Growth Fraction of Myelocytes In Normal Human Granulopoiesis

Abstract. The labelling index (LI) of myelocytes (M) after flash labelling of normal human bone marrow cells with [³H]-thymidine ([³H]TdR) is always lower that the LI obtained for myeloblasts (MB) and for promyelocytes (PM). This fact can be interpreted in two ways: it may mean that the duration of the G₁ phase of the cell cycle is longer in M than in MB or PM, or it may mean that the proportion of cells in cycle, i.e., the growth fraction (GF), is lower in the M population than in MB or PM. the evolution of the LI and of the mean number of grains per cell was monitored in [³H]TdR-labelled normal bone marrow during in vitro incubation for 50 hr. the generation time, measured by the halving time of the mean number of grains per cell after flash labelling, was similar for M to that for MB and PM. During continuous labelling, the LI of MB and PM reached 1 and the LI value for M never rose to more than 50% of the values recorded for MB and PM after 30 hr. These findings give support to the second hypothesis, i.e., a lower GF in the M population. Good correlation was found between the LI of M and the proportion of mature polymorphonuclear cells in the bone marrow of normal subjects and of patients with chronic benign neutropenia or hyperleucocytosis. Variations in the M growth fraction could be a medium-term (2-3 days) regulatory factor in granulocyte production.     

An unusual labeling index pattern of neutrophilic granulocytes after a 3H-thymidine flash labeling injection

After flash labeling of neutrophilic granulocyte precursors with 3H-Thymidine in 3 hematologically normal subjects the postinjection labeling indices (LI) and the labeling intensities of venous blood neutrophils were determined. The LI pattern during 18 days after injection showed a reproducible triphasic course of the LI curves: (a) a low "plateau", (b) a steep rise, (c) an undulating decrease until the end of the experiments. On the basis of these findings a simultaneous "at random" and "first in--first out" release of neutrophils from the postmitotic bone marrow compartment to the blood was suggested. Moreover, the intravascular half-life time of the neutrophils was determined as 9.3 h, the corresponding mean circulation time was 14 h. From the undulating course of the LI curves and the labeling intensity curves the duration of the mean neutrophil precursor cell cycle time in the mitotic bone marrow compartment was estimated to range between 19 and 24 h.     

CYTOKINETICS OF TUMOUR AND ENDOTHELIAL CELLS AND VASCULARIZATION OF LUNG METASTASES IN C3H/HE MICE

A model of lung metastases was developed using intravenous injection of tumour cell aggregates of spontaneous C3H/He mammary tumours in syngeneic mice. the growth rate of lung tumours decreased with increasing tumour volume, with mean host survival of 46 days. the cytokinetics of individual tumours ranging between 0.004 and 4.2 mm³ in volume were studied. the labelling index (LI) ranged between 12 and 17%, the DNA synthesis time (T_s) being 9–10 hr. the growth fraction (GF) ranged between 26 and 38%. the cell cycle time (T_c) was found to be 18–19 hr. the LI and the GF decreased with increasing tumour volume doubling time (T_d). No correlation was found between the tumour volume and T_c. the LI of endothelial cells within these tumours, ranging between 0.004 and 4.2 mm³ in volume was 14–15% and endothelial cell proliferation was not affected by tumour growth. Vascular parameters were also determined for these tumours as a function of tumour volume. Vascular volume increased with increasing tumour size while the percentage of capillary vessels decreased. the cellular volume to capillary volume ratio increased with increasing tumour volume. Necrosis was observed in 0.27 mm³ tumours and increased with increasing tumour size. The results from these studies suggested that the age-dependent decrease in proliferative activity of tumour cells growing in the lung is related to change in effective vascularity.     

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.     

