Cheek Pouch Epithelium Research Articles

Iron deficiency depresses cell proliferation in hamster cheek pouch epithelium

Iron deficiency anaemia was induced in hamsters by feeding a low iron diet coupled with weekly bleeding. To assess cell proliferation, the stathmokinetic agent vinblastine sulphate was administered and cell birth rates were calculated from cumulative mitotic indices. The rate was significantly reduced in epithelium from iron-deficient animals. The uptake of tritiated thymidine [( 3H]TdR) was also significantly reduced in these animals. Results of both stathmokinetic and labelling experiments indicate that cell production in the cheek pouch epithelium of iron-deficient animals is impaired.

Low Level X-Radiation Effects on Carcinogenesis by 7,12-Dimethylbenz(a)anthracene in Syrian Hamster Cheek Pouch Epithelium: Acute vs Fractionated Radiation Dose Studies

Studies examined the effects of acute and fractionated low to moderate level X-ray exposures on hamster cheek pouch carcinogenesis in vivo by 7,12-dimethylbenz(a)anthracene (DMBA). Animals were grouped by treatment as follows: acute doses of 0.85-3.40 Gy X rays; 17 once weekly doses of 0.01-0.20 Gy X rays (fractionated radiation); topical DMBA for 10 weeks; DMBA plus fractionated radiation starting together; DMBA plus acute radiation in Week 1 or 10 of DMBA treatments; and sham irradiation, DMBA vehicle, or anesthesia controls. After 44 weeks, hamsters were sacrificed, and their cheek pouches were excised, serially sectioned, and examined by light microscopy for histopathology. No histologic changes were observed in radiation-only hamsters. Carcinoma incidences in DMBA-only groups ranged from 45 to 60%. Carcinoma incidences were greater in groups receiving DMBA plus fractionated radiation than in groups receiving either acute radiation + DMBA or DMBA alone. Carcinoma incidences in acute radiation plus DMBA groups were lower than those in DMBA-only groups. These results suggest complex interactions between radiation and DMBA, perhaps with radiogenic cell killing being a principal factor in acute radiation + DMBA groups, and reciprocal additive or synergistic effects of radiation and DMBA on cancer induction and manifestation in fractionated radiation + DMBA groups.

Effect of smokeless tobacco on the replication of herpes simplex virus in vitro and on production of viral lesions in hamster cheek pouch

Previous experiments have shown that combination of herpes simplex virus (HSV) infection and simulated snuff-dipping in hamster buccal pouches enhances the development of micro-invasive squamous cell carcinoma in cheek pouch epithelium. The effect has now been determined of waterextractable components of snuff (snuff-extract) on the growth and the cell-lysing activity of HSV. Various dilutions of snuff-extract in tissue culture medium significantly inhibited the growth of HSV in Vero cell monolayers by inhibiting the viral DNA replication. Moreover, HSV was inactivated and its cell-lysing activity lost when it was incubated with snuff-extract in cell-free condition. Snuff also had a similar anti-herpetic effect in vivo: HSV infection of pouch tissues followed by simulated snuff-dipping resulted in significant inhibition of viral growth. Thus snuff interferes with the DNA synthesis and cytolytic activity of HSV in vitro and in vivo, and this in turn, may increase its oncogenic capacity.

Keratin protein profile in squamous cell carcinoma as induced in the hamster cheek pouch with DMBA (preliminary report).

The purpose of this study is to compare the keratin profile in DMBA-induced squamous cell carcinoma with that in normal hamster cheek pouch epithelium. The pattern of keratin expression in these two groups was studied by the methods of SDS-PAGE and immunoblot analysis on the electrophoretic keratin protein. A study of the specific changes of this protein were revealed as an essential step for analyzing the consequent changes in keratin expression that occurs in the course of development of a fully malignant lesion.

The development of the Syrian hamster cheek pouch.

