Tumorigenesis of rat mammary epithelial cells by N-nitroso-N-methylurea in anin vitro system: Characterization of the microtumors

Summary Chemical carcinogenesis is a lengthy process that involves the rather loosely defined stages of initiation, promotion, and progression. Several model systems of mammary carcinogenesis have been designed to elucidate the mechanisms of chemical carcinogenesis. Most of these systems have included animal models. While organ specific chemical carcinogenesis can be initiated in these systems, the subsequent stages of promotion and progression are difficult to study in detail. Investigations onin vitro carcinogenesis have shown transformation of mammalian cells in culture; the transformational event, however, is difficult to discern within the monolayer culture. We have recently reported the development of anin vitro carcinogenesis system that allows both the initiation as well as the progression of mammary cells in a collagen gel matrix culture system. The cells transformed by a chemical carcinogen develop into discernible microtumors with the three dimensions of a collagen gel culture. Isolation of these microtumors from the collagen gel an subsequent culture in monolayer has produced cells capable of colony formation in soft agar. The present study further characterizes these microtumors originatedin vitro by analysis of cell growth kinetics versus parallel control cells. In addition, flow cytometric and cytogenetic studies have been performed to investigate the chromosomal stability of these cells. It was also observed that the microtumors, producedin vitro from mammary epithelial cells of an inbred strain of rats, show the ability to form tumors upon transplantation into the fat pad of syngeneic hosts.     

Effect of molybdenum supplementation onN-nitroso-N-methylurea-induced mammary carcinogenesis and molybdenum excretion in rats

Molybdenum (Mo) supplementation reduces the incidence of nitrosamine-induced tumors in the esophagus and forestomach of laboratory animals, and the incidence of mammary cancer in female rats induced byN-nitroso-N-methylurea (NMU). The present study was conducted to evaluate the effect of graded amounts of Mo on NMU-induced mammary carcinogenesis, and on the excretion of Mo and copper (Cu). Female Sprague-Dawley rats aged 5 wk were givenad libitum a low-Mo (0.026 mg/kg) diet and deionized water. After 15 d, a single SC injection of 50 mg NMU/kg body wt was administered to each of 30 rats in groups 2–5. Eight rats in group 1 served as untreated control. One week after the carcinogen treatment, 0.1, 1.0, or 10 mg Mo from sodium molybdate were added to each liter of drinking water for groups 3, 4, and 5, respectively. Groups 1 and 2 did not receive any Mo supplementation. After the rats had been Mosupplemented for 38, 67, and 85 d, 48-h urine and fecal samples were collected from the same 48 rats, and Mo and Cu were determined. Molybdenum seemed to have little effect on Cu excretion. At each time interval, animals fed 0 or 0.1 mg Mo/L excreted more Mo in feces than in urine, whereas rats fed 1 and 10 mg Mo/L water excreted more Mo in urine than in feces, which indicates that Mo absorption was not easily saturated as the amount of Mo increased. However, the liver became saturated with Mo when 0.1–1 mg Mo/L was fed. The total number of palpable tumors per group 101 d after NMU administration was 109, 115, 101, and 81, and the total carcinomas per group were 92, 96, 86, and 65 for the animals in groups 2–5, respectively. The results indicate that supplemental Mo in the amount of 10 mg/L of drinking water inhibited mammary carcinogenesis.     

Mammary gland morphogenesis in vitro: Extracellular requirements for the formation of tubules in collagen gels by a cloned rat mammary epithelial cell line

Summary The mechanism of induction of tubular outgrowths in vitro on floating collagen gels and the influence of extracellular factors on this process have been investigated using the clonal rat mammary epithelial cell line, Rama 25. Growth of Rama 25 on such floating gels causes their contraction. Contraction of the gel is accompanied by a 10-fold increase in the number of cells per unit area, a change in cell shape, and a convolution of the epithelial cell sheet. Gels folded over manually show an 11-times higher incidence of tubules along the folds than on the flat surface. Tubular formation begins when cords of cells develop from local proliferations of the cell sheet and become canalized. Tubules follow wrinkles in the gel and branch to yield monopodial, dichotomous, or lobular architecture. Hydrocortisone and insulin, in the presence of serum, stimulate both narrow and thick tubular structures on folded gels, whereas extra additions of 1 ng/ml cholera toxin or 100 ng/ml epidermal growth factor preferentially stimulate thick tubular structures. Floating glutaraldehyde-fixed gels, very thick collagen gels, and collagen gels prepared on the top of rigid steel grids fail to support the formation of tubules, suggesting that flexibility and access of the medium to basal surfaces are important to their genesis. Incorporation of hyaluronic acid into the gel matrix preferentially inhibits the thick tubular outgrowths. Thus, the branching tubular structures generated by Rama 25 can be influenced in different ways by various extracellular factors in the medium and in the gel. During the course of this work E. J. Ormerod was in receipt of a Ludwig Research Studentship.     

