Cell kinetic studies in the murine ventral tongue epithelium: thymidine metabolism studies and circadian rhythm determination.

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.

Cell kinetic studies in murine ventral tongue epithelium: cell cycle progression studies using double labelling techniques.

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.

Cell kinetic studies in the murine ventral tongue epithelium: the effects of repeated exposure to keratinocyte growth factor.

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.

Cell kinetic studies in the murine ventral tongue epithelium: mucositis induced by radiation and its protection by pretreatment with keratinocyte growth factor (KGF).

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.

Epithelial cell proliferative activity and oral cancer progression.

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 effects of repeated doses of keratinocyte growth factor on cell proliferation in the cellular hierarchy of the crypts of the murine small intestine.

Keratinocyte growth factor (KGF) administered on a daily basis for 3 or more days can result in dramatic changes in tissue architecture, particularly the thickness in oral epithelia, and can afford protection against the cytotoxic effects of radiation on the clonogenic stem cells in the crypts. This protection of intestinal stem cells (increased numbers of surviving crypts) is reflected in an increased survival of animals exposed to a lethal dose of irradiation. The mechanisms underlying these effects are not clear. The present experiments were designed to investigate the nature of any proliferative changes induced in the crypts of the small intestine by protracted exposure to KGF. Tritiated thymidine or bromodeoxyuridine labeling showed statistically significant increases in labeling in the stem cell zone of the crypt, with a concomitant reduction in labeling in the upper regions of the crypt corresponding to the late-dividing transit population. The increase in labeling in the lower regions of the crypt was also observed with Ki-67 staining, but the reduction in the upper regions of the crypt seen with tritiated thymidine was not observed with Ki-67. Metaphase arrest data suggest that the rate of progression through the cell cycle is essentially the same in KGF-treated animals as in controls, but there is a statistically significant increase in the number of mitotic events per crypt. Double labeling studies suggest that, at certain times of the day, there is a greater influx into S phase than efflux. The data overall indicate that KGF induces some complex proliferative changes in the intestinal crypts and are consistent with the hypothesis that the radioprotection may be afforded, at least in part, by a KGF-induced increase in stem cell numbers and/or increases in the number of stem cells in the S phase of the cell cycle. This alteration in the homeostasis of the crypt is compensated for by a foreshortening of the dividing transit lineage.

In vivo administration of genistein has no effect on small intestinal epithelial proliferation and apoptosis, but a modest effect on clonogen survival.

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.

Maintenance of functional stem cells in isolated and cultured adult intestinal epithelium.

We have previously described a method for the primary culture of adult large intestinal epithelium, suggesting that stem cells had survived both the isolation and the culture procedures. However, as no markers for such cells exist, confirmation of stem cell survival is difficult-only the functional properties can be used to define them. Unfortunately, many of these (e.g., differentiation, crypt regeneration) do not occur in culture, probably due to suboptimal conditions. To address this problem both freshly isolated and cultured small and large intestinal crypts were grown subcutaneously in an immunocompromized mouse. All initially formed cysts lined by a simple epithelium which gradually became multicellular and formed invaginations containing many mitoses and apoptoses. Epithelial differentiation, as assayed by Goblet cell mucin production, was also apparent. Mucin maturation was also typical of the normal intestine. The lumen was frequently filled with mucin and apoptotic bodies. Interestingly, in grafts displaying pronounced crypt-like morphology the regions of proliferation were situated toward the base of the structure and the Goblet cells toward the lumen, i.e., a typical crypt-like morphology. Hence, functional adult stem cells appear to survive isolation and tissue culture, permitting organotypic regeneration, possibly involving homeobox gene expression. This may now allow direct stem cell characterization and experimental manipulation, such as transfection, and may ultimately permit transplantation and therapeutic gene therapy.

Effect of cyclosporin A on endothelin synthesis by cultured human renal cortical epithelial cells.

We report here for the first time that human renal proximal tubular cells secrete endothelin, clear evidence of de-novo endothelin synthesis by these cells and the effect of cyclosporin A (CsA) on endothelin synthesis both in short-term (24 h) and medium-term (5-day) culture. Human renal cortical epithelial cells were cultured and shown to possess proximal tubular characteristics. These cells produced endothelin in culture in a time-dependent manner, as measured by radioimmunoassay (291.6 +/- 51.4 pg/well/24 h). Furthermore, endothelin production by these cells was significantly decreased by up to 80% by cycloheximide (1051.8 +/- 54.9 pg/mg cell protein/24 h versus 253.2 +/- 12.6 pg/mg cell protein/24 h), showing that these cells actively synthesize endothelin. In short-term culture (24 h), CsA significantly inhibited endothelin synthesis at a medium concentration of 10,000 micrograms/l. No change in endothelin synthesis was seen at lower CsA concentrations. In contrast, over a 5-day period, a non-significant increase in endothelin synthesis was observed at CsA concentrations of 2000 micrograms/l (152.5 +/- 20.4%); however, cell growth was significantly decreased at this concentration (71.33 +/- 6.39%). Using a newly developed two-site immunoradiometric assay specific for endothelin-1 (ET-1), we demonstrate that ET-1 is the major endothelin isoform produced by human renal proximal tubular cells.

The absorption of arsenic and its relation to carcinoma.

A test dose of arsenic was given to four patients with arsenical carcinoma. Blood arsenic levels, urinary and faecal excretion were measured and compared with those of three normal control subjects. Blood levels and urinary excretion were lower in the carcinoma subjects, suggesting increased storage. Abnormally high retention of arsenic, either medicinal or environmental, may be an individual metabolic trait and may be an important factor in carcinogenesis.