The objective of this study was to provide a detailed account of the morphogenesis and early cytodifferentiation of the hamster cheek pouch. Although the newborn "cheek pouch" is used for in vitro studies of the effects of retinoids and carcinogens, its rudimentary structure has not been adequately described. Complete paraffin serial sections of the heads of 14- and 15-day fetuses were cut in three planes to determine the location and shape of the earliest pouch rudiments. Complete paraffin serial sections were prepared from pouch rudiments dissected from hamsters at birth and at daily intervals from 3 to 12 days postnatal. Semithin Epon sections were examined by light microscopy and ultrathin sections by transmission electron microscopy. The pouch can appear in the fetus as two solid epithelial ingrowths from the lining of the oral cavity. They are the margins of an ingrowing sheet of oral epithelium which becomes leaflike at about the time of birth, as it grows caudad into the tissue of the cheek. The central cells of the ingrowth accumulate large quantities of glycogen before differentiating as a stratum spinosum 5 days after birth. Within the stratum spinosum, groups of cells containing keratohyalin granules initiate the stratum granulosum. Keratinized cells appear within the stratum granulosum areas. Spaces appear between keratinized cells, and the spaces coalesce to form the pouch cavity between 7 and 12 days postnatal. Soon afterward, this cavity opens to the oral cavity to make a pouch, and the ultrastructure of the cheek pouch epithelium closely resembles that of the adult.

ハムスター口くうがん形成過程における免疫組織化学的研究 Ｉ きょうのう粘膜がんにおけるケラチンの局在について

The distribution of intracellular keratin was studied in dysplasia and squamous cell carcinomas of the cheek pouch mucosa of the hamster induced by the application of a 0.5% DMBA mineral oil. The hamster cheek pouchs were excised, and placed in a cold aqueous solution of 1% acetic acid for 1 hour while stirring. The pouches were rinsed and placed in a shallow pan of distilled water. The epithelium was then gently freed from the underlying connective tissue with the use of wooden chopsticks. With this technique the epithelium was easily separated from the connective tissue. anti-keratin antibody was induced in rabbits against total keratin extracts from the squamous epithelium of the cheek pouch mucosa. The serum was tested by the indirect immunoperoxidase method (PAP method). Chilled acetonefixed, paraffin-embedded tissues were employed.Anti-keratin antibody against total keratin was directed toward cytoplasmic antigen of keratinocytes of spinous and granular layers of the hamster cheek pouch epithelium. No labeling could be detected in the basal cell layer. In the dysplastic lesions and squamous cell carcinomas the expression of keratin antigens was markedly diminished or not detected.The present study suggests that the difference of the staining intensities of keratin among the normal epithelium, dysplasia and squamous cell carcinoma is a valuable indicator of the malignant transformation in the cheek pouch mucosa.

Low-Level X-Radiation Effects on Functional Vascular Changes in Syrian Hamster Cheek Pouch Epithelium during Hydrocarbon Carcinogenesis

Effects of repeated low-level X radiation on functional microvascular changes in hamster cheek pouch epithelium during and following carcinogenesis by 7,12-dimethylbenz[a]anthracene (DMBA) were studied. Prior studies showed enhancement of such carcinogenesis by repeated 20 rad head and neck X-radiation exposures, and it was proposed that one possible mechanism was radiogenic alteration of the functional microvasculature in a manner which favored subsequent tumor development. Hamsters were treated with either radiation, DMBA, radiation + DMBA, or no treatment. Animals were sacrificed at 3-week intervals from 0 to 39 weeks after treatments began. Pouch vascular volume and permeability changes were studied by fractional distributions of radiotracers and were analyzed by a variety of statistical methods which explored the vascular parameters, treatment types, elapsed time, presence of the carcinogen, and histopathologic changes. All treatments resulted in significant changes in vascular volume with time, while only DMBA treatments alone resulted in significant changes in vascular permeability with time. Prior to the appearances of frank neoplasms, volumetric changes in DMBA only and radiation only groups were similar, while volume changes in DMBA + radiation groups increased slowly to a peak later than in other groups and then declined steadily to levels similar to the radiation only group. As in prior studies, there were significant vascular volume differences between DMBA and DMBA + radiation groups of tumor-bearing cheek pouches. DMBA maxima were significantly higher than those of DMBA + radiation. Radiation significantly affected DMBA-associated vascular volume and permeability changes during carcinogenesis. Several possible explanations for the relationship of these changes to the enhancement of DMBA carcinogenesis include: radiation blocking normal capillary proliferative and/or dilatory responses to inflammation secondary to neoplastic changes; radiation-induced focal increases in the pericapillary connective tissue histohematic barrier, stimulating angiogenesis but reducing nutrient diffusion; radiation exposures sensitizing vascular endothelium to subsequent angiogenic stimulation from premalignant tissues; DMBA vascular and epithelial effects partially or completely blocking radiation effects on epithelial and/or endothelial cells; and radiation damage to vessel walls partially or fully inhibiting normal physiologic mechanisms of repairing DMBA damage to the vessels.

