Margot M. Ip

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. I. Regulation of proliferation by hormones and growth factors

SummaryA serum-free primary culture system has been developed which allows for three-dimensional growth and differentiation of normal rat mammary epithelial cells (RMECs) within an extracellular matrix preparation. RMECs were isolated from mamary glands of immature 50- to 60-d-old rats and the organoids embedded within a reconstituted basement membrane matrix prepared from the Engelbreth-Holm-Swarm sarcoma. Cells grown in a serum-free media consisting of phenol red-free Dulbeccos modified Eagles medium-F12 culture medium containing 10 μg/ml insulin, 1 μg/ml prolactin, 1 μg/ml progesterone, 1 μg/ml hydrocortisone, 10 ng/ml epidermal growth factor (EGF), 1 mg/ml fatty-acid-free bovine serum albumin (BSA), 5 μg/ml transferrin, and 5 μM ascorbic acid proliferated extensively (15- to 20-fold increase in cell number as quantitated using the MTT dye assay) over a 2- to 3-wk culture period and remained viable for months in culture. Several types of colonies were observed including the alveolarlike budding cluster which predominates at later times in culture, units with no or various degrees of ductal-like projections, stellate colonies, and two-and three-dimensional web units. Optimal proliferation required insulin, prolactin, progesterone, EGF, and bovine serum albumin. Hydrocortisone was not required for proliferation, but the colonies developing in its absence were morphologically altered, with a high frequency of colonies that formed an extensively branched network with many fine projections. Cell proliferation was also dependent on substratum, with significantly less growth and development occurring in RMECs grown within a type I collagen gel matrix compared to RMECs grown within the reconstituted basement membrane. In conjunction with other studies demonstrating extensive differentiation as well as proliferation, it is concluded that this model should prove to be an improtant tool to study the hormonal regulation of the growth and development of rat mammary cells.

Mammary organoids from immature virgin rats undergo ductal and alveolar morphogenesis when grown within a reconstituted basement membrane

We have recently described a primary culture system which allows for extensive proliferation and functional differentiation of immature mammary epithelial cells. Herein, these findings are extended to demonstrate that a distinct pattern of ductal and alveolar morphogenesis can be induced within the mammary organoids isolated from virgin female rats and cultured within an Engelbreth-Holm-Swarm sarcoma-derived reconstituted basement membrane under defined serum-free conditions. The lobular and multilobular organoids that emerged resemble the alveoli of the mammary gland in gross form, multicellular architecture, and cytologic and functional differentiation, while the ductal organoids expressed characteristics typical of mammary gland ducts in vivo. The epithelial cells within the alveolar- and duct-like organoids displayed the capability of secreting two morphologically distinct milk products, casein and lipid, into the luminal compartment. The expression of histiotypic morphogenesis and mammary-specific functional differentiation by the cultured mammary organoids proceeded in the absence of a morphologically distinct basal lamina. We illustrate that development highly reminiscent of that which naturally occurs in the mammary gland in vivo can be induced and supported in vitro under defined serum-free conditions. In addition, the methodologies are available to simultaneously monitor mammary organoid morphogenesis, growth, and functional differentiation. This system should serve as a unique model in which the regulation of branching morphogenesis, development, gene expression, and transformation can be examined.

Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions.

SummaryA serum-free primary culture system is described which allows normal rat mammary epithelial cells (RMECs) embedded within a reconstituted basement membrane to undergo extensive growth and functional differentiation as detected by synthesis and secretion of the milk products casein and lipid. RMECs isolated from mammary glands of immature virgin rats were seeded within an extracellular matrix preparation derived from the Engelbreth-Holm-Swarm sarcoma and cultured in a serum-free medium consisting of Dulbeccos modified Eagles medium-F12 containing insulin, prolactin, progesterone, hydrocortisone, epidermal growth factor, bovine serum albumin, transferrin, and ascorbic acid. Casein synthesis and secretion were documented at the electron microscopic level as well as by an enzyme-linked immunosorbent assay (ELISA) assay using a polyclonal antibody against total rat caseins. Numerous secretory vesicles with casein micelles were noted near the apical surface of the RMECs, and secreted casein was observed in the lumen. These ultrastructural data were confirmed by the ELISA assay which showed that microgram amounts of casein per well were synthesized by the RMECs and that the amount of casein increased with time in culture. Using immunoblot analysis it was demonstrated that the full complement of casein proteins was synthesized. In addition to casein protein, β-casein mRNA levels were shown to increase with time. Synthesized lipid was detected at both the light and electron microscopic levels. Phase contrast photomicrographs demonstrated extensive intracellular lipid accumulation within the ductal and lobuloalveolarlike colonies, and at the electron micrograph level, lipid droplets were predominantly localized near the apical surface of the RMECs. The lipid nature of these droplets was verified by oil red O staining. Results from this study demonstrate that RMECs from immature virgin rats proliferate extensively and rapidly develop the capacity to synthesize and secrete casein and lipid when grown within a reconstituted basement membrane under defined serum-free conditions. This unique system should thus serve as an excellent model in which the regulation of mammary development and gene expression can be investigated.

Regulation of rat natural killing: II. Inhibition of cytolysis and activation by inhibitors of lipoxygenase: Possible role of leukotrienes

Rat splenic natural killer (NK) cell activity against 51Cr-labeled YAC-1 or TMT-081 tumor cells can be augmented by culturing at 37 degrees C for 18 hr. Inhibitors of the lipoxygenase pathway of arachidonic acid metabolism, NDGA, alpha-phenanthroline, quercetin, ETYA, BW755C, esculetin, and timegadine, inhibited this NK activation and also inhibited NK cytotoxicity when added directly to the NK assay. However, there was a partial loss of sensitivity of activated NK cells to suppression by NDGA, BW755C, and esculetin. Indomethacin failed to reverse the inhibition of NK activation caused by NDGA. However, LTB4 and LTC4 (0.01 microgram/ml) were able to reverse the inhibitory effect of NDGA on NK activation. Furthermore, spleen cells cultured for 18 hr synthesized detectable amount of LTC4 in their supernatants. NDGA inhibited the LTC4 synthesis in a dose-dependent manner. These data therefore suggest that leukotrienes are responsible for NK activation, and lipoxygenase activity is essential for NK cytolytic activity.

Effect of dietary polyunsaturated fat and 7,12-dimethylbenz(a)-anthracene on rat splenic natural killer cells and prostaglandin E synthesis.

SummaryDietary polyunsaturated fat has been shown to stimulate mammary tumorigenesis induced in rats by 7,12-dimethylbenz(a)anthracene (DMBA). Studies were undertaken to investigate the effect of polyunsaturated fat and DMBA on splenic natural killer (NK) activity and prostaglandin E (PGE) synthesis. In a first experiment, splenic NK activity at 33, 55, 75, and 110 days of age was measured in Sprague-Dawley rats fed 0.5% low fat (LF), 5% normal fat (NF), or 20% high fat (HF) corn oil diets from 23 days of age. At 55 days of age, half of the rats from the 75 and 110 day age groups were given 5 mg DMBA. Ten days after the initiation of the diets splenic NK activity against YAC-1 lymphoma was decreased from 50