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A formula is given, and proved, for the expected position of the highest labelled cell in an individual crypt column, if the labelling index distribution is known.

In the rat small bowel mucosa significant variation was found in both the labelling and the mitotic indices with time of day. The zenith and the nadir of labelling and mitotic activity coincided at 15.00 and 02.00 hours respectively. Small changes were found in the "cut-off" position, but this variation in proliferative compartment size was insufficient to account for the comparatively wider fluctuations in proliferative indices. Measurements of the rate of entry into mitosis, using metaphase arrest with vincristine at three widely separated times during the day, showed no significant change. Changes in the growth fraction or in the birth rate as measured cannot account for diurnal variation in the proliferative activity of the small bowel mucosa. We propose a hypothesis which involves diurnal fluctuations in the transit times through G1 and through G2.

Cell kinetics in the jejunal crypt of the male Wistar rat were studied using autoradiographic techniques with tritiated thymidine and a stathmokinetic technique with vincristine. The migration rate measured by following the movement of the 50% peak on the labelling index distribution curve with time after injection of tritiated thymidine gave a value of 1-43 +/- 0-14 (SE) cell positions per hour, compared with a value from a cumulative birth rate of 1-78 cell positions per hour. Tht crypt column length was 32-9 +/- 0-2 cells and the column count was 22-3 +/- 0-2. This measurement gave a total crypt population of 734 cells, compared with an estimate of 650 +/- l from direct observation of squashed, microdissected crypts. In each crypt 22-5 +/- 0-5 mitoses were present, and the crypt cell production rate was 32 cells per crypt per hour; this latter value was confirmed using two independent techniques. The crypt growth fraction calculated from the durations of phases of the cell cycle and the labelling index was 0-62. A value of 0-61 was found from the labelling index distribution curve. As assessed from crypt squashes, there were 403 proliferating cells per crypt.

Organ culture, using human colorectal mucosa and tumours, is a good system in which to test a new stathmokinetic agent such as vindesine. Using this system we have found that vindesine has similar metaphase-arresting properties to vincristine, including at least a 6-fold dose response difference in its ability to arrest mitosis in mucosa and tumour, mucosa being the more sensitive. Vindesine is a satisfactory stathmokinetic agent, but in view of its greater cost offers no particular advantages over vincristine.

The intraperitoneal administration of large doses of quinacrine in rats results in a state of enteromegaly affecting mainly the distal small bowel, caecum and proximal colon. This enteromegaly is associated with mucosal crypt hyperplasia, and hypertrophy and hyperplasia of the Muscularis propria. In order to investigate the changes in the intestinal mucosal crypts, morphometry and a metaphase-arrest experiment with vincristine were undertaken on a group of rats given 12 mg of quinacrine hydrochloride by intraperitoneal injection daily for 5 days 2 weeks previously, and comparisons drawn with a group of control animals. In the quinacrine-treated animals there was marked enteromegaly affecting the distal small bowel, caecum and proximal colon, and in these segments there was clear crypt hyperplasia. Proximal and distal to the dilated bowel hyperplasia was not seen. No consistent pattern of change in crypt-cell birth rate was evident. The mechanisms by which quinacrine may effect kinetic and morphometric changes in the intestinal crypts are considered.

The development of an organ-culture system for rat colonic mucosa has enabled a direct assessment of the effect of epidermal growth factor (EGF) on cell division. An augmented mitotic index (AIm) has been employed to identify changes in cell proliferation. Explants of colonic mucosa from four animals were maintained in a medium containing serum for five days. On the fifth day of culture, half of the explants received fresh medium containing EGF (40 ng/ml) and the remainder (controls) fresh medium only. At 6, 12, 24 and 48 hr thereafter groups of both experimental and control explants received the metaphase-arresting drug vincristine (4 micrograms/ml) for 3 hr prior to fixation. The proportions of vincristine-arrested metaphases within the explants were determined. Analysis of the data indicates that when serum is present exogenous EGF exerts a trophic effect which increases with time (P less than 0.001). In a second experiment colonic explants from four animals were maintained for five days in a serum-free medium and were then divided into groups, each of which received one of a range of concentrations of EGF. The AIm was determined for each group after 36 hr. It was found that increasing concentrations of EGF produce a small but significant increase in cell proliferation (P less than 0.01). This effect, however, was less pronounced than that seen when serum was present. These results suggest that EGF has a trophic action on the colon and interacts with additional factors found in serum.

The effect of a single injection of hydroxyurea (HU) on cell population kinetics in the jejunal crypt of the rat was studied using autoradiography with tritiated thymidine and metaphase arrest with vincristine. HU appeared to act selectively on cells in the S phase producing inhibition of DNA synthesis and cell death. The deficit in proliferating cells was made good by a decrease in cell cycle time and an increase in growth fraction. Particular attention was paid to the basal, slowly cycling (and possibly clonogenic) crypt cells; early in the recovery sequence an increase in cell production rate was found in the base of the crypt. It is proposed that basal crypt cells, having survived cycle-specific insult because of long cell cycle times, proceed to repopulated the depleted proliferative compartment.

Short-term organ culture, using a technique to preserve epithelial/stromal interaction and metabolism, is a useful technique for carrying out kinetic studies on human colorectal carcinoma and adjacent normal mucosa, providing initial perturbations of proliferative indices are allowed to settle. Tumours require 3.0 micrograms/ml vincristine for complete metaphase arrest compared with mucosa, which needs 0.5 microgram/ml, a 6-fold difference. Using a stathmokinetic technique, the birth rate of tumour cells is 10.21 cells/1000 cells per hr, compared with 7.73 cells/1000 cells per hr for mucosa, a statistically significant difference (P less than 0.01).

Pulse-labelling with tritiated thymidine and a fraction of labelled mitoses experiments have been performed in order to investigate the proliferative changes induced at various sites in the hyperplastic small-intestinal mucosa of rats previously subjected to resection of 70% of the small intestine. Proliferative activity in the colon was also studied. In the distal ileum there is a significant reduction in cell cycle time (Tc) of cells at all levels within the crypt and the growth fraction falls. In the jejunum and proximal ileum the crypts contain an increased number of proliferating cells, but as the size of the maturation zone is also increased, there is no significant alteration in the relative number of proliferating cells per crypt. Nor does the distribution of proliferating cells in these crypts seem to alter. There is no general reduction in Tc at these sites, but there does appear to be a significant reduction in Tc on the part of the cells in the stem-cell zone at the crypt base. In neither proximal nor distal colon was there any significant proliferative change apparent after small-intestinal resection.