Rat C-6 Glioma Cells Maintained In an Organ Culture System: A Study of Kinetic Parameters

Rat C-6 glioma cells were grown on a sponge foam matrix in an organ culture system and the cell cycle parameters, including the growth fraction (GF), were assessed after autoradiography. the zones of growth consisted of a compact upper layer (UL) at the gaseous interface, a central necrotic layer and a deeper lower layer (LL) which invaded the matrix. the fraction of continuously labeled mitoses (FCLM) was similar in both the UL and LL cells. the derivatives of the FCLM curves obtained in three experiments gave an average modal T_G2 of 5 hr. A mathematical model relating GF, T_G2, T_C and labeling index as a function of time, LI(t), was devised for cells in a steady state exposed continuously to tritiated thymidine and was applied to data obtained from UL cells. A mean GF of 9% (range: 8–10%) and a mean cell cycle time (T_C') of 27 hr (range: 13–47 hr) were obtained. the mean T_S was calculated to be 11 hr (range: 8–16 hr) by the method of grain counts per mitotic figure or grain index (GI). Knowledge of T_S permitted alternative calculation of the cell cycle time from the equation T_S/T_C= LI(0)/GF: this gave a mean cell cycle time (T_C) of 29 hr (range: 20–45 hr). Except for the GF, the cell kinetics were comparable to those of the same cell line grown in monolayer culture. the GF in the in vitro system described is in the lower range reported in some human malignant gliomas in vivo.     

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.     

REGENERATION OF UTERINE EPITHELIUM AFTER EXPERIMENTAL ABLATION IN THE RAT

Regeneration of the uterine luminal epithelium was studied after its mechanical removal in progesterone-primed rats, leaving one control horn intact. Pulse labelling with [³H]TdR during regeneration, showed a rapid peak of labelling index in remaining glands. A differentiated and highly labelled luminal epithelium reappeared at 34 hr, thereafter showing a rapidly declining LI. After initial depletion, the glandular cell population size was restored within 64 hr, whereas luminal epithelium cell numbers became stabilized at about half normal level. Grain counts after prelabelling showed more rapid dilution in gland cells of stripped uterine horns, indicating accelerated cycling of previously dividing cells. Thymidine labelling indices also showed that, after removal of the epithelium, almost all gland cells became rapidly committed to divide. On average, less than two cell cycles were necessary to restore stable glandular and epithelial population sizes. Numbers of labelled cells were also drastically increased in myometrium and serosa of treated horns. This suggests a non-specific mechanism for stimulation of mitotic activity after ablation of epithelium.     

Proliferation kinetics of plasma cells and of normal haemopoietic cells in multiple myeloma

DNA synthesis time (Ts) and 3H thymidine (TdR) labelling index (LI) of bone marrow (BM) myelomatous plasma cells (PC) and of the residual haemopoietic cell population (RHCP) were measured by in vitro quantitative 14C-TdR autoradiography in five patients with multiple myeloma (MM) in different phases of disease (three at presentation and two at relapse) and in one patient with solitary extra-osseous myeloma. One other patient with plasma cell leukaemia (PCL) was studied during an initial relapse phase and later during the leukaemic terminal phase. PC Ts was 18.8 +/- 3.7 (from 13.3 to 25.0) hr and PC LI was 2.5 +/- 1.8% (from 1.0 to 6.3%). In the case of PCL, circulating PC had a Ts of 14.4 hr and a LI of 3.1. From these experimental measurements, the fractional turnover rate (FTR-percentage of cells produced per unit time) and the potential doubling time (Td) of BMPC were calculated assuming that all BMPC were in a steady-state at the time of the study. BMPC FTR was 3.53 +/- 2.3% cells per day (from 1.2 to 6.72) and BMPC Td was 46.8 +/- 27.5 days (from 15.0 to 75.4). Comparison with results obtained in BM blasts of children with acute lymphoblastic leukaemia (ALL) indicated that BMPC had a lower proliferative activity (P less than 0.001), although BMPC Ts was not significantly different. In two patients a tumour doubling time of 6 and 13 months was determined by clinical follow up. Comparison of this parameter with Td showed a cell loss factor of more than 90% in both patients. Kinetic data relative to RHCP showed slight variations with respect to those found in normal subjects, with a general tendency towards a prolongation of Ts and a reduction of LI.     