Role of serum and hormones during the growth and development of rat mammary tumor epithelial cells in collagen gel culture

Summary The characteristics of hormone-dependent rat mammary tumors in response to serum and hormones were determined in collagen gel matrix culture. Epithelial cells from 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary adenocarcinomas were embedded in collagen gel and the effect of estrogen, progesterone, prolactin, insulin, and serum was tested. The total cell number and [³H]thymidine incorporation were used to determine the growth pattern of the cells in culture. It was found that in medium containing 20% porcine serum and supplemented with insulin, estrogen, progesterone and prolactin, both the cell number and [³H]thymidine labeling index increased with time, after an initial lag. Serum seemed to be essential to maintain growth of the tumor cells, because hormones alone, in the absence of serum, were unable to sustain growth of the cells. When estrogen, progesterone, prolactin, and insulin were tested individually in the presence of 20% porcine serum, only estrogen demonstrated a significant stimulatory effect.     

Collagen gel culture of rat mammary tumor cells as an assay system for determination of therapeutic efficacy of chemotherapeutic agents

Summary Collagen gel culture of rat mammary epithelial cells was used as an in vitro assay system for determination of the therapeutic efficacy of three cytotoxic agents commonly used in the treatment of human breast cancer, namely 5-fluorouracil (5-FU), methotrexate, and Adriamyin (ADR). The same three drugs were also evaluated in vivo, and a good correlation was obtained between the results in these two systems. A 9-d culture was shown to be more reliable than a 12-d culture, because nondrug-related cell mortality became a confounding factor after 12 d. Although further experiments are necessary, it is suggested that collagen gel culture may well prove to be a useful assay system for determination of sensitivity of tumor cells to cytotoxic drugs with possible clinical applications in the choice of treatment modality administered to cancer patients.     

Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels

Summary Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture. This investigation was supported by Grants CA05388 and CA09041 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and by cancer research funds of the University of California.     

Cultureof bovine mammary epithelial cells on collagen gels

A method for the isolation of lobules of acini from bovine mammary gland and their storage in liquid nitrogen is described. After further dissociation of freshly prepared or frozen lobules, clumps of cells are obtained which attach to collagen gels and give rise to colonies which, on morphological criteria, appear predominantly epithelial. Storage for up to 6 months did not adversely affect viability. Increase in colony area involved cell division, was more rapid in air than in 95% oxygen and was enhanced by fetal calf serum.     

Ultrastructural features of bovine mammary epithelial cells grown on collagen gels

Bovine mammary epithelial cells cultured on floating gels of rat tail collagen showed two principal cell types, columnar and squamous, with ultrastructural features resembling secretory and myoepithelial cells respectively. Cultures of freshly prepared cells released alpha-lactalbumin into the culture medium and in some cases contained fat droplets, although these did not appear to be released. No ultrastructural evidence of casein synthesis was observed. A notable feature was the failure to secrete a continuous basement membrane. Intermediate filaments were present in abundance in squamous epithelial cells.     

Isolation and characterization ofPenicillium funiculosum mutants with enhanced glucose oxidase production

After the mutagenesis ofPenicillium funiculosum with UV light andN-nitroso-N-methylurea, 83 of 2237 grown colonies were surrounded with increased zones of glucose oxidase diffusion. Analysis of the glucose oxidase activity of selected mutant strains grown in submerged cultures allowed 18 mutant strains to be obtained whose glucose oxidase activity was 5–153% higher (in a medium with glucose) and 4–83% higher (in a medium with sucrose) than that of the parent strain. Two of these mutant strains, UV6.31 and NMU95-132, possessed high glucose oxidase activity when grown in media with glucose or sucrose and produced large amounts of mycelia. The active and morphologically stable mutantP. funiculosum NMU95-132 was chosen for further selection work.     

