Stem cell characteristics of transplanted rat mammary clonogens.

Rat mammary glands contain a subpopulation of clonogenic epithelial cells with large proliferation and differentiation potentials. When transplanted, the clonogens in monodispersed rat mammary epithelial cell suspensions give rise to either alveolar units (AUs) or ductal units (DUs) depending on the nature of the hormonal milieu in the graft recipient. Clonogens are also the primary cells of origin of mammary cancer following exposure to ionizing radiation or chemical carcinogens. Given the other stem cell characteristics of mammary clonogens, it would be expected that the primary AUs and DUs to which they give rise when grafted and hormonally stimulated (a) would be derived from the same clonogenic cell subpopulation, (b) would contain all of the functionally differentiated cell types of homologous parts of comparably stimulated mammary glands in situ, and (c) would also contain clonogen subpopulations capable when subtransplanted of giving rise to secondary AUs and DUs of similar cell composition. The current experiments were designed to test these expectations. The data are discussed in the context of results of previous studies with this and other experimental models. The results further support the conclusion that rat mammary clonogens are multipotent mammary stem cells.

Evidence that carcinogenesis involves an imbalance between epigenetic high-frequency initiation and suppression of promotion.

Evidence is presented in support of the hypothesis that cancer development depends on an imbalance between highly frequent epigenetic initiation and suppression of promotion of the initiated cells. When irradiated clonogenic mammary epithelial cells are transplanted and hormonally stimulated, they give rise to clonal glandular structures within which carcinomas may arise. In the current study, the cancer incidence in grafts of approximately 13 7-Gy-irradiated clonogens per site indicated that at least 1 of approximately 95 clonogens was radiogenically initiated. A similar initiation frequency had been seen in grafts of approximately 5 methylnitrosourea (MNU)-treated clonogens. Such initiation is thus far more frequent than specific locus mutations. In sites grafted with larger cell inocula, cancer incidences per clonogen were suppressed inversely as the numbers of irradiated or MNU-treated clonogens per graft increased. Addition of unirradiated cells to small irradiated graft inocula also suppressed progression. Radiation and MNU thus produce quantitatively, and perhaps qualitatively, similar carcinogenesis-related sequelae in mammary clonogens.

Age and radiation sensitivity of rat mammary clonogenic cells.

The relative risk of breast cancer is very high among women who were exposed to ionizing radiation during or before puberty. In the current studies, the surviving fractions of clonogenic mammary cells of groups of virgin rats were estimated after single exposures to 137Cs gamma rays at intervals from 1 to 12 weeks after birth. The radiosensitivity of clonogens from prepubertal rats was high and changed with the onset of puberty at between 4 and 6 weeks of age. By this time, the increase in the size of the clonogenic cell subpopulation was slowing and differentiation of terminal mammary end buds and alveolar structures was occurring. Analysis of the relationship of clonogen survival and radiation dose according to the alpha/beta model showed that the exponential alpha D term predominated at the second and fourth weeks of age. By the eighth week of age, the beta D2 term had come to predominate and the survival curve had a pronounced initial convex shoulder. Further experiments are required to determine whether there is an association between the high sensitivity of the prepubertal and pubertal mammary clonogens to radiation killing and a high susceptibility to radiogenic initiation of cancer.

Difference in the response of neu and ras oncogene-induced rat mammary carcinomas to early and late ovariectomy.

Rat mammary carcinomas were induced by directly inserting activated neu or ras genes into in situ rat mammary ductal cells using replication-defective retroviral vectors. neu was over 200 times more potent than ras in inducing rat mammary carcinomas. Ovariectomy 2 days postinfection dramatically reduced the occurrence of carcinomas induced by neu and extended their latency. In general, early ovariectomy had much less effect on the occurrence of carcinomas induced by ras and had no significant effect on their latency. Carcinomas induced by neu in ovariectomized rats had down-regulated estrogen receptor and progesterone receptor, while those induced by ras had only down-regulated progesterone receptor. Fully progressed mammary carcinomas in intact rats induced by both neu and ras had a similar response to ovariectomy, with an approximate regression rate of 60%. These data suggest that the activation of ras, but not neu, can replace at least some functions performed by ovarian hormones in the early phases of mammary carcinogenesis. These data also suggest a role for antiestrogen drug therapy in the prevention of neu-associated breast cancer.

Overcoming the activity of mammary carcinoma suppressor gene in Copenhagen rats by v-H-ras oncogene transfer into mammary epithelial cells in situ.

Susceptibility to mammary cancer in rats is genetically controlled by both susceptibility and suppressor genes. The Copenhagen (COP) rat strain is highly resistant to both spontaneous and induced mammary carcinogenesis. The resistant trait is due to the inheritance of an autosomal dominant allele termed mammary carcinoma suppressor (mcs) gene. To test whether the activity of mcs gene can suppress the transforming potential of an activated oncogene, we introduced v-H-ras oncogene into COP mammary epithelial cells in situ using a replication-defective retroviral vector. v-H-ras transfer caused the rapid development of mammary carcinomas at high multiplicities. Hormonal promotion further increased the penetrance of the activated ras gene. Compared with the mammary carcinoma-susceptible Wistar Furth (WF) rat strain, tumor development in the COP rat followed analogous kinetics. However, COP tumors were more differentiated and less locally invasive than were WF tumors. The possible role of the mcs gene in mammary differentiation is discussed.

Frequent induction of mammary carcinomas following neu oncogene transfer into in situ mammary epithelial cells of susceptible and resistant rat strains.

Varying results have been reported on the role of neu oncogene in mammary carcinogenesis. In order to further address this issue, the activated neu oncogene was introduced into mammary epithelial cells in situ of both mammary carcinoma-susceptible Wistar Furth and resistant Copenhagen rats by infusing replication-defective recombinant retroviruses carrying the neu oncogene into the mammary gland lumen. At the highest virus titer tested, very high numbers of mammary carcinomas developed within 2 weeks in all exposed glands in both rat strains. When the virus titer was reduced, however, individual tumors occurred with varying latencies. In addition, not all of the neu-infected mammary cells progressed to form mammary carcinomas. These results suggest that while neu is a potent mammary transforming gene, either other events in addition to neu expression may be required for full malignant transformation or not all mammary ductal epithelial cells are able to be neoplastically transformed.

Carcinoma induction following direct in situ transfer of v-Ha-ras into rat mammary epithelial cells using replication-defective retrovirus vectors.

Chemically induced mammary carcinomas often contain the activated Ha-ras oncogene. The role of this oncogene in the multistage process of carcinogenesis remains undefined. In order to model the role of ras in mammary carcinogenesis, gene transfer into adult rat mammary epithelial cells was accomplished by infusing helper-free, replication-defective retrovirus vectors into the central duct of each gland. In the initial experiments, the beta-galactosidase reporter gene was used to optimize the efficiency of this in situ gene transfer method. Stable infection of greater than 0.1% of mammary cells could be achieved following exposure to the beta-galactosidase gene-expressing vector. v-Ha-ras was then introduced into in situ adult rat mammary epithelial cells using this method. Cellular infection frequencies of less than 1% resulted in the frequent and rapid appearance of mammary carcinomas without any further treatment. Tumors arising following v-Ha-ras oncogene transfer resembled those induced by chemical carcinogens in both the kinetics of their development and histopathological spectrum. These observations support the hypothesis that ras activation can act as an initiation event in chemically induced mammary carcinogenesis. However, only a small percentage of v-Ha-ras infected cells, even with hormonal promotion, were neoplastically transformed, suggesting that ras-driven transformation is not a one-step event.

Irradiation and prolactin effects on rat mammary carcinogenesis: intrasplenic pituitary and estrone capsule implants.

The implantation of silicone capsules that contained estrone and that were adjacent to grafts of anterior pituitary tissue in the spleens of adrenalectomized glucocorticoid-deficient inbred F344 rats resulted in high circulating prolactin (Prl) levels without the untoward effects of chronic hyperestrogenism or of grafts of Prl-secreting pituitary tumors. All peripheral serum estrone titers were below the titers in sera of proestrous untreated intact rats. Peripheral serum estrone and Prl levels were, however, a function of capsule surface area over the capsule sizes tested (12-74 mm2); the elevated Prl levels persisted for as long as 700 days. In adrenalectomized glucocorticoid-deficient female rats, both 5 Gy gamma-irradiation alone and intrasplenic pituitary-estrone implants alone induced mammary carcinomas; the combination of these treatments induced a greater incidence of first carcinomas and reduced first carcinoma latency. There were, however, no marked differences in tumor incidence or latency due to differences in estrone capsule size. Finally, ovariectomy reduced first carcinoma risk in irradiated, pituitary-estrone-implanted rats but did not change the time of maximum risk. Ovarian secretory activity thus persisted in such rats and ovarian hormones synergized with Prl in mammary carcinoma induction.