TUMOUR CELL PROLIFERATION IN RELATION TO THE VASCULATURE

The proliferation pattern of a transplantable mouse mammary carcinoma has been studied in relation to its macroscopic and microscopic structure. No significant differences were seen in the labelling or mitotic indices or in the percentage labelled mitoses curves for the peripheral 2-0 mm rim or for the central tumour core. When these parameters were scored for cells classified according to their position in relation to capillaries or to necrotic regions, marked differences were observed in all the parameters. Higher labelling and mitotic indices and higher grain counts were seen adjacent to the capillaries. These appear to result from a shorter cell cycle duration and a higher growth fraction. The variation in cell cycle is mainly due to a change in the duration of G₁.     

A study of diurnal proliferative activity in tumour and small intestine of C3H mice bearing a transplanted mammary carcinoma

Diurnal changes in proliferative activity were investigated in tumour and small intestinal epithelium of mice bearing a transplanted mammary carcinoma. In addition to mitotic and labelling index studies, the metaphase-arrest technique with vincristine (VCR) was employed. In the tumour there was no clear evidence of a significant diurnal rhythm in proliferative activity but in the small intestinal epithelium such a rhythm was clearly demonstrated. A higher cell production rate (kB) measured by metaphase-arrest and higher labelling and mitotic indices were seen in the mid to late part of the dark period. The peak mitotic index was seen 3 to 6 h after the labelling peak in the small intestine. The basal third of the crypt which is believed to include the stem cell compartment of this tissue showed larger diurnal fluctuations in both labelling index and kB than the rest of the proliferative compartment.     

Mitotic and Labelling Activity In Normal Human Epidermis In Vivo

Labelling and mitotic indices were studied in the epidermis of twenty-eight young men. A mean labelling index of 5.5% was found from the whole study and a mean mitotic index of 0.06%. Mitotic index particularly was extremely variable; indices between 0.002 and 0.438% were found in individual biopsies. In the first two of three experiments in which mitotic index at 09.00 hours was compared with that at 15.00 hours, significant differences were found (15.00 hours > 09.00 hours by a factor of 2.6, P < 0.001). However, in the third such experiment no such difference was found, suggesting that the timing and occurrence of diurnal rhythms of mitotic activity may not be consistent in normal human epidermis. In the one experiment in which it was investigated, a significantly higher mitotic index was found at 21.00 hours compared to 09.00 and 15.00 hours. Labelling index did not vary significantly at 09.00, 15.00 or 21.00 hours. However, labelling index did show a significant pattern of change over a 12-month period in two groups of subjects; peaks of labelling were seen in July and troughs in January. Very high ratios of labelled: mitotic cells were found, the median ratio for the whole study being ninety-eight labelled: one mitotic cell. This finding supports the possibility that not all labelled cells subsequently go on to divide in normal human epidermis.     

Mitotic and labelling activity in normal human epidermis in vivo

Labelling and mitotic indices were studied in the epidermis of twenty-eight young men. A mean labelling index of 5.5% was found from the whole study and a mean mitotic index of 0.06%. Mitotic index particularly was extremely variable; indices between 0.002 and 0.438% were found in individual biopsies. In the first two of three experiments in which mitotic index at 09.00 hours was compared with that at 15.00 hours, significant differences were found (15.00 hours greater than 09.00 hours by a factor of 2.6, P less than 0.001). However, in the third such experiment no such difference was found, suggesting that the timing and occurrence of diurnal rhythms of mitotic activity may not be consistent in normal human epidermis. In the one experiment in which it was investigated, a significantly higher mitotic index was found at 21.00 hours compared to 09.00 and 15.00 hours. Labelling index did not vary significantly at 09.00, 15.00 or 21.00 hours. However, labelling index did show a significant pattern of change over a 12-month period in two groups of subjects; peaks of labelling were seen in July and troughs in January. Very high ratios of labelled: mitotic cells were found, the median ratio for the whole study being ninety-eight labelled: one mitotic cell. This finding supports the possibility that not all labelled cells subsequently go on to divide in normal human epidermis.     

CELL POPULATION KINETICS OF PLUCKED AND UNPLUCKED MOUSE SKIN I. UNIRRADIATED SKIN

A detailed study of the cellular proliferation kinetics in interfollicular plucked and unplucked mouse skin has been made in Swiss albino mice, using tritiated thymidine autoradiography. Diurnal variations in mitotic and labelling indices were demonstrated in both systems.
The mean cell cycle times for unplucked and plucked skin were estimated by four different methods and found to be 100 ± 10 and 47 ± 3 hr respectively. Most of the difference was due to the shortening of G₁ phase after plucking. Repeated labelling at intervals shorter than the DNA synthesis times resulted in all the basal layer cells becoming labelled, so that the growth fraction was unity, in unplucked and plucked skin.
A well-defined second wave of labelled mitoses was seen at about 100 hr after labelling the unplucked (i.e. normal) mouse skin.
A double labelling technique using ¹⁴C-TdR and ³H-TdR with a single layer of emulsion gave reasonable values for the duration of the DNA synthesis phase.     

A Stochastic Model For Cell Populations With Circadian Rhythms

A mathematical model for cell kinetics, based on a random walk, is developed. the model allows variations with time of the rates of passage of proliferating cells through the four phases of the mitotic cycle. Circadian variations in the mitotic and labelling indices of the Syrian hamster cheek pouch epithelium have previously been observed, and the random walk model has been used to simulate this phenomenon. Assuming that all basal cells are proliferative and that these cells leave the basal layer randomly throughout the mitotic cycle to become differentiated cells, it was found that the experimentally observed circadian rhythms of the mitotic and labelling indices could be reproduced in the model by postulating a circadian rhythm in the rate of passage of cells through the G₁ and S phases only. Moreover, the growth activity of cells in both the G₁ and S phases appears to reach a peak during the dark hours of the light-dark cycle, and to fall off rapidly in the early hours of daylight. the postulate of Møller, Larsen & Faber (1974) that injection of the animals with tritiated thymidine causes a shortening of the G₂ phase duration has been qualitatively confirmed by using the random walk model to simulate the FLM and MI curves after injection with tritiated thymidine.     

Influence of the Beta-Adrenergic Antagonists Propranolol, Practolol and Oxprenolol On the Proliferation of Rat Jejunal Crypt Cells

Abstract. Beta-adrenergic blockade by quite large doses of propranolol, practolol and oxprenolol, once or continuously applied, does not influence jejunal crypt-cell proliferation in the rat. After a single i.p. injection of 20 mg/kg propranolol or practolol and even of 100 mg/kg practolol, the mitotic index, the labelling index and the duration of the S phase do not differ between treated and untreated control animals nor between animals treated with the different drugs. Continuous application of 30 mg/kg/d propranolol, practolol or oxprenolol for 7 or 14 days does not affect the mitotic and labelling indices either, nor does it change the duration of the cycle of the jejunal crypt cells and its phases as determined by the percent labelled mitoses method. These results are in contrast to those reported previously by Tutton & Helme (1974).     

Influence of the beta-adrenergic antagonists propranolol, practolol and oxprenolol on the proliferation of rat jejunal crypt cells

Beta-adrenergic blockade by quite large doses of propranolol, practolol and oxprenolol, once or continuously applied, does not influence jejunal crypt-cell proliferation in the rat. After a single i.p. injection of 20 mg/kg propranolol or practolol and even of 100 mg/kg practolol, the mitotic index, the labelling index and the duration of the S phase do not differ between treated and untreated control animals nor between animals treated with the different drugs. Continuous application of 30 mg/kg/d propranolol, practolol or oxprenolol for 7 or 14 days does not affect the mitotic and labelling indices either, nor does it change the duration of the cycle of the jejunal crypt cells and its phases as determined by the percent labelled mitoses method. These results are in contrast to those reported previously by Tutton & Helme (1974).     

Differential proliferation of rat aortic and mesenteric smooth muscle cells in culture.

Smooth muscle cells (SMC) from various arterial origins have been successfully maintained in culture. The present study evaluates the proliferative activity of aortic and mesenteric SMC in culture. Aortic and mesenteric SMC were obtained from male Wistar rats by explant and enzyme digestion techniques, respectively. Vascular SMC obtained by either method exhibited a characteristic hill-and-valley growth pattern in culture after confluence and were positively labelled with either anti-smooth muscle actin or myosin by an indirect immunofluorescent method. The rate of incorporation of thymidine into DNA and cell number counting were used as indices of proliferation in vitro. Vascular SMC from passages 4-33 were first synchronized with either Dullbecco's Modified Eagle's Medium (DME) or Ham's F-12 medium, supplemented with insulin-transferring-selenium (ITS), for 72 hours. SMC were then stimulated with 10% bovine serum for either 24 or 72 hours with the former processed for scintillation counting, the latter for cell number determination. The incorporation of tritiated thymidine into DNA following a 2 hour incubation was determined by scintillation counting after perchloric acid extraction. In terms of cell numbers, proliferative responses to bovine serum were determined by Coulter counting. Autoradiography was also carried out in some cultures to determine both thymidine and mitotic labelling indices. The rate of thymidine incorporation in aortic cells was 2-3 fold higher than in mesenteric cells. Aortic and mesenteric SMC lines exhibited similar cell cycle intervals in terms of total duration and individuals cycle parameters. However, the total thymidine index was higher in the aortic than mesenteric SMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of intestinal cell proliferation after guanethidine-induced sympathectomy. II. Percentage labelled mitoses studies

Neonatal administration of guanethidine-sulfate results in an alteration of the cell proliferative pattern of the small intestinal epithelium of the young adult rat. Sympathectomy with guanethidine has previously been shown to depress mitotic, labelling, and total cellular migration indices while increasing the generation cycle time (Tc) of small intestinal crypt cells as measured by a stathmokinetic method. The present study showed that the G1, S and G2 phases of the crypt cell cycle are altered by sympathectomy, G1 accounting for most of the increase in Tc. In addition, the percentage of [3H]-thymidine labelled crypt cells is reduced and the duration of crypt cell transit is lengthened by guanethidine-induced sympathectomy.     

Epidermal cell kinetics in the nude mouse

The epidermal cell kinetics in nude mice is investigated by determining the mitotic rate and the mitotic count, the H3Tdr labelling index, and the proportion of basal cells in the different cell cycle phases by flow cytometry. The mitotic duration was calculated. The parameter values of the epidermal cell kinetics of the nude mouse are largely similar to those of the hairless mouse.     

The cell cycle of low passage and high passage human diploid fibroblasts

Using mitotic selection, synchronous populations of passage 25 and passage 50 WI38 cells were obtained. Thymidine incorporation, mitotic index, and labeling index were determined. No great differences could be seen in the organization of the cell cycle of low passage and high passage cells. Although labeling indices for passage 25 and passage 50 exponential cells were 83% and 37%, respectively, 75% of all mitotically selected cells were found to enter S phase, regardless of passage number. Thus, the loss of late passage diploid cell cultures does not occur from alteration of the cell cycle in the majority of rapidly proliferating cells.     

Kinetic study of the hepatic regeneration in the rat after exeresis of the left lobe only (Hepatectomy of a third of the mass)]

Regeneration is induced after exeresis of the left liver lobe lone (i.e. 1/3 of the normal liver mass). Cell divisions are less numerous than after the usual 2/3 hepatectomy; they appear later and the slope of the first labelling and mitotic indices is much less steep. However, the same circadian rhythm of divisions is also observed and the duration of the cell cycle phases (G2, S and M) are the same whatever the size of the hepatectomy.     

Mitotic cycle kinetics of root meristems of isolated barley embryos and intact seedlings. Labelling of nuclei by3H- thymidine and its cytogenetic consequences

The duration of the mitotic cycle and its individual phases was estimated in root meristems of isolated barley embryos and intact barley seedlings by means of pulse labelling with³H-thymidine and construction of labelled mitoses curve. The duration of the whole mitotic cycle in the cell population of root meristems of isolated barley embryos cultivated in the aerated liquid complete medium is 12.2 h. The mitotic cycle time of root meristems of intact barley seedlings, oultived in Petri dishes on wet blotting paper is 9.2 h. Most of root meristem cells belong to the fraction of rapidly proliferating cells, but this fraction exerts a high degree of variability by itself. Pulse treatment by³H-thymidine in our experimental conditions (74 kBq ml^-1 - or 2 μCi ml^-1, exposure 0.5 h) did not induoe any chromosomal aberrations in unlabelled cells and only a very low frequency of chromosomal aberrations in labelled cells. Measuring the cell population kinetics by pulse labelling with³H-thymidine can be used simultaneously with the study of induction of ohromosomal aberrations by mutagens.     

Circadian rhythms in mouse epidermal basal cell proliferation. Variations in compartment size, flux and phase duration.

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G2 phase DNA content were measured by flow cytometry of isolated basal cells, and the [3H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G2 phase and traversed the G2 phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G2 phase and mitosis. Circadian variations in the duration of the S phase, the G2 phase and mitosis were clearly demonstrated.     

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.     

Cultured human tumour cells may be arrested in all stages of the cycle during stationary phase: demonstration of quiescent cells in G1, S and G2 phase

Six human colon carcinoma cell lines were induced to enter stationary phase of growth by nutrient deprivation and cell crowding. Growth kinetics parameters (cell number, flow cytometric analysis of DNA distribution, and labelling and mitotic indices) were measured sequentially for all lines during the various stages of in vitro growth. Our results demonstrated that a substantial fraction of cells (9-18%) were located in G2 phase when they changed from an exponential to a stationary mode of growth. Moreover, a large number of cells in stationary phase of growth had an S-phase DNA content, as determined by flow cytometry, but failed to incorporate radioactive DNA precursors (up to 15-fold difference). To substantiate these findings, cells in stationary phase of growth were induced to enter exponential growth by re-seeding in fresh medium at a lower density. Subsequently observed changes in DNA-compartment distribution, and in labelling and mitotic indices were those expected from cells that had been arrested at different stages of the cycle during their previous stationary phase. Thus, the non-proliferating quiescent state (Q), traditionally located 'somewhere' in G1 phase, appears to be composed also of cells that can be arrested at other stages of the cycle (Qs and QG2). Although the proportion of such cells is rather small, their contribution to the growth kinetics behaviour of human in vivo tumours will become apparent following 'recruiting' or 'synchronizing' clinical manoeuvres and will prevent the formation of a clear-cut wave of synchronized cells.     

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.     

Cellular proliferation during cicatrization of the skin of 7-day chick embryos cultured in vitro

A cell proliferation study during in vitro wound healing of dorsal thoraco-lumbar skin of 7-day chick embryos was performed by pulse labelling using a single isotope (tritiated thymidine). The unoperated (controls) and operated explants were incubated in the radioactive medium (1 microCi/ml tritiated thymidine) 1 h prior to fixation and where fixed 1 h (start control), 48, 72 and 96 h after the excision. Mean labelling and mitotic indices of the unwounded epidermis were, respectively, 18.22% and 2.66% at 7 days, and 7.03% and 0.88% at 11 days (7 days + 96 h). 72 h after the excision, the labelling and mitotic indices of wounded epidermis increased, on average, respectively to 212,5% and 220% with respect to those of the controls, in the proximal zones near the inner edge on the wound. The labelling and mitotic indices in the dermis were, respectively, 27.95% and 3.63% at 7 days and 7.65% and 1.30% at 11 days. 72 h after the excision, the labelling and mitotic indices of the operated dermis increased, on average, respectively to 220% and 130% with respect to those of the controls in the centre and the proximal zones of the wound. The increase of the labelling index of the operated integument persisted for a maximum of 24 h, between 48 to 72 hours after the excision.     

Administration of caerulein to rats promotes antral epithelial cell renewal

Summary The growth-promoting effect of caerulein on antral gastric mucosa was explored using Wistar rats. Implanted osmotic minipumps were used to administer submaximal doses of either caerulein or saline to normal rats for up to 4 days. In one group, reflux of bile and pancreatic juice into the stomach was avoided by previous surgical diversion of the distal common bile duct to the jejunum. DNA synthetic and mitotic activity in the antrum epithelium were estimated by ³H-thymidine pulse labelling and autoradiography during the administration of the peptide. The rate of cell migration was determined in animals killed 1, 2 and 3 days after the ³H-thymidine pulse. Administration of caerulein to normal rats provoked significant increases in both labelling and mitotic indices, and a significant acceleration of the upward cell migration in the glandular tubes. In the animals with distal diversion of bile and pancreatic secretions both labelling and mitotic indices were also increased over control values under the effect of the peptide. These data indicate that administration of caerulein stimulates cell proliferation in the antral gastric mucosa. This effect cannot be explained through increased reflux of pancreaticobiliary secretions in the stomach.