 decreased the mean cycle time to 7.3 hr in transfused mice and increased expansion of the progenitor cells. The effects of erythropoietin on cell proliferation were prompt: a significant increase of incorporation of ³H-TdR into DNA occurred within 2 hr of injection. Erythroblasts were absent from the spleens of transfused, irradiated bone marrow injected mice; however, erythroblasts appeared by 72 hr and 48 hr following EPO injection either 2 days or 5 days after transplantation respectively. Increased uptake of radioactive iron in spleen after erythropoietin injection preceded the appearance of erythroblasts by 2 and 1 days when erythropoietin was injected either 2 or 5 days after marrow transplantation respectively. The increase in cellular proliferation induced by erythropoietin in transfused irradiated mice injected with bone marrow equivalent to 0.35 femoral shaft was manifested as an increase of the total DNA content in the spleen by 119 μg (11.9 × 10⁶ cells) within 48 hr of injection. The cellular increment produced by EPO injection on day 5 to mice given 0.05 femoral shaft consisted mainly of undifferentiated mononuclear cells, most of which were labelled, with erythroblasts comprising only one quarter of the increment. Erythropoietin inactivated by mild acid hydrolysis failed to increase cellular proliferation.     

CYTOKINETICS OF RAT TRACHEAL EPITHELIUM STIMULATED BY MECHANICAL TRAUMA

Mild abrasion of rat tracheal epithelium results in irreversible damage to the superficial cells and stimulates the viable basal cells to participate in a nearly synchronous wave of DNA synthesis and mitosis. For the growth population as a whole, DNA synthesis started at 14 hr after injury and persisted for 16 hr. The duration of S in individual cells was determined autoradiographically by identifying the time at which a second pulse of DNA precursor (¹⁴C-TdR) was no longer incorporated by cells labelled with ³H-TdR at the onset of S. S was found to be 8–9 hr long. It was also determined that cells entering S at later times synthesized DNA for the same 8–9 hr period. T_G2 was calculated to be 21/2–31/2 hr by subtraction of T_s and 1/2T_M from the period from onset of DNA synthesis to metaphase. By making a second denuding lesion adjacent to the first injury, the cells were stimulated through at least another period of S. At the peak of the second wave of DNA synthesis (50 hr after injury) ¹⁴C-TdR was present in the same cells which had incorporated ³H-TdR administered at the mid-point of the preceding synthetic phase. The 28-hr interval between these two peaks of synthesis is the measure of cell cycle duration for these regenerating tracheal epithelial cells.     

CIRCADIAN RHYTHMS OF PRESUMPTIVE STEM CELLS IN THREE DIFFERENT EPITHELIA OF THE MOUSE

Variation in the percentage of labelled cells (LI), mitoses (MI) and apoptosis (AI: i.e. shrinkage necrosis) have been studied throughout a 24 hr period (40 min after labelling with ³H-TdR) for tongue epithelium, epidermis and intestinal epithelium in the mouse. A room with reversed light cycle was used to obtain data for half of the 24 hr period. All three tissues showed marked variations in LI with peak values between 24.00 and 03.00 hours. In the intestine a maximum value for MI was observed 3-6 hr after that for LI and with a maximum value for AI slightly later. In all three epithelia the circadian rhythm was most striking in cells at positions which can be correlated with presumptive stem cell activity; e.g. in the crypts the labelling and mitotic peaks reflecting a circadian rhythm were most clearly distinguishable at the basal part of the crypts. These observations are discussed in relation to the validity of various proliferative models.     

THE INFLUENCE OF INJECTED TRITIATED THYMIDINE ON THE MITOTIC CIRCADIAN RHYTHM IN THE EPITHELIUM OF THE HAMSTER CHEEK POUCH*

The initial effect of an injection of TdR-5-³H (1 μCi/g body weight; 6 Ci/mmol) in the cheek pouch epithelium of the Syrian hamster is an increase in the mitotic index. The increase is observed 1–5 hr after injection, depending upon the time of day when the injection is given, and is followed by compensatory variations in mitotic index. This deviation from the normal circadian rhythm in the mitotic index appears to depend on the fraction of G₂-cells at the time of injection. The main effect is a shortening of t_g2. No effect is observed after injection of non-radioactive TdR or isotonic saline. The results of the present experiment emphasize that unexpected results may be obtained when using mitotic indices from animals labelled with ³H-TdR, as well as the risks of using the PLM-method in a partially synchronized system.     