Isolation and characterization of membrane-associated proteoglycans from normal and malignant human mammary epithelial cells

The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [³H]glucosamine and [³⁵S]Na₂SO₄ by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate - Di-OS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose - Di-4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose - Di-6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose - Gdn-HCl guanidine hydrochloride - WGA wheat germ agglutinin     

In vitro culture of Geigeria aspera Harv.

Callus-, root-, shoot-, and rooted shoot cultures of Geigeria aspera were established on Murashige and Skoog's nutrient medium. Organogenesis of roots and shoots was induced with treatments containing BA or KN in combination with NAA, IAA, IBA or 2, 4-D. Rooted plantlets were successfully transplanted into soil.     

Casein production during differentiation of mammary epithelial cells in collagen gel culture

Mouse mammary epithelial cells cultivated on floating collagen gels secrete, as judged by immunoblotting, the full array of caseins found in mouse milk. The secreted caseins are all phosphorylated and have estimated minimum molecular weights (MWs) of 45, 40, 27, and 23 kD in SDS-PAGE. Intracellular caseins of epithelia from collagen gel cultivation or from lactating mammary glands are a combination of mature caseins identical with the secreted molecules and novel caseins whose apparent size in SDS-PAGE is different from the secreted molecules. The novel caseins were shown to be non-phosphorylated species apparently insufficiently mature for secretion. Our data indicate that, with regard to casein expression, cultivation of mouse mammary epithelia on collagen gels essentially duplicates their behavior in the lactating mouse mammary glands.     

In vitro culture of Aloe Barbadensis Mill.: Micropropagation from vegetative meristems

A method for rapid and highly effective plant micropropagation from vegetative meristems was established for Aloe barbadensis Mill. Plant micropropagation was achieved culturing apices on medium containing 1.1 M 2,4-dichlorophenoxyacetic acid and 2.3 M kinetin for 15–30 days. High morphogenetic ability was maintained by transferring explants (after 60 days) on media containing 0.11 M 2,4-dichlorophenoxyacetic acid and 2.2 M 6-benzylaminopurine.     

Calcium regulation of normal human mammary epithelial cell growth in culture

Summary The concentration of Ca⁺⁺ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca⁺⁺ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able, to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of α-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca⁺⁺ to less than 0.08 mM. The reduction of Ca⁺⁺ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca⁺⁺ on growth and differentiation were specific (Mg⁺⁺ and Mn⁺⁺ variations were without effect) and reversible and in many respects resembled Ca⁺⁺ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca⁺⁺-induced coupling and uncoupling of proliferation and the program of differentiation, we propose that Ca⁺⁺ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu. This study was supported by grants NIH-CA18175 and CA36399 and an institutional grant from the United Foundation of Greater Detroit.     

In vitro cytodifferentiation of perinatal rat islet cells within a tridimensional matrix of collagen

Summary Cell suspensions prepared by collagenase digestion of pancreases obtained from rat fetuses (21.5 d old) and newborns (2.5 d old) were mixed with a collagen solution and inoculated on a collagen base layer. At the onset of the culture, most acinar cells became necrotic, whereas other epithelial cells proliferated. Most of the cell clusters arranged themselves into simple polarozed structures composed of epithelial cells forming hollow spheres, and from these budded neoformed endocrine islets. Scarce fibroblasts were located close to these structures. Immunocytochemical localization of insulin and glucagon, as well as ultrastructural characteristics of the cell types revealed an intrainsular distribution similar to the in vivo localization. Tridimensional matrix of collagen offers, to perinatal pancreatic cells in culture, an environment close to the in vivo conditions: cells reorganize themselves in tissuelike structures and cell interactions concerned in the cytodifferentiation of pancreatic islets occur. This system allows for the study of undifferentiated epithelial cells—the presumed stem cells—differentiating and differentiated endocrine cells in the same preparation. B.A. is supported by a doctoral scholarship from the Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture, Brussels. This work was supported by grants from the Fonds National de la Recherche Scientifique, Brussels, and from Petrofina S.A., Brussels.     

