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Keratinocyte growth factor (KGF) stimulates proliferation and differentiation in various epithelial systems. Three daily subcutaneous injections of 125 microg of this protein into mice induce dramatic changes in the histology and histometric measurements of the ventral tongue epithelium. The thickness of the epithelium is increased two-fold and the number of cells beneath a 1-mm length of the surface is increased 1.6-fold. KGF also induces a four-fold increase in the number of S phase cells labelled with tritiated thymidine in the basal layer on the third day after KGF administration. The increase in thickness and cellularity persist for at least 4 days after the end of the KGF injections. However, there is a dramatic fall in the number of S phase cells detected by 3HTdR pulse labelling 2 days after the end of the KGF treatment. There are indications that by 7 days after the 3-day regimen of KGF treatment, both thickness and cellularity have fallen back to near control levels. Continued exposure to KGF over a period of 7 days does not result in any further increases in thickness, cellularity or proliferation. In fact, the proliferation decreases on the fifth, sixth and seventh days of KGF injection to control values on day 7. These changes in the epithelium following KGF treatment suggest that the thicker and more cellular epithelium may be more able to cope with an exposure to a cytotoxic agent and hence be protected in comparison with normal or vehicle-treated epithelium.

Accurate, predictive assessment of the behaviour and progression of oral cancers and precancers remains elusive in clinical practice. Archival tissue specimens from 10 previously treated patients with oral lesions of known clinical outcome (3 years post-treatment) were re-examined histopathologically, and proliferative cell labelling indices (LIs) determined for Ki67, cyclin A and histone mRNA cell cycle markers. While histone mRNA labelling ultimately proved unreliable, both Ki67 and cyclin A LIs demonstrated a clear trend for enhanced labelling to occur in increasingly dysplastic and neoplastic tissue, with particular emphasis on suprabasal labelling in abnormal tissue. Perhaps of greatest significance was the observation of increased LIs and suprabasal labelling in lesions with poor clinical outcome, such as patients developing recurrent disease or cervical lymph node metastasis. Measurement of cell proliferative activity in individual oral epithelial dysplastic lesions or invasive squamous cell carcinomas may thus provide unique, predictive information on clinical outcome.

The dorsal and ventral epithelia on the murine tongue exhibit very pronounced circadian rhythms in terms of the cell cycle. These rhythms are such that three injections of tritiated thymidine 3 h apart spanning the circadian peak in S phase cells labelled between 40 and 50% of the basal cells. Injection of bromodeoxyuridine generally gave slightly lower labelling indices. Approximately the same proportion (54% of the basal cells) could be accumulated in metaphase over a 24-h period using vincristine as a stathmokinetic agent. The experiments reported here using mouse ventral tongue epithelium use double-labelling approaches to address the question: what proportion of the approximately 50% of the basal cells that are proliferating have a 24-h cell cycle and can therefore be labelled by a similar labelling protocol the following day? The results suggest a heterogeneity amongst the proliferating basal cells, similar to the heterogeneity proposed for the dorsal tongue epithelium. Although not all the basal component has been accounted for, the data presented here suggest that about 20% of the basal cells may have a cell cycle time of 24 h, about 30% appear to have a longer cell cycle time (48 or 72 h), while about 20% of the basal cells appear to be postmitotic maturing G1 cells, awaiting the appropriate signals for migration into the suprabasal layer.

The oral mucosa is a rapidly replacing body tissue that has received relatively little attention in terms of defining its cell kinetics and cellular organization. The tissue is sensitive to the effects of cytotoxic agents, the consequence of which can be stem cell death with the subsequent development of ulcers and the symptoms of oral mucositis. There is considerable interest in designing strategies to protect oral stem cells and, hence, reduce the mucositis side-effects in cancer therapy patients. Here we present details of a new histometric approach designed to investigate the changing patterns in cellularity in the ventral tongue mucosa. This initial paper in a series of four papers presents observations on the changing patterns in the labelling index following tritiated thymidine administration, which suggest a delayed uptake of tritiated thymidine from a long-term intracellular thymidine pool, a phenomenon that will complicate cell kinetic interpretations in a variety of experimental situations. We also provide data on the changing pattern of mitotic activity through a 24-h period (circadian rhythms). Using vincristine-induced stathmokinesis, the data indicate that 54% of the basal cells divide each day and that there is a high degree of synchrony in mitotic activity with a mitotic peak occurring around 13.00 h. The mitotic circadian peak occurs 9-12 h after the circadian peak in DNA synthesis. The data presented here and in the subsequent papers could be interpreted to indicate that basal cells of BDF1 mice have an average turnover time of about 26-44 h with some cells cycling once a day and others with a 2- or 3-day cell cycle time.

Radiation kills or reduces reproductive capacity of proliferating cells, including stem cells. In the oral mucosae this can result in a decline in the number of cells in the tissue which, if severe enough, will result in the formation of an ulcer when the cellularity essentially reaches zero. We have used histometric measurements of cellularity following exposure to radiation in mouse ventral tongue epithelium as a model for oral mucositis (ulcer development). Here we provide further measurements of cellularity changes in the basal layer and in the epithelium as a whole at various times following 15, 20 or 25 Gy doses. The protective effects of prior treatment with keratinocyte growth factor (KGF) are also investigated. 20 Gy of 300 kV X-rays has become our standard reference dose and the changes in cellularity seen following this dose are highly reproducible, with minimum values being observed 6 days following irradiation. A higher dose results in a greater reduction of cellularity, although the minimum value also occurs at 6 days. A lower dose (15 Gy) results in a much shallower curve, with a minimum value being observed about 1 day earlier. These changes in cellularity can be related to the less sensitive index of mucositis, namely epithelial thickness. There is also a sharp peak in proliferation about 1 day after the minimum in cellularity, i.e. on day 7. The peak following a lower dose of radiation occurs a little earlier and, following the higher dose, the peak tends to be broader. Previous work and data presented in the preceding paper in this series has shown that KGF, given over a period of 3 days, results in a dramatic increase in epithelial thickness in oral mucosa, including the ventral tongue. As a result of the increased cellularity induced by KGF given before radiation, a delay in the fall in cellularity results, which is the consequence of the increased number of cells in the epithelium at the beginning of the study.

The soya metabolite genistein possesses anti-proliferative, pro-apoptotic activities in intestinal epithelial cells in vitro and may reduce epithelial cancer incidence. This could involve cell cycle arrest/apoptosis in the proliferative or clonogenic cells. We investigated the effects of genistein on the small intestinal epithelium in vivo. No effect on the number or distribution of proliferative cells in the crypts was detected. Similarly, no change in spontaneous apoptotic cell incidence or the characteristic stem cell apoptotic response following low dose irradiation was observed. Genistein afforded a modest decrease in clonogen radiosensitivity. Hence, using a range of dosing protocols, sub-cutaneous administration of genistein for periods of up to 1 week did not alter intestinal epithelial homeostasis.