MEASUREMENT OF THE DURATION OF DNA SYNTHESIS IN ERYTHROID CELLS OF THE BONE MARROW USING A DOUBLE LABELLING AUTORADIOGRAPHIC TECHNIQUE DESIGNED SPECIFICALLY FOR USE WITH HAEMOPOIETIC TISSUES

This paper describes a double labelling autoradiographic technique for use with haemopoietic tissues. In involves two photographic emulsions separated by a thin piece of mica on which the cells have been smeared. In this way the autoradiographic grains due to tritium and carbon-14 appear above and below the cells respectively. Applying the method to bone marrow normoblasts of young rats, the average duration of DNA synthesis (t_s) for the pro- and early normoblasts taken together is found to be 5.1 hr and the mean cell cycle time (t_c) to be 8.2 hr. For the intermediate normoblasts, the corresponding figures are 6.3 hr and 15.7 hr. Average values for all dividing normoblasts in the bone marrow are 5.8 hr and 12.8 hr respectively for t_s and t_c. The average duration of mitosis is 32 min.     

Negative effect of iodide on the survival of newly divided epithelial cells in chronically stimulated rat thyroid

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 microCi/g). Some animals were killed 1 hr or 24 hr after [3H]TdR injection (i.e. on day -2 and -1, day 0 corresponding to the restoration of a normal iodine diet); the other animals were killed after different delay periods and following [3H]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 0 of iodide refeeding (3000 +/- 330 ng/ml) and remained at this level until day 8. Plasma PBI was very low on day 0, remained so until day 4 and greatly increased on day 8. Plasma iodide was also very low on day 0, but markedly increased on day 1, then did not vary significantly until day 43 of iodine refeeding. Thyroid weight, elevated on day 0, decreased relatively quickly until day 30, then more slowly until day 73. The [3H]TdR labelling index (LI) of the thyroid epithelial cells (TEC) was high on day 0 (56 +/- 3 labelled cells/10,000 cells), and 24 hr thereafter increased to 104 +/- 3, by division of the labelled cells. On day 1 of iodine refeeding, the LI had abruptly decreased to about half this value and then remained stable for 3 more days. Between day 4 and day 16, a progressive decline in the LI, (by about 3-4 per day), was observed. The LI showed no further modification, up to day 73, the longest period investigated. The decrease in LI occurred without any significant changes in the labelling intensity (grain count) of the remaining labelled cells between day 1 and 16, this indicates that no cell division took place during this period. The data are therefore interpreted as showing a biphasic elimination after iodide refeeding, of cells that were actively proliferating during the goitrous state.(ABSTRACT TRUNCATED AT 400 WORDS)     

骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖的促进作用

本文通过制备小鼠骨髓内皮细胞无血清条件培养液(serum-free murine bone marrow endothelial cell conditioned medium, mBMEC-CM),经超滤分为分子量>10 kDa组分和<10 kDa组分,分别观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞集落生成的影响。用Wright’S Giemsa染色计数内皮细胞集落及检测骨髓内皮细胞的vWF,通过[3H]- TdR掺入量,观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞增殖的影响,并用分子杂交方法检测内皮细胞表达的细胞因子,从几个方面来研究mBMEC-CM对骨髓内皮细胞增殖的作用。结果显示,骨髓内皮细胞vWF 检测阳性。mBMEC-CM原液及其分子量>10 kDa组分能刺激骨髓内皮细胞集落增殖,且能明显增加骨髓内皮细胞[3H]-TdR 掺入量;分子量<10 kDa组分对骨髓内皮细胞集落增殖无明显刺激作用,也不能增加骨髓内皮细胞[3H]-TdR掺入量。外源加入IL-6、IL-11、SCF、GM-CSF、VEGF、bFGF 6种细胞因子能明显刺激骨髓内皮细胞集落增殖,SCF、VEGF、bFGF能明显增加骨髓内皮细胞[3H]-TdR掺入量。Atlas array膜杂交实验显示骨髓内皮细胞内源性表达GM-CSF、SCF、MSP-1、endothelin-2、thymosin β10、connective tissue GF、PDGF-A chain、MIP-2α、PlGF、neutrophil activating protein ENA-78、INF-γ、IL-1、IL-6、IL-13、IL-11、inhibin-α等细胞因子的mRNA。上述结果提示,骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖具有促进作用。     

