Comparative clastogenic sensitivity of respiratory tract cells to gamma rays.

To understand the relationships between exposure and damage to different cell populations in the respiratory tract, methods were developed to culture deep-lung fibroblasts and epithelial cells from the nose, trachea and deep lungs. Female F-344 Fischer and male Wistar rats were exposed to 1-5 Gy of 60Co gamma rays at a dose rate of 0.4 Gy/min. Cells were isolated for short-term culture, and the incidences of binucleated cells and micronuclei were determined. The incidences of micronuclei were determined in cytochalasin-B-induced binucleated cells at 72 h for nasal and tracheal tissue and 96 h for deep-lung fibroblasts and epithelial cells. Maximum frequencies of binucleated cells were found in the control nonirradiated cells at these harvest times, and the frequencies were not significantly affected at these harvest times by radiation exposure. No significant differences were found in the frequencies of micronuclei induced in the nasal epithelial cells isolated from female F-344 Fischer or male Wistar rats. Fibroblasts cultured in different media and isolated from either female F-344 Fischer or male Wistar rats also showed a similar frequency of micronuclei. Over the doses tested, the frequency of micronuclei in the respiratory tract cells increased linearly with the dose. The slopes were 92.2 +/- 9.2, 76.2 +/- 7.9, 32.8 +/- 2.4 and 28.7 +/- 3.4 micronuclei/1000 binucleated cells/Gy for deep-lung epithelial cells, deep-lung fibroblasts, tracheal epithelial cells and nasal epithelial cells, respectively. Deep-lung epithelial or fibroblast cells were about two to three times as sensitive for elastogenic damage as nasal and tracheal epithelial cells. The measurement of micronuclei in isolated respiratory tract cells is very useful in assessing cytogenetic damage induced in different cell types by radiation.

Lung cancer response following inhaled radon in the A/J and C57BL/6J mouse.

A/J and C57BL/6J mice, strains which differ in susceptibility to chemical-induced lung tumours, were examined for differential sensitivity to radon-induced lung cancer. Sixty weeks after exposure to 952 WLM radon (3.34 J h1 m-3), no lung tumours were found in the control, sham- (one dust-) exposed, or radon-exposed C57BL/6J mice. In contrast, each of the A/J groups had a substantial number of tumours. Although there were no differences in overall lung tumour incidence or multiplicity associated with radon and ore dust exposure in A/J mice, distinct differences in tumour size and histopathological appearance were noted. The ratio of lung adenoma-to-carcinoma found in control A/J mice was 10:1, whereas the ratio in one dust-exposed animals was 10:8 and that in radon-exposed animals was 8:6. Based on these data, particulates appear to modify the promotion-progression of lung cancer in this strain. In addition, the appearance of two very large carcinomas histologically resembling Clara-cell-derived tumours only in the radon-exposed A/J mice and the possibility of alpha-particle-induced killing of target cells in the radon-exposed animals suggests that A/J but not the C57BL/6J, mice may have genes that modify susceptibility to radon-induced lung cancer.

An investigation of rat mammary healthy and R3230AC tumor tissues and cells by means of solid-state 13C NMR.

The first results are shown of a low temperature 13C solid-state, cross-polarization magic angle spinning (CPMAS) NMR study of R3230AC rat mammary carcinoma transplanted into female Fischer rats. Intact, healthy mammary tissues and tumor tissues, quickly frozen at -78 degrees C after excision, were examined, as were normal epithelial cells and tumor cells extracted from these tissues. The experiments were performed at -100 degrees C and -40 degrees C. The solid-state 13C NMR spectrum of the healthy tissue is dominated by the triacylglycerols present in the adipose tissue. The solid-state spectra of the other compounds differ significantly from the spectrum of the healthy tissue, and are dominated by the phospholipids and the large molecular weight proteins, for a major part present in the membranes. The spectra of the tumor tissues and the tumor cells were very similar, in accordance with the fact that the tumor tissues consisted of 80-90% tumor cells. The spectrum of the normal cells shows the same general features as those of the tumor tissue and tumor cells, but also exhibits some significant differences. The main difference at -100 degrees C is that, in the tumor samples, the relative intensity of the resonance line arising at 30 ppm, which is due to methylene chains, is reduced. At -40 degrees C this intensity is further reduced in the tumor samples, whereas the spectrum of the normal cells remains unaltered. It is tentatively concluded that the spectral differences between the normal and the malignant cells are due to changes in mobilities occurring in the cells after tumorigenesis, and that compared with normal epithelial cells the tumor cells contain an increased amount of mobile methylene chains. These differences virtually disappear if the samples are preserved at temperatures equal to or above 4 degrees C, emphasizing the importance of sample preservation at low temperatures, and hence of the solid-state NMR approach to investigate structures and dynamics in cells before and after tumor formation.

Proliferation-independent growth factor modulation of the radiation sensitivity of human prostate cells.