In vitro culture of Ginkgo

Summary Ginkgo biloba L. is an important landscape tree, is resistant to insect, fungi and other pests, and produces a number of chemicals that have pharmaceutical properties (termed ginkgolides). Studies were initiated to establish an in vitro culture protocol for Ginkgo. Explants (intact embryos, embryos with cotyledons removed, and cotyledon tissue) were removed from disinfested seeds and cultured on Murashige and Skoog minimal organics medium with various combinations of either 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) and either kinetin or benzyladenine (BA). Cultures were incubated in the light and morphological development was recorded. Both embryo and cotyledon explants produced callus (cotyledon tissue produced the most callus). Ginkgolides A and B were detected in callus tissue extracts. Intact embryo cultures initiated on media with 2,4-D plus NAA for 5 wk produced shoots and roots when transferred to media with 4.5 μM 2,4-D alone for an additional 5 wk. Plants were transferred from the 2,4-D media to pots and maintained in the greenhouse.     

Effects of 1alpha,25-dihydroxycholecalciferol and cortisol on the growth and differentiation of primary cultures of mouse mammary epithelial cells in collagen gel

We examined the effects of 1alpha,25-dihydroxycholecalciferol (1,25-DHCC) and the glucocorticoid, cortisol, on primary mouse mammary epithelial cells in collagen gel cell culture systems. Physiological low concentrations (10(-11)-10(-9) M) of 1,25-DHCC stimulated growth of the cells in a collagen gel matrix culture in serum-free DMEM+Ham's F12 (1:1) medium containing BSA, EGF and cholera toxin, and the cell number reached 1.8-fold the control after 6 d in culture. In contrast, supraphysiological concentrations (10(-8)-10(-7) M) of 1,25-DHCC suppressed cell growth. Cortisol produced similar, but smaller, dose-dependent effects. The addition of serum to the culture medium masked the stimulatory effect of 1,25-DHCC and both the stimulatory and inhibitory effects of cortisol. 1,25-DHCC also affected casein synthesis by cells cultured in a serum-free floating collagen gel culture containing prolactin, insulin and cortisol, enhancing synthesis at low concentrations (10(-11)-10(-9) M) and inhibiting it above 10(-8) M. In the absence of cortisol, no detectable change in casein synthesis was induced by 1,25-DHCC. These results suggest a physiological role for 1,25-DHCC in stimulating both growth and differentiation of mouse mammary epithelial cells, though 1,25-DHCC does not substitute for glucocorticoids in the differentiation of the cells.     

Reconstruction of the skin in three-dimensional collagen gel matrix culture

Summary The skin comprises three layers: epidermis, dermis, and hypodermis. We report here on a skin, reconstructed in vitro, that is composed of all three layers. The topmost layer, epidermis, was exposed to air by a new method. The exposure induced an extensive proliferation, and differentiation, i.e. keratinization was eventually observed in the cultured epidermal cells. Skin thus cultured will be a useful graft of transplantation and provide an ideal model system in which to study diseases of the skin.     

In vitro culture of sugarcane infected with maize streak virus (MSV)

Young buds and leaf tissues of sugarcane cv. R 574 (a Saccharum hybrid), healthy or infected with Maize Streak Virus (MSV) were cultured in vitro. The success rate of the cultures from infected material is lower than that from healthy material. The symptoms of the disease were displayed on most of the plantlets produced from buds but only on 6% of the plantlets regenerated from calli. The indirect double-antibody sandwich ELISA technique using a monoclonal antibody makes it possible to identify virus-infected plantlets. In vitro, the infection results in the reduced growth of shoots.     

In vitro plant regeneration via somatic embryogenesis from root culture of some rhizomatous irises

A method for plant regeneration of Iris via somatic embryogenesis is described. Root and leaf pieces from in vitro-grown plants of several genotypes of rhizomatous Iris sp. were cultured in vitro. Callus induction occurred only on root cultures incubated under low light intensity (35 mol m^-2 s^-1) on two induction media containing 2,4-D (4.5 or 22.5 M), NAA (5.4 M) and kinetin (0.5 M). Somatic embryos developed after transfer of callus onto four regeneration media containing 9 or 22 M BA, or 5 M kinetin and 2 M TIBA or 9 M BA and 4 M TIBA. Plantlets could be obtained from these somatic embryos. Genotypic differences were found both in callus induction and somatic embryo formation, with I. pseudacorus responding better than I. versicolor or I. setosa. Cytological analysis performed on root tips of 80 regenerated plants revealed that two of the I. pseudacorus regenerants were tetraploid.Abbreviations 2,4-D dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - BA 6-benzyladenine - TIBA 2,3,5-triiodobenzoic acid - IBA indolebutyric acid