Effects of iron deficiency on respiration of hamster cheek-pouch epithelium in vitro

Iron-deficiency anaemia was induced in hamsters by feeding a low-iron diet coupled with weekly bleeding. Respiration, assessed by measuring oxygen uptake, was significantly reduced ( p < 0.002) in cheek-pouch epithelia from iron-deficient animals. To relate data on oxygen consumption to cell numbers, the cell densities of the basal and maturation compartments were measured. The cell density of the maturation compartment in iron-deficient animals was significantly reduced ( p < 0.05), whereas that of the basal cell compartment was marginally higher than in control animals. However, the cell density of the tissue as a whole was similar in both groups. Thus, the reduced oxygen consumption in iron deficiency was not due to a reduction in cell numbers. Although there was a significant reduction in epithelial volume in the iron-deficient group, this was not sufficient to account for the reduced oxygen uptake in cheek-pouch epithelia from these animals. It is concluded that iron-deficiency anaemia affects cell respiration directly, probably by interfering with the amounts and/or functions of iron-containing enzymes or cytochromes in the electron-transport chain.

Orcadian variation in mitotic influx in a keratinized epithelium

An in vivo study of the hamster cheek pouch epithelium using the stathmokinetic technique (Colcemid) demonstrated a circadian variation in mitotic influx. Based on measurements of all nucleated epithelial cells the diurnal mean was estimated in two separate experiments as 0.34%/h +/- 0.02 (SE) and 0.27%/h +/- 0.02 (SE) respectively. 3HTdR was injected in the latter study (a double labelling experiment). The significant difference between the two experiments is, however, probably due to biological variations. The maximal values for the mitotic rate were found during the light (resting) period, as were the maximal values for the mitotic index. The mean mitotic influx for the 'light period' was estimated as 0.5-0.4%/h, and for the 'dark period' as 0.2%/h. Independent analyses demonstrated the necessity of a circadian-dependent correction of the 1 and 4 h values of accumulated metaphases. The 1 h value was significantly too high during the light as well as the dark period. The 4 h value was found to be too low, but only significantly so during the dark period. Basing the estimation of mitotic rate on the 3 h accumulation value produced only very similar results to those found by using all four accumulation periods. The use of overlapping experiments proved that only cells entering mitosis after Colcemid application were arrested, so that when arrested metaphases were counted the accumulation line was correctly drawn through the origin. In the latter study (the double labelling experiment) both S- (Møller and Keiding 1982) and mitotic influx were estimated, the estimates being 0.55%/h +/- 0.03 (SE) and 0.27%/h +/- 0.02 (SE) respectively. Even considering possible methodological problems, the discrepancy between the S efflux and the mitotic influx indicates cell death and/or differentiation from G2.

Circadian-stage dependence of methotrexate in a keratinized epithelium. An in-vivo study using flow cytometry on the hamster cheek pouch epithelium.

The partially synchronized cell system of the hamster cheek pouch epithelium shows a characteristic diurnal rhythm of cell proliferation. Bolus injections of methotrexate (Mtx) in both lethal (10 g/m2) and non-lethal (2 g/m2) doses were found to inhibit cell-cycle progression primarily by impairing the G1/S transition. The results were obtained by flow cytometric DNA analysis. The inhibitory effect of Mtx manifested itself as a relative decrease of the S fraction (drug-effector phase), and was found to be dependent both on the dose and on the time of the day it was given. A bolus injection of Mtx was given either at 1200 hr (when a minimal number of cells are in S phase) or at 0200 hr (when a maximum number of cells are in S phase). The greatest cumulative decrease in S fraction was seen when the injection was given at 1200 hr. The time between injection and the effect (seen as a decrease in S fraction) was independent of the time of the Mtx injection, but seemed instead to be related to the natural diurnal period of increasing flux from G1 to S phase (at the onset of the dark period). The main effect (the relative decrease in S fraction) was repeated during the following 24-hr period, pointing to a protracted effect of Mtx on G1 cells. G1 cells affected by the initial high Mtx plasma concentration seem to be responsible for the reduced influx into S phase in both the first and second 24-hr period. In earlier toxicological studies, the survival rate of hamsters was dependent on the time of injection and was highest after injection at 1200 hr. Thus maximum cytokinetic effect on epithelial cells was found at the time of the day when there was a minimum lethal effect on the animal.

Effect of methotrexate on cells in a keratinized epithelium with an active thymidine salvage pathway assessed by autoradiography.

In the partially synchronized cell system of the hamster cheek pouch epithelium, the inhibitory effect of a bolus injection of methotrexate (Mtx) (2 g/m2, injected at 1200 hr) was analysed by means of both autoradiography and flow cytometry (FCM) in a 21-hr experiment. For autoradiography [3H]TdR and [3H]UdR were used as tracers for salvage and de novo pathways of thymidylate (TMP) synthesis, respectively. For FCM no tracers were injected. The autoradiographic studies demonstrated an active TdR salvage pathway for DNA synthesis, not affected by the impaired de novo TMP synthesis. The blocked de novo TMP synthesis was partially released 7 hr after Mtx injection, but it had not totally recovered at the end of the experiment. The decrease in the fraction of S-phase cells detected about 10 hr after Mtx injection by autoradiographic labelling with [3H]TdR and by FCM was found to be caused by a decrease in the number of cells entering S phase. However, Mtx did not influence the salvage TMP synthesis rate of cells entering S phase.

Effects of the anti‐inflammatory agents indomethacin, metiamide and acetylsalicylic acid on the induction of hyperplasia in hamster cheek‐pouch epithelium and ear epidermis

Previous studies have shown that agents such as indomethacin and hydrocortisone act to reduce the level of epidermal hyperplasia produced by various chemical, but little information is available about the effects of such anti-inflammatory agents on the induction of hyperplasia in oral mucosa. Hamster cheek-pouch epithelium and ear epidermis were treated with indomethacin, metiamide and acetylsalicylic acid prior to treatment with turpentine, podophyllin or TPA. The levels of hyperplasia induced were monitored by rates of epithelial glycolysis, protein synthesis and mitosis. The response of the metabolic assays to epinephrine was also examined. Alone, turpentine, podophyllin and TPA each caused a 3-5 fold increase in the metabolic assays and loss of the normal tissue response to epinephrine. Tissues pretreated with indomethacin showed significant reductions in the levels of hyperplasia produced and they retained a normal response to epinephrine. No reduction in hyperplasia was observed following pretreatment with acetylsalicylic acid, an alternative inhibitor or prostaglandin synthesis or with metiamide, a histamine blocker. The results indicate that the production of epithelial hyperplasia by turpentine, podophyllin and TPA can be inhibited by indomethacin.

Desmosomes in hamster cheek pouch epithelium: their quantitative characterization during epithelial differentiation.

