Satyabrata Nandi

Growth of cultured cells using collagen as substrate.

Publisher Summary This chapter discusses the growth of cultured cells using collagen as substrate. Collagen is a major constituent of the extracellular matrix in vivo and is used as a substrate for cultured cells. Collagen is a component of extracellular matrices and basement membrane. Collagen occurs in four chemically and genetically distinct forms: type I, found in bone, tendon, and skin; type II, found in cartilage and eye; type III, associated with type I in skin, blood vessels, and smooth muscle; and type IV, found in basement membranes. Collagen used as a substrate in culture work, in the form of either thin film of dried collagen or hydrated collagen gel, usually consists of type I found in acid extract of rat tail. Collagen enhances the growth of cultured cells more than the conventional plastic or glass culture dish. High adhesion collagen substrate enhances neuronal survival and neuritic outgrowth while slowing down non-neuronal proliferation. Three-dimensional expression of cultured mammary epithelial cells in the collagen gel matrix provides conditions such as those found in vivo for the study of mammogenesis, lactogenesis, and carcinogenesis. This culture system may be applied to the studies of transformation, morphology, and cloning of mammary epithelium, proliferation, and differentiation.

Cellular expression of estrogen and progesterone receptors in mammary glands: regulation by hormones, development and aging

At present, there is an extensive body of literature documenting the participation of estrogen receptors (ER) and progesterone receptors (PR) in mammary gene expression. Yet, the precise roles of these receptors in regulating mammary development, carcinogenesis and the growth of a subset of tumors still remain unclear. Mammary glands are composed of various cell types with different developmental potentials. Further, ultimately, that it is their mutual interactions which dictate the behavior of mammary epithelial cells. Therefore, to resolve the roles of ER and PR in normal mammary growth, differentiation and carcinogenesis, analyses for the expression of these receptors at the level of individual cell types is of paramount importance. Accordingly, in the present studies using immunolocalization techniques, we document the ontogeny and cellular distribution of ER and PR during mammary development and in response to ovarian hormones and aging. In addition, we discuss the potential biological significances of the expression patterns of ER and PR during various physiological states. We believe that the observations reported here should provide a conceptual framework(s) for elucidating the roles of ER and PR in normal and neoplastic mammary tissues.

Short-term exposure to pregnancy levels of estrogen prevents mammary carcinogenesis

It is well established that pregnancy early in life reduces the risk of breast cancer in women and that this effect is universal. This phenomenon of parity protection against mammary cancer is also observed in rodents. Earlier studies have demonstrated that short-term administration of estradiol (E) in combination with progesterone mimics the protective effect of parity in rats. In this study, the lowest effective E dosage for preventing mammary cancer was determined. Rats were injected with N-methyl-N-nitrosourea at 7 weeks of age; 2 weeks later, the rats were subjected to sustained treatment with 20 μg, 100 μg, 200 μg, or 30 mg of E in silastic capsules for 3 weeks. Treatments with 100 μg, 200 μg, and 30 mg of E resulted in serum levels of E equivalent to those of pregnancy and were highly effective in preventing mammary cancer. E treatment (20 μg) did not result in pregnancy levels of E and was not effective in reducing the mammary cancer incidence. In another set of experiments, we determined the effect of different durations of E with or without progesterone treatments on mammary carcinogenesis. These experiments indicate that a period as short as one-third the period of gestation is sufficient to induce protection against mammary carcinogenesis. The pioneering aspect of our study in contrast to long-term estrogen exposure, which is thought to increase the risk of breast cancer, is that short-term sustained treatments with pregnancy levels of E can induce protection against frank mammary cancer.

Method for culturing mammary epithelial cells in a rat tail collagen gel matrix

Mammary glands are enzymatically dissociated and the resulting tissue digest enriched for epithelial cells by isopycnic banding on a density gradient of Percoll. The cells are embedded within a rat tail collagen gel matrix and fed with the appropriate medium. Growth and differentiation are superior in such a system when compared to culture on plastic, using identical media.

Mammary Phenotypic Expression Induced in Epidermal Cells by Embryonic Mammary Mesenchyme