STUDY OF CELL DEATH IN FRIEND LEUKAEMIA

Cell death of splenic Friend leukaemic cells has been studied in vivo, using ¹²⁵I-UdR and ³H-TdR pulse labelling. The evolution of the splenic specific activity has been measured by autoradiography and external counting during 40 hr after injection of the labelled precursor. These two techniques show the existence of a large reutilization of ³H-TdR (50%), which is measurable as soon as 7 hr after the injection. The DNA turnover rate is rapid, 83-8 % of the splenic cellular DNA being renewed per day. These results confirm that most of the cells produced in the Friend leukaemic spleen are rapidly lost; they also demonstrate that this cell loss is mainly due to a massive death, which occurs in proerythroblastic and erythroblastic compartments after one or two cell divisions. Friend leukaemic cells, which are characterized by a limited capacity of proliferation and a short lifespan, do not appear to be malignant.     

DIFFERENT ACTION OF SUICIDAL DOSES OF TRITIATED THYMIDINE AND HYDROXYUREA ON MURINE HAEMOPOIETIC CELLS

In order to gather information on the factors that cause the different action of suicidal doses of tritiated thymidine (³H-TdR) and of hydroxyurea on murine stem cells, the incorporation of ³H-TdR into DNA of bone marrow and spleen cells has been studied. Continuous death of labelled cells after suicidal ³H-TdR is indicated by a more pronounced decline of total DNA-bound radioactivity in bone marrow and spleen cells compared to that in control animals which had received tracer doses of ³H-TdR. Extensive and rapid loss of DNA-bound radioactivity occurred in ³H-TdR labelled animals after hydroxyurea treatment indicating an instantaneous and highly effective killing of labelled cells. After double labelling of DNA with ³H-TdR and ¹²⁵iodo-deoxyuridine (¹²⁵I-UdR), the decline of the ratio of DNA-bound ¹²⁵I to DNA-bound ³H after suicidal ³H-TdR indicates prolonged tritium reutilization. Following hydroxyurea, reutilization was completed within the first 12 hr after drug administration. These findings explain in part the slow recovery of different stem cell compartments after suicidal ³H-TdR on the basis of protracted tritium reutilization as compared to the fast recovery which follows the rapid action of hydroxyurea.     

Characteristics of the isoprenaline stimulated proliferative response of rat submaxillary gland.

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using 3H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMWa) waves gave a mean Gis transit time of 21-2 hr, mean Gis +S transit time of 27-0 hr, and mean Gis + S + G2 transit time of 35-8 hr for a single injection of isoprenaline, where Gis is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the Gis phase dropped from 18-1 to 1-3 while the other variances remained unchanged. The estimated proliferative fraction was 0-24 after a single injection of isoprenaline, and 0.31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated 3H-TdR injections, were 0-21 and 0-36 respectively.     

G2 Sub-Population In Mouse Liver Induced Into Mitosis By Lead Acetate

A single intracardiac dose of lead acetate (40 μ lead/g body weight) induced a 25-fold increase in mitosis of mouse hepatocytes 5 hr after injection, as determined by autoradiography. the prompt appearance of a mitotic wave and the relatively large number of mitoses suggest that the mitotic cells were derived from a hepatocyte sub-population arrested in the G₂ phase. the injection of lead also stimulated a small increase in labeled hepatocytes within 6 hr. Analysis of grain counts gave no evidence for unscheduled DNA synthesis. the incremental labeled cells may have originated from a small fraction of the G₁ population that was ready to enter the S phase without the usual pre-synthetic delay.     

Thymidine metabolism and the monitoring of DNA synthesis in insects

The thymidine degradation pathway established for other organisms is confirmed in insects. When ³H-TdR is used as a marker for DNA synthesis in developing silkmoths, some is incorporated into DNA and some degraded to compounds not incorporated into DNA. After a single injection, ³H-TdR is rapidly cleared from haemolymph and other tissue, resulting in, at most, a 4 hr pulse. In wing tissue, detection of DNA synthesis is possible for a maximum of 4 hr after injection of precursor and for 6 hr in vitro. Continuous monitoring of DNA synthesis can be attained by perfusion, which maintains high levels of circulating ³H-TdR.