The survival of human prostatic epithelial cells irradiated in different physiological states is reported. Exponentially growing cells and contact-inhibited cells grown and irradiated in the presence of the growth factors epidermal growth factor (EGF) and bovine pituitary extract (bPE) had overlapping radiation dose-cell survival curves. However, when EGF and bPE were removed from exponentially growing cells before irradiation, an increase in radiosensitivity was observed if the cells were replated into medium containing growth factors (EGF and bPE) immediately after irradiation. Treating cells with the nonspecific growth factor receptor antagonist suramin had similar effects as did growth factor deprivation. In contrast, when growth factor-deprived cells were maintained in this same medium for 12 h postirradiation, an increase in radiation survival was observed. This increase in survival is attributed to the repair of potentially lethal damage (PLD). Both the increase in radiosensitivity induced by deprivation of growth factor before irradiation and the repair of PLD caused by deprivation of growth factor after irradiation were independent of changes in cellular proliferation.

The effects of goitrogenesis, involution, and goitrogenic rechallenge on the clonogenic cell content of the rat thyroid.

A fraction of enzymatically monodispersed rat thyrocytes from untreated animals clonally proliferate into thyroid follicular units following transplantation into the subcutaneous fat pads of syngeneic recipients. During the induction of experimental goiters in rats either with 3-amino-1,2,4-triazole/iodine sufficient diet or KClO4/Remington low iodine diet, the clonogenic fractions of cells from aminotriazole goiters decreased to 1.9 x 10(-4) and KClO4 goiters to 9.8 x 10(-5) as compared to 5.8 x 10(-3) for cells from age-matched controls during the growth phase of goitrogenesis. With continued aminotriazole treatment after thyroid hyperplasia had ceased, the clonogenic fraction increased to 2.0 x 10(-3) while continued KClO4 treatment had little further effect. The changes in the clonogenic fraction induced by both regimens were reversed during involution; goitrogenic rechallenge of involuted glands led to changes in the clonogenic fraction similar to that noted during the initial challenge. The clonogenic fractions of cells from aminotriazole goiters were greater than that of cells from KClO4 goiters at all time points examined despite similar TSH levels in situ. We conclude that the rat thyroid contains a hierarchy of cells with different proliferative capacities and that the clonogenic thyrocytes possess many of the attributes of a stem-cell.

The plateau phase rat goiter contains a sub-population of TSH-responsive follicular cells capable of proliferation following transplantation.

In the rat, chronic TSH stimulation leads to self-limited thyroid hyperplasia, goitrogenesis, and TSH-responsive thyroid tumors. The current studies were aimed at clarifying the mechanism by which hormone-responsive, proliferating follicular cells arise in quiescent plateau phase rat goiters. Enzymatically monodispersed rat thyrocytes from early plateau phase and involuting goiters were analyzed for the capacity to form thyroid follicular units after transplantation into syngeneic recipients. Goiters induced with two different regimens contained substantial numbers of cells capable of proliferating into functioning thyroid follicular tissue after transplantation. The clonogenic fractions of cells from goiters induced by 3-amino-1,2,4-triazole or KClO4/Remington low iodine diet were significantly lower than that of cells from control glands. Furthermore, the clonogenic fraction of cells from the KClO4 goiters was also found to be significantly less than that of cells from aminotriazole goiters despite similar TSH levels in the cell donors. The hormone responsiveness of the clonogenic goiter cells and the histological appearance and functional capacity of the thyroid tissue which arose after grafting were indistinguishable from controls. We conclude that the capacity to clonally proliferate into follicular units is a specific trait which characterizes a unique subset of follicular cells and speculate that the hormone-responsive tumors which typically appear in the chronically stimulated rat thyroid originate from cells within this subset.

On the cells of origin of radiogenic thyroid cancer: new studies based on an old idea.

We have presented evidence that the functional thyroid follicles (follicular units, FU) which are formed in grafts of monodispersed rat thyroid cells, and hence the thyroid tumors which later develop in such grafts, are clonal in origin. Transplantation assays indicate that the clonogens comprise approximately 1% of the cells in monodispersed suspensions of normal thyroid tissue. Carcinogenesis studies show that neoplastic initiation of thyroid clonogens by radiation is a common event. Promotion-progression to cancer from radiation initiated clonogens has, however, been shown to be inversely related to the total grafted thyroid cell number; i.e. more tumors develop per irradiated clonogen in grafts of small cell numbers than of large cell numbers. Recent studies have been designed to investigate: a) whether the cell number-dependent inhibition of promotion-progression is mediated by remote hormonal feed-back, local cell-cell interactions, or both; b) the cell population kinetics of the clonogen subpopulation during goitrogenesis and goiter involution; and c) the effect of prolonged exposure to high levels of TSH (thyrotropin) on the capacity of the clonogens to give rise to functional FU. The results indicate that local cell-cell interactions play an important role in the cell number-dependent suppression of neoplastic promotion-progression. They also show that if sufficient thyroid cells are grafted, the thyroid-pituitary axis can be reestablished in thyroidectomized rats fed normal diets. In such animals given iodine deficient diets, the FU that develop in the thyroid grafts shift their secretory pattern to increase the ratio of T3 (triiodothyronine) to T4 (thyroxine), and thus conserve the available iodine. Finally, the clonogenic subpopulation is conserved during both goitrogenesis and goiter involution. When they are transplanted to thyroidectomized recipients, clonogens from two types of goiters form FU that are morphologically indistinguishable from those that develop in grafts of normal thyroid clonogens. Furthermore, the secretion of T3 and T4 by such grafts is dependent on the grafted clonogen number, and hence FU formation, and not on the total number of thyroid cells transplanted. We conclude that the thyroid clonogens, the presumptive cancer progenitor cells, have many of the characteristics of stem cells.