Simulation of cell proliferation in mouse oral epithelium, and the action of epidermal growth factor: evidence for a high degree of synchronization of the stem cells.

Computer simulation has been carried out to help to determine the cell-proliferative mechanisms underlying data gathered from a double-labelling experiment on the dorsal tongue of the mouse. Good fits to the data have been obtained by assuming that there is a high degree of synchrony in the stem cells, which have a 24-h cell cycle time, and that daughters of these cells undergo two further divisions, with mean cell cycle times of 48 h, before differentiating. This results in one-seventh of proliferative cells being stem cells, which ties in well with the concept of epidermal proliferative units. There is no need to assume that S-phase duration changes diurnally. The administration of epidermal growth factor seems to increase the degree of synchrony. In such systems, the influx to S-phase and the efflux from it have very sudden short peaks, which it is impossible to observe unless observations are taken very frequently. There are therefore implications for the designs of experiments that attempt to study diurnal rhythms or the effect of factors that disturb the normal proliferative pattern of cells.

In vitro labelling studies and the measurement of epithelial cell proliferative activity in the human oral cavity.

Samples (110) of human mandibular gingiva and buccal mucosa, harvested from patients undergoing third-molar surgery, were subjected to in vitro labelling with tritiated thymidine, bromodeoxyuridine, or a sequential double-labelling technique comprising tritiated thymidine followed by bromodeoxyuridine, in order to determine the efficacy of a new incubation labelling technique, and to characterize S-phase labelling indices in human oral mucosa. Whilst, there were no demonstrable differences in labelling indices obtained by single thymidine, single bromodeoxyuridine or double labelling, there was a significant difference between anatomical sites, with higher S phase labelling observed in buccal mucosa (mean LI 11.7) than mandibular gingiva (mean LI 8.5; P<0.01). There was, however, no significant correlation between individual labelling indices and patient age, sex or the time of day when tissue was harvested. The in vitro labelling technique provides a reliable and quantifiable method of characterizing proliferative labelling indices in the human oral cavity. Further investigation is being carried out to profile wider age and anatomical ranges and to utilize the double-labelling technique to calculate S-phase durations and cell-cycle times. These profiles may have a future role in the assessment of oral mucous membrane disease.

Mapping dynamic epithelial cell proliferative activity within the oral cavity of man: a new insight into carcinogenesis?

Our aim was to characterize epithelial cell proliferative activity within the oral cavity and to find out if there were differences between sites with high and low incidence of cancer. A total of 105 samples of clinically normal mucosa were harvested from various intra-oral sites. Excised specimens were incubated in vitro with tritiated thymidine and bromodeoxyuridine to 'double label' cells undergoing DNA synthesis, and enable calculation of the duration of S phase and estimation of variables of cell flux to and from S. Mean labelling indices (percentage of cells within the S phase of the cell cycle) were highest in the floor of mouth (12.3%) and ventral tongue (10.1%), while activity was lowest in the dorsum of tongue (4.3%) and the palate (7.2%), P<0.001. In general, both cell influx and the duration of S increased proportionally to the labelling index. Sites with a high incidence of cancer were characterized by high labelling indices, increased cell influx and a prolonged S phase.

Characterization of epithelial cell activity in patients with oral cancer.

Accurate, predictive assessment of the clinical behaviour and progression of individual oral cancers and premalignant lesions requires reproducible and quantitative analyses of diseased tissue. In this paper we describe the use of in vitro double labelling (sequential tritiated thymidine and bromodeoxyuridine staining of proliferating epithelial cells) to calculate S phase labelling indices (LIs), estimation of S phase duration (tS), and measurement of variables of flux to and from S for excised specimens of oral squamous cell carcinoma, premalignant lesions, and clinically normal mucosa from patients with oral cancer. There was a significant increase in mean LIs in buccal mucosa leukoplakias (14.5%) compared with normal mucosa (10.3%); P = 0.03. LIs were also increased in patients with cancers of the floor of mouth and ventral tongue but neither these changes nor alterations in flux parameters or S Phase durations were significant. Twenty-one kinetic profiles of dysplastic and malignant tissue were compared with conventional histopathological results, however, and these showed a 2.2% increase in LIs with each increase in grade of dysplasia (P = 0.004) and a 12% increase in LIs with each reduction in tumour differentiation (P = 0.02).

Tritiated thymidine and bromodeoxyuridine double-labelling studies on growth factors and oral epithelial proliferation in the mouse.