Desmosomes in stratified squamous epithelia appear to exhibit quantitative alterations during differentiation. In this work we use stereological and other morphometric methods to quantify these structures in epithelial cells from defined basal, spinous and granular strata. Hamster cheek pouch mucosa from five animals was processed for electron microscopy using strictly standardized techniques and a stratified random sampling procedure was used to obtain micrographs of cells from basal, spinous and granular layers. Stereological intersection counting techniques were used to determine for each layer the relative surface area of plasma membrane occupied by desmosomes (Ss), the number of desmosomes per unit surface area of plasma membrane (Ns), the mean individual desmosomal diameter (delta) and the mean individual desmosomal surface area (s). In addition, estimates of nuclear volume were obtained by direct measurement of nuclear profiles and volume-to-surface ratios were obtained by a combination of point and intersection counting, which enabled estimates for the volume (Vcell) and plasma membrane surface area (SPM) of the 'average' cell within each stratum to be acquired. Using this information, it was then possible to calculate both the total surface area (S) and the number (N) of desmosomes on the plasma membranes of average cells. The parameters Ss and Ns showed progressive increases between basal and granular layers, whereas values for delta and s were lower in granular cells when compared with basal and spinous cells. The parameters Vcell, SPM, S and N all increased progressively and significantly during differentiation. Between basal and granular layers, the mean cell volume and surface area had each increased approximately threefold, whereas the surface area and number of desmosomes on the average cell plasma membrane had increased approximately seven- and eleven-fold, respectively. Granular cells thus possess more numerous desmosomes, which occupy a greater proportion of the plasma membrane area but which are individually smaller, when compared with basal and spinous layers.

Touch domes and Merkel cells in hamster cheek pouch epithelium

Minute dome-shaped elevations on the epithelial surface of hamster buccal pouch were counted in full-thickness whole mounts of detached pouch epithelium. Domes were examined by light and electron microscopy. Each dome consisted of thickened epithelium containing numerous Merkel cells with associated unmyelinated terminal axons. The pouch domes have structural characteristics in common with Haarscheiben, or touch domes, described in hair-bearing skin in man and other mammals and must not be confused with focal hyperplastic or precancerous lesions.

Effects of calcium ions on hamster cheek pouch epithelium grown in vitro.

Epithelial outgrowths from hamster cheek pouch explants were cultured for 14 days in media containing calcium concentrations of 0.05 mM, 0.075 mM, 0.1 mM, 0.25 mM, 0.75 mM or 1.45 mM (control). Compared with controls, the cultures grown at lower calcium concentrations (0.25-0.75 mM), exhibited higher mitotic indices and an increase in outgrowth size. The mitotic index of cultures at 0.25 mM calcium concentration was 30 +/- 2.26, compared to 24 +/- 2.24 for controls. Increase in size of the outgrowths was also observed at 0.25 mM calcium concentration compared with those grown in control medium. The epithelial outgrowths grown in control media exhibited cell stratification not observed at lower calcium concentrations. These studies indicate that lower calcium concentration in the media (0.25-0.75 mM) increased epithelial cell proliferation and inhibited keratinization.

Stereological methods for quantifying cell surface specializations in epithelia, including a concept for counting desmosomes and hemidesmosomes.

We present stereological procedures for morphometric evaluation of intercellular junctions appearing in ultrathin sections of epithelia. A model representing desmosomes and hemidesmosomes as flat, circular discs differentiated within the plasma membrane of epithelial cells forms a basis for estimating number per unit volume of tissue or per unit area of cell surface. Methods for estimating relative surfaces are also provided. Consideration is given to problems of tissue sampling, varying feature size and finite section thickness. The procedures are improvements over earlier estimates of number per unit area of section or per unit length of membrane. Worked examples of calculations are included and methods are illustrated further by reference to quantitative studies of hamster cheek pouch epithelium during experimental carcinogenesis.

A quantitative ultrastructural analysis of changes in hamster cheek-pouch epithelium treated with vitamin A.

The ultrastructural changes induced by the topical application of retinol acetate on hamster cheek pouch epithelium were evaluated using stereological analysis. Electron micrographs were prepared of the basal and superficial regions of the nucleated cell layer of the epithelium obtained from 3 treated and 3 control animals and examined at two levels of magnification. A total of 528 micrographs were analyzed using a coherent double lattice test system. Although the mean thickness of the nucleated cell layer did not change significantly after 10 days of treatment with retinol acetate the formation of keratinized squames was completely inhibited. This was paralleled by significant changes in the volume density of a number of organelles in both the basal and superficial strata. Rough endoplasmic reticulum increased significantly whereas filaments, which maintained a constant diameter of approximately 9 nm, keratohyalin granules and membrane-coating granules decreased in both strata. Desmosomes also showed a significant decrease in numerical area density in the treated tissues. In contrast, no changes were observed in the volume density of the Golgi apparatus, free ribosomes or mitochondria in the treated epithelium. It is concluded that this treatment provides an epithelium lacking all features of keratinization and may be a useful model for examining metabolic activities specifically associated with keratinization.

Circadian variations in influx and efflux of the S phase in a partially synchronized cell system. Double-labelling with [3H]thymidine in the epithelium of the hamster cheek pouch.

By means of a double-labelling experiment, circadian variations in the kinetic parameters of the S phase of the hamster cheek pouch epithelium were studied. The evaluation of the experiment included a recently developed correction for deviations from the strict pulse interpretation of the labelling technique. Pronounced circadian variations were found in S phase influx and efflux; the diurnal mean of both was estimated as 0.5%/hr, when based on measurements of all nucleated epithelial cells. Variations in S phase influx seem mainly responsible for the diurnal variation in cell proliferation, although diurnal variation in DNA synthesis rate, and thus in mean transit time, was also found. The increases in LI and influx were closely correlated and related to the beginning of the dark period. A circadian variation in cell number was also observed.

A quantitative study of lamina densa alterations in hamster cheek pouch carcinogenesis.

Lesions produced by topical application of 0.5 per cent. DMBA to the hamster cheek pouch epithelium were classified as hyperplasia, dysplasia and carcinoma groups using strict histological criteria. Untreated epithelium served as a control. Tissue samples from five animals in each group were processed for electron microscopy and electron micrographs from the epithelial-connective tissue junction were obtained from 5 blocks per animal. The micrographs were subjected to stereological intersection counting to determine the relative surface (SSLD, BM) of lamina densa which was in normal relationship to the basal cell plasma membranes. Quantitative results indicated a progressive loss of lamina densa during carcinogenesis and this was accompanied by the extrusion of pseudopodia from the basal cells through the gaps. The pseudopodia were frequently related to peripheral cytoplasmic microfilaments. Quantitative data confirmed the progressive nature of this loss, with values for SSLD,BM being of the order of 98 per cent., 88 per cent., 76 per cent, and 42 per cent. for normal epithelium and for the hyperplastic, dysplastic and carcinomatous lesions respectively. The loss of lamina densa is discussed in relation to the specificity of the response and to the development of features indicative of motility in transforming cells.

Langerhans Cell Function Dictates Induction of Contact Hypersensitivity or Unresponsiveness to DNFB in Syrian Hamsters

The relationship between distribution and function of Langerhans cells within the epidermis and the capacity of cutaneous surfaces to promote the induction of contact hypersensitivity to DNFB have been examined in inbred Syrian hamsters. In a manner very similar to previous findings in mice, the results indicate that hamster cutaneous surfaces deficient in normally functioning Langerhans cells, naturally (cheek pouch epithelium) or artificially (after perturbation with ultraviolet light), are inefficient at promoting DNFB sensitization. Instead, DNFB applied to these regions of skin results in the induction of a state of specific unresponsiveness. Viable lymphoid cells from unresponsive hamsters can transfer the unresponsiveness to naive hamsters suggesting that active suppression is at least partly responsible, probably mediated by T lymphocytes.