MECHANISM OF EOSINOPHILIA

Eosinophil leucocyte production was studied in the bone marrow of normal rats and rats given single injections of Trichinella spiralis larvae which stimulated eosinopoiesis. the development sequence of eosinophils in the bone marrow was based on morphological criteria combined with studies of the extent of eosinophil labelling after injections of tritiated thymidine. the proliferative compartment contained at least three recognizable steps in eosinophil development which were defined.
There was a delay of 23 hr after injection of larvae before the proportion of eosinophils in the bone marrow increased and it had doubled by 49 hr. the mitotic index increased by a factor of 3 after stimulation. Estimates of the cell cycle parameters were made for marrow eosinophils 1-3 days after stimulation, using the technique of analysing labelled mitoses. the results were compared with a similar group of normal rats, and were processed by using a computer program. Marrow eosinophil cell cycle time was 30 hr in normal rats and 9 hr in stimulated rats, and this acceleration was associated with a reduced spread of cell cycle times. the number of eosinophil cell divisions and the transit times for each compartment in normal and stimulated rats were estimated. This showed that the stimulus may have resulted in five or six additional divisions among the youngest eosinophils in the dividing compartment. From these figures an outline of eosinopoiesis in the marrow of normal and stimulated rats is proposed.     

Autoradiographic studies of glial proliferation in different areas of the brain of the 14-day-old rat

Cell cycle parameters, as well as the mode of proliferation of glial cells, in four different areas of the brain of the 14-day-old rat (cortex, corpus callosum, nucleus caudatus putamen and commissura anterior) were studied using different cell kinetic methods after injection of [3H]TdR and/or [14C]TdR. The duration of the S phase (tS) was found to be about 10 hr and that of the cycle time (tC) about 20 hr, tG2 is less than 2 hr and t(G2 + M) about 4 hr. These values are valid for glial cells in all four brain areas studied. However, the labelling index (LI) of the glial cells differs by a factor of 3, between 1.8 and 5.4% in the different brain areas. Accordingly, the growth fraction of the glial cell population in the four areas varies between 0.04 and 0.12. Glial cells (astrocytes as well as oligodendrocytes) proliferate according to a steady state system. Furthermore, the proliferation of glial cells is associated with continuous cell loss. After each mitosis about 3% of the daughter cells become pyknotic and die. In addition, a permanent exchange of glial cells occurs between the proliferating and non-proliferating pool.     

Cell population kinetics of thyroid follicular cells in infant rats

Abstract. T cell population kinetics of thyroid follicular cells in rats were studied by means of autoradiography and a statmokinetic technique. During the first fortnight after birth no significant changes in the mitotic index (MI) and labelling index (LI) were found. In the next 2 weeks a constant decrease in the number of proliferating cells occurs. In 10-day old animals 40% of the follicular cells were in the cell cycle (GF); 3.25 ± 0.77 (SEM) % in the S phase and 0.18 ± 0.04% in mitoses (MI). Day–night changes in the LI and mitotic rate (MR) indicated a peak value at 13.30 hours with a lowest value at 22.30 hours. The mean LI and MR averaged over the whole 24 hr were 3.1 ± 0.1% and 122.2 ± 18.1%, respectively. In 10-day old animals, using the fraction of labelled mitoses (FLM) method the median cell cycle time ( T _C) was 79 hr and the phase durations were T _G1—64.6 hr, T _s—8.2 hr and T _G2—5.1 hr. The decrease in the number of proliferating cells with the age of the animals is considered to be a result of both cell cycle prolongation and in growth fraction reduction.     