Mouse tongue epithelium is characterized by a circadian variation in the number of cells undergoing DNA synthesis. Groups of male BDF1 mice were followed over 48 h and a double-labelling method with tritiated thymidine and bromodeoxyuridine used to determine S-phase labelling indices, together with cell influx to and cell efflux from S, at 4-hourly time points. Control animals exhibited diurnal peaks in labelling index at 03:00 with trough activity 12 h later at 15:00. Cell influx peaked at 23:00 with troughs occurring between 11:00 to 15:00. Peak cell efflux occurred at 07:00 with trough activity at 19:00. Animals injected with epidermal growth factor at 05:00 demonstrated a significant fall in both influx and efflux throughout the 48-h period (P < 0.001), but with preservation of labelling indices, suggesting a slower transit of cells through S-phase, whereas epidermal growth factor injected at 15:00 only produced a significant rise in cell-efflux values. Adrenergic stimulation by intravenous phenylephrine/isoprenaline injection at both 05:00 and 15:00 resulted in a significant rise in cell efflux (P < 0.001), although there was also a rise in labelling index in the 15:00 group (P < 0.001). Animals injected with calmodulin at 05:00 demonstrated a significant reduction in labelling index throughout the 48-h period (P < 0.001), but maintained control values for cell influx and efflux, suggesting faster transit of cells through S. Calmodulin injection at 15:00 produced only a significant reduction in cell influx (P < 0.001). Administration of exogenous growth factors significantly alters the normal rhythmical proliferation of oral epithelial cells in a mouse model. These effects appear to be both growth factor- and time-dependent, and may have both physiological and pathological implications.

Colonic epithelial cell proliferation in hereditary non-polyposis colorectal cancer.

BACKGROUND: Despite the recent discovery of four genes responsible for up to 90% of all cases of hereditary non-polyposis colorectal cancer (HNPCC), there will still be families in whom predictive testing is not possible. A phenotypic biomarker would therefore be useful. An upwards shift of the proliferative compartment in colonic crypts is reported to be one of the earliest changes in premalignant mucosa. AIMS: To assess the role of crypt cell proliferation as a phenotypic biomarker in HNPCC. PATIENTS: Thirty five patients at 50% risk of carrying the HNPCC gene (21 of whom subsequently underwent predictive testing and hence gene carrier status was known) and 18 controls. METHODS: Crypt cell proliferation was measured at five sites in the colon using two different techniques. Labelling index was determined using the monoclonal antibody MIB1 and whole crypt mitotic index was measured using the microdissection and crypt squash technique. The distribution of proliferating cells within the crypts was also assessed. RESULTS: There were no significant differences in the total labelling index or mean number of mitoses per crypt, nor in the distribution of proliferating cells within the crypt, between the study and control groups at any site. When the 21 patients in whom gene carrier status was known were analysed separately there were no significant differences in the measured indices of proliferation between the HNPCC gene carriers and non-gene carriers. CONCLUSION: Crypt cell proliferation is not a discriminative marker of gene carriage in HNPCC.

Sulindac enhances cell proliferation in DMH-treated mouse colonic mucosa.

In a previous study we reported that the NSAID sulindac had a marked inhibitory effect on the development of colonic tumours in mice treated with the carcinogen 1,2-dimethylhydrazine (DMH). In this study we examined the effects of sulindac in respect of cell-kinetic changes in mouse colonic mucosa as determined by flash labelling with the thymidine analogue bromodeoxyuridine (BrdUrd) at varying intervals during the process of colonic carcinogenesis. We also investigated the possibility that these changes may be modulated by misoprostol a prostaglandin E1 analogue. Four groups of 36 mice each were treated for 18 weeks with the following drug/s respectively: (1) DMH; (2) DMH and sulindac; (3) DMH, sulindac and misoprostol; and (4) DMH and misoprostol. Three animals from each group were killed each week between the sixth week and the eighteenth week after the start of the experiment. A 1-h flash label technique was employed and paraffin sections of colonic mucosa were examined. For each animal a total of 50 perfect axially cut crypts were chosen and the following parameters determined: crypt length, labelling index and labelling index distribution: the data were analysed using the computer program GLIM. For each of the four groups, crypt lengths increased significantly with the duration of treatment with no significant difference between the groups. In sulindac-treated animals the labelling index for all positions increased with duration of treatment whereas for animals not treated with sulindac there was no significant difference in labelling index with respect to duration of treatment. The administration of misoprostol did not appear to significantly alter the effects of sulindac. It is postulated that the observed increase in cell proliferation could be a compensatory phenomenon occurring secondary to loss of crypt epithelial cells by apoptosis induced by sulindac. Also the finding of an increase in labelling index mediated by a chemopreventive agent indirectly questions the rationale behind the therapeutic manipulation of crypt cell proliferation in order to reduce the risk of colon cancer.