The goal of this research was to establish methods for inducing mammary epithelial differentiation from nonmammary epithelium. For this purpose, mid-ventral or dorsal epidermis (skin epithelium; SKE) from 13-day rat or mouse embryos was associated with 13-day embryonic mouse mammary mesenchyme (mammary gland mesenchyme; MGM) (mouse MGM+rat or mouse SKE). The resultant MGM+SKE recombinants as well as controls (homotypic mouse mammary recombinants, homotypic mouse skin recombinants and mouse mammary mesenchyme by itself) were grafted under the renal capsule of syngeneic or athymic female nude mouse hosts. Most female hosts were induced to undergo lactogenesis by grafting an adult pituitary which elicited a state of hyperprolactinemia. Tissue recombinants of mouse MGM+rat or mouse SKE grown for 1 month in vivo formed a hair-bearing keratinized skin from which mammary ductal structures extended into the mesenchyme. The ducts were composed of columnar luminal epithelial cells as well as basal, actin-positive myoepithelial cells. When grown in pituitary-grafted hosts, the ductal epithelial cells expressed casein and alpha-lactalbumin as judged by immunocytochemistry. The expression of caseins in MGM+SKE recombinants was confirmed by Western blot. The epithelial cells in mouse MGM+rat SKE recombinants expressing milk proteins were shown to be rat cells while the surrounding connective tissue was composed of mouse cells based upon staining with Hoechst dye 33258. Using mammary-specific markers, these studies confirmed the earlier morphological studies of Propper and unequivocally demonstrated for the first time that embryonic mammary mesenchyme can induce morphological and functional mammary differentiation from nonmammary epithelium.

Recent Studies of the Hormonal Influence in Mouse Mammary Tumorigenesis

This paper reviews the hormonal factors involved in the events leading to formation of mammary tumors in mice. Studies on the process of tumorigenesis have been facilitated by the existence of a recognizable preneoplastic state the hyperplastic alveolar nodule in the mouse mammary gland. In C3H/Crgl high-tumor bearing mice the hormonal control of various phases of mammary gland development was analyzed by treating hypophysectomized-ovariectomized-adrenalectomized mice with various combinations of ovarian adrenocortical and adenohypophyseal hormones. The minimum hormonal combination effective in the induction of ductal and alveolar development is estrogen + a C-21 steroid (luteoid or corticoid) + an adenohypophyseal factor (somatotropin or mammotropin). However mammary development in virgin C3H/Crgl mice is attributed to estrogen corticoids and somatotropin as mammotropin and progesterone are not found in virgin mice. In breeding mice progesterone and mammotropin contribute to the mammogenic hormonal milieu. In all phases of mammary development and function in this strain somatotropin and mammotropin are interchangeable. In the presence of alveolar mammogenic combination of estrogen corticoids and somatotropin hyperplastic alveolar nodules develop in virgin mice while in breeding mice the contributory hormonal factors are progesterone and mammotropin. Estrogen is not needed in the maintenance (although it is needed in the formation) of nodules it is present in normal intact mice. In A/Crgl virgin female mice which do not normally develop mammary tumors the hormonal milieu was found to be favorable to tumor formation from transplanted nodules. The main difference between mouse strains with a low or a high tumor incidence among virgin females is in the hormones capable of supporting nodule formation. There may be a relationship between phenotypic expression of the inherited hormonal influence and sensitivity of the mammary gland to somatotropin-containing hormonal combinations.

Hormone-induced protection of mammary tumorigenesis in genetically engineered mouse models

IntroductionThe experiments reported here address the question of whether a short-term hormone treatment can prevent mammary tumorigenesis in two different genetically engineered mouse models.MethodsTwo mouse models, the p53-null mammary epithelial transplant and the c-neu mouse, were exposed to estrogen and progesterone for 2 and 3 weeks, respectively, and followed for development of mammary tumors.ResultsIn the p53-null mammary transplant model, a 2-week exposure to estrogen and progesterone during the immediate post-pubertal stage (2 to 4 weeks after transplantation) of mammary development decreased mammary tumorigenesis by 70 to 88%. At 45 weeks after transplantation, analysis of whole mounts of the mammary outgrowths demonstrated the presence of premalignant hyperplasias in both control and hormone-treated glands, indicating that the hormone treatment strongly affects the rate of premalignant progression. One possible mechanism for the decrease in mammary tumorigenesis may be an altered proliferation activity as the bromodeoxyuridine labeling index was decreased by 85% in the mammary glands of hormone-treated mice. The same short-term exposure administered to mature mice at a time of premalignant development also decreased mammary tumorigenesis by 60%. A role for stroma and/or systemic mediated changes induced by the short-term hormone (estrogen/progesterone) treatment was demonstrated by an experiment in which the p53-null mammary epithelial cells were transplanted into the cleared mammary fat pads of previously treated mice. In such mice, the tumor-producing capabilities of the mammary cells were also decreased by 60% compared with the same cells transplanted into unexposed mice. In the second set of experiments using the activated Her-2/neu transgenic mouse model, short-term estradiol or estradiol plus progesterone treatment decreased mammary tumor incidence by 67% and 63%, and tumor multiplicity by 91% and 88%, respectively. The growth rate of tumors arising in the hormone-treated activated Her-2/neu mice was significantly lower than tumors arising in non-hormone treated mice.ConclusionBecause these experiments were performed in model systems that mimic many essential elements of human breast cancer, the results strengthen the rationale for translating this prevention strategy to humans at high risk for developing breast cancer.

Inhibition of N-methyl-N-nitrosourea-induced mammary carcinogenesis by molecular iodine (I2) but not by iodide (I − ) treatment Evidence that I2 prevents cancer promotion

We analyzed the effect of molecular iodine (I2), potassium iodide (KI) and a subclinical concentration of thyroxine (T4) on the induction and promotion of mammary cancer induced by N-methyl-N-nitrosourea. Virgin Sprague-Dawley rats received short or continuous treatment. Continuous I2 treated rats exhibited a strong and persistent reduction in mammary cancer incidence (30%) compared to controls (72.7%). Interruption of short or long term treatments resulted in a higher incidence in mammary cancer compared to the control groups. The protective effect of I2 was correlated with the highest expression of the I-/Cl- transporter pendrin and with the lowest levels of lipoperoxidation expression in mammary glands. Triiodothyronine serum levels and Na+/I- symporter, lactoperoxidase, or p53 expression did not show any changes. In conclusion continuous I2 treatment has a potent antineoplastic effect on the progression of mammary cancer and its effect may be related to a decrease in the oxidative cell environment.

Maintenance of normal rat mammary epithelial cells by insulin and insulin-like growth factor 1☆

Normal rat mammary epithelial cells were cultured within a rat tail collagen gel matrix formed under improved conditions for controlling pH and osmolarity. Under these conditions, growth can be maintained for up to 3 weeks with a 10- to 15-fold increase in cell number. The cells grow in response to prolactin, progesterone, epidermal growth factor, and cholera toxin, in a medium of DME: Hams F12 supplemented with BSA and insulin at 10 micrograms/ml. When the insulin concentration was reduced to more physiological levels (10 ng/ml) the cells did not grow. However, at these more physiological concentrations it could be shown that insulin had a concentration-dependent effect on the maintenance of the cells with an optimum concentration around 25 ng/ml. The cells could be maintained in hormone-supplemented medium with low levels of insulin in a quiescent state for up to 14 days. The high levels of insulin needed for optimal growth could be replaced by insulin-like growth factor 1 (IGF-1) at much lower concentrations (25-50 ng/ml). The superphysiological level of insulin required for optimum growth is probably due to its acting weakly through an IGF-1-mediated growth-promoting mechanism. Insulins effect on cell maintenance occurs at physiological levels and may better reflect its role in mammary cell growth.

The hormones responsible for lactogenesis in BALB/cCrgl mice.

Abstract In an attempt to determine the endocrine factors responsible for lactogenesis, various combinations of hormones were administered to female BALB cCrgl mice whose mammay glands had been stimulated to lobulo-alveolar development by exogenous hormones. This stimulation was accomplished by treatment with estrogen and progesterone for 15 days before hypophysectomy-ovariectomy (doubly operated) or hypophysectomy-ovariectomy-adrenalectomy (triply operated) followed by a postoperative treatment with a mammogenic combination for 5 days. Various possible lactogenic combinations were then administered for a final 5-day period. Final treatment with either cortisol (125 μg) + mammotropin or cortisol (125 μg) + somatotropin resulted in lactogenesis in triply operated mice. However, the same amount of mammotropin or somatotropin given with much higher daily doses of corticosterone (1 mg) was ineffective. In doubly operated mice, milk secretion followed the administration of corticotropin + mammotropin, but not of corticotropin + somatotropin. Full lactogenesis occurred in triply operated mice following final treatment with either corticosterone + mammotropin + somatotropin or cortisol + mammotropin + somatotropin, but the amount of corticosterone required was 8 times that of cortisol. In doubly operated mice, a combination of corticotropin + mammotropin + somatotropin was also capable of inducing full secretion. Although daily administration of corticosterone (1 mg) + mammotropin (1 mg) + somatotropin (0.1 mg) was highly effective in inducing secretion, reversal of the ratio of mammotropin to somatotropin resulted in poor lactogenesis. Provided that the daily dose of corticosterone was maintained at 1 mg, some secretion occurred in all mice with the administration of as little as 0.55 mg mammotropin + 0.1 mg somatotropin. However, if the corticosterone dose was reduced to 250–500 μg/day, little or no lactogenesis occurred even when the quantity of protein hormone was increased. The addition of other hormones (aldosterone, dehydro-corticosterone, thyroxine, insulin) had little or no influence on the lactogenic potency of corticosterone + mammotropin + somatotropin. In view of these findings and of the inability of mammotropin to induce premature secretion in pregnant mice when corticoids are effective in so doing, it is suggested that the corticotropin-glucocorticoid axis may be of fundamental importance in triggering lactation in mice.