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.     

Rabbit bone marrow suppressor cells block the production or release of a soluble bone marrow growth factor

Fc gamma-receptor (Fc gamma R)-bearing cells from normal rabbit bone marrow suppressed the constitutive proliferation rates of the remaining, Fc gamma R-, cells. In the absence of the suppressive influences of Fc gamma R+ cells, cells in the Fc gamma R- population spontaneously elaborated a soluble growth factor (GF) which induced the proliferation of unseparated bone marrow cells. To examine regulation by Fc gamma R+ bone marrow cells, graded numbers of the Fc gamma R+ cells were mixed with constant numbers of the FcR- cells. At 24 hr, supernates were collected and tested for GF activity. The Fc gamma R+ suppressor cells efficiently and in a dose-dependent fashion blocked GF production or release. The GF was nondialyzable and relatively heat stable. Supernates with GF activity also had colony-stimulating factor activity, but were negative in assays modified from murine interleukin 1 (IL-1) and IL-2 assays. Regulation of GF production or release represents a new function for bone marrow suppressor cells.     

Immediate bromodeoxyuridine labelling of unseparated human bone marrow cells ex vivo is superior to labelling after routine laboratory processing

It is important to evaluate the proliferation of bone marrow cells in several disease conditions and during treatment of patients with for example cytokines. Labelling with bromodeoxyuridine (BrdUrd), immunocytochemical staining with anti-BrdUrd antibody and analysis by flow cytometry provides a reliable and reproducible technique for estimation of the fraction of cells that incorporated BrdUrd into DNA during S-phase. We have compared immediate BrdUrd labelling of unseparated bone marrow cells with the previously used labelling in the laboratory after routine separation of the mononuclear cells. Bone marrow aspirates from seven lymphoma patients without bone marrow involvement were studied with these two methods. We found higher BrdUrd labelling indices (LI) in the mononuclear cells, when cells were labelled immediately. A large variation in LI was found between patients. Our results suggest that ex vivo BrdUrd labelling of bone marrow cells should be performed immediately after aspiration and before separation, because these data are closer to values reported from in vivo labelling with BrdUrd.     

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.     

PCNA Ki-67 dissociation in childhood acute lymphoblastic leukaemia. An Immunofluorescent Laser Confocal Scanning Microscopical study.

Cell proliferation rates of diagnostic marrow aspirate cells of 21 children with Acute Lymphoblastic Leukaemia and 16 controls were compared using immunocytochemical labelling of PCNA and Ki-67 antigen as assessed by Confocal Laser Scanning Microscopy. The results showed an unexpected, highly significant degree of dissociation between PCNA and Ki-67 expression in ALL blasts. The PCNA labelling indices of ALL patients were significantly increased (mean 44, range 24-77) compared with normal reactive marrow cells (mean 13.8, range 4-26) (p<0.000001, Mann Whitney U two tailed test), suggesting an abnormal commitment to proliferation. Ki-67 expression was raised to a lesser extent in ALL cells (mean 14.8, range 1.2-35) when compared to non-malignant proliferations (mean 6.6, range 1.7-25) (p < 0.02). PCNA/Ki-67 LI ratios in ALL (mean 7, range 1.1-35) were higher than in controls (mean 2.7, range 1.04-6.5, p<0.09). As cell proliferation rates actually achieved in the bone marrow do not differ as strongly as suggested by the extreme difference in PCNA labelling, a pathological dissociation of PCNA / Ki-67 expression exists, suggesting immortalisation.     

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.