Effects of gastrointestinal peptides on azoxymethane-treated colonic mucosa in vitro.

An organ-culture system has been used to investigate the effect of certain gastrointestinal peptides on the morphology and cell proliferation of explants of azoxymethane (AOM)-treated colonic mucosa. Our aim was to ascertain whether such factors play a direct part in the maintenance of hyperplastic changes in the large intestine. Explants of AOM-treated colonic mucosa from 15 animals were maintained in a serum-free medium in the presence of either gastrin-17 (250 pg/ml and 250 ng/ml), peptide YY (80 pmol/l and 160 pmol/l) epidermal growth factor (EGF) (10 ng/ml and 100 ng/ml) or the C-terminal fragment of glucagon-37 (30 pmol/l) for a period of up to 7 days. Other explants (controls) received fresh medium only each day. After 1, 2, 3, 5 and 7 days of culture both experimental and control explants received vincristine (4 micrograms/ml) for 3 h prior to fixation. The proportion of vincristine-arrested metaphases within the explants was determined together with crypt length. Neither gastrin nor peptide YY was found to influence cell division at either concentration. Despite an initial inhibitory effect, both concentrations of EGF exerted a trophic effect which increased with time. The glucagon-37 fragment caused an immediate increase in proliferation which then declined as time progressed. None of these factors, however, were able to maintain the hyperplastic changes seen in the pre-culture samples of AOM-treated mucosae.

What statistics should we teach medical undergraduates and graduates?

It is suggested that the emphasis on teaching statistics to medical undergraduates has usually been quite wrong: that the courses have become much too long, too detailed, and irrelevant to the needs of the majority. Examples are given which may help to introduce the basic concepts which all medical (and dental) undergraduates require, and which form a basis for the more traditional teaching of analytical methods to the appropriate subset who proceed to undertake medical research.

The effect of sulindac on colonic tumour formation in dimethylhydrazine-treated mice.

Dimethylhydrazine has been used to produce colonic tumours in mice. If sulindac, a non-steroidal anti-inflammatory drug, is administered simultaneously fewer microadenomata and fewer macroscopic tumours are produced. Those which do appear are comparable in size to the ones in the mice which do not receive sulindac. Sulindac therefore appears to exert an anti-tumour influence at the stage between dysplasia and the formation of microadenomata.

A protective effect of sulindac against chemically-induced primary colonic tumours in mice.

Sulindac, a non-steroidal anti-inflammatory drug, has been reported to lead to tumour regression in cases of human polyposis coli. We have investigated the effects of this drug on the growth of 1,2-dimethylhydrazine (DMH)-induced mouse colonic tumours. In one experiment, DMH and oral sulindac were administered concurrently to a group of mice for a period of up to 24 weeks, while a control group of animals received DMH only for the same period. Sulindac caused a significant reduction in both the number of mice with colonic tumours and the number of tumours per mouse. In a second experiment, two groups of mice which had already been treated with DMH for 17 weeks received either sulindac or not for 78 days. In this experiment sulindac had no effect. These results demonstrate that sulindac has a protective effect against the chemical induction of colonic tumours in mice, but does not cause the regression of established tumours.

Verapamil sensitizes normal and neoplastic rodent intestinal tissues to the stathmokinetic effect of vincristine in vivo.

A morphological method has been developed allowing measurement of the effect on intestinal epithelia of vincristine. In routinely prepared tissue sections the proportion of mitotic events progressing beyond metaphase is counted by microscopy. When estimated over a range of doses of vincristine this post-metaphase index (PMI) can be used to compare the sensitivity of differing intact tissues. Intestinal tumours were induced in rats by chemical carcinogenesis. Administration of vincristine in the presence or absence of verapamil was performed in these tumour-bearing animals. Sections were prepared from colonic and small-bowel tumours and from normal mucosa. The results show that verapamil increases the sensitivity of the tissues studied to vincristine. A dose dependent effect of verapamil on vincristine sensitisation was demonstrated in colonic tissues. These findings indicate a shared pharmacological property between the resistance of primary tumour tissue and the multidrug-resistance phenotype.

A comparison of crypt-cell proliferation in rat colonic mucosa in vivo and in vitro.

The successful development of a long-term organ culture system has made it possible to perform experiments on rat colonic mucosa in vitro. However, the effect of trauma or the withdrawal of trophic influences in culture may result in the disturbance of proliferation within the tissue. In this paper we describe an investigation designed to characterise the culture system by a comparison of the proliferative parameters in vitro with those in vivo. Stathmokinetic experiments were performed in vivo and in vitro to estimate cell birth rate. Mitotic indices were also calculated. The in vivo birth rate (7.8 +/- 0.8 cells/1000 cells/hour) and the in vitro birth rate for the whole explant (7.7 +/- 0.5 cells/1000 cells/hour) were found to be similar. A study of crypts in the centre and at the edge of the cultured explants, however, indicated that proliferation at the two sites differed markedly, the birth rate at the edge (7.5 +/- 0.9 cells/1000 cells/hour) being approximately twice that at the centre (3.2 +/- 0.9 cells/1000 cells/hour). Provided that this topological difference in proliferation within the explant is recognised the in vitro model, in particular the basal level found at the centre, may still be regarded as a valid system for studying tissue responses to carcinogens and trophic factors.

Presence of epidermal growth factor receptor as an indicator of poor prognosis in patients with breast cancer.

Epidermal growth factor receptors are present in some breast cancers in man, and there is an inverse relation to oestrogen receptor state. We assessed the presence of epidermal growth factor receptors as a single prognostic indicator in a series of breast tumours by comparing this with the Bloom and Richardson scores for these tumours. One hundred and eight ductal tumours were examined for epidermal growth factor receptors by radioligand binding. There was a significant (p less than 0.01) correlation between the presence of the growth factor receptor and poor prognosis as assessed by the Bloom and Richardson score, suggesting that epidermal growth factor receptor state could be a useful prognostic marker. Epidermal growth factor receptor state was not significantly correlated with the lymph node state but showed a tendency to be associated with large tumours.

Demonstration of vincristine resistance in primary intestinal neoplasms in the rat by the 'post-metaphase index'.

A method is described enabling the direct measurement of vincristine resistance in intact tissues in vivo by morphological study. Using the metaphase arresting properties of the drug, counts were made of escaping anaphase and telophase mitotic figures at a range of doses. The proportion of post-metaphase mitotic figures is called the post-metaphase index (PMI). In 95 primary intestinal tumours induced by dimethylhydrazine (DMH) in rats, an increase in resistance to vincristine was shown over normal mucosa (P less than 0.001). The data were analysed by computer modelling and a linear relationship is demonstrated between the logit of the post-metaphase index, and log dose of vincristine. To achieve a PMI of 1% the fitted lines show an enhanced vincristine dose requirement over normal mucosa of 6 times in colonic tumours, and 8 times in small intestinal tumours. Non-neoplastic mucosa from the DMH-treated animals requires an enhanced dose of vincristine of 1.5 times, compared with normal mucosa, to achieve a PMI of 1%. Given current interest in the mechanism of vincristine resistance in cell lines this new approach provides a technique for assessing the resistance of solid tumours, both in vivo and in vitro, and for subsequent experimental manipulation.

Transplantation of a segment of ileum to the external abdominal wall: an animal model of intestinal mucosal hyperplasia.

When a segment of small intestine is transplanted to the external abdominal wall in rats adaptive changes occur in the exposed mucosa. These probably represent an extreme example of a physiological response to one type of trophic influence--the effect of mechanical trauma. The nature of the changes has been studied at 7 weeks after externalization using simple morphometry and a number of cytokinetic techniques (thymidine labelling, vincristine-induced metaphase arrest and the fraction-of-labelled-mitoses method), and comparisons drawn with the normal ileum. The exteriorized mucosa showed marked villus atrophy and hyperplasia of the crypts to three times normal size as a result of increases both in crypt length and crypt circumference. Neither metaplastic nor dysplastic epithelial abnormalities were observed. Crypt-cell production rate doubled in the hyperplastic crypts due to an increase in the size of the proliferation zone within the crypt, and the distribution of proliferating cells within the crypt changed. But cell cycle times were prolonged and more maturing cells were retained in the hyperplastic crypts. The potential usefulness of this model, particularly in carcinogenicity studies is considered.

Vindesine as a stathmokinetic agent in human rectal tumours in organ culture.

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.

Aspects of statistics in studies of cell proliferation. IV. Simulation.

Computer simulation of an experiment can help to reveal how effective that experiment might be in estimating cell kinetic parameters, or in elucidating the nature of a proliferative response to a perturbation of the steady state, such as a carcinogenic or therapeutic treatment. Writing a simulation also clarifies one's thoughts about possible mechanisms of the proliferative response.

Aspects of statistics in studies of cell proliferation. II. Standard errors of estimated parameters.

Statistical methods such as regression analysis are often used to estimate cell kinetic parameters, such as cell birth rate or tumour growth rate. The standard errors which the statistical procedures provide may be considerable underestimates of the true uncertainty surrounding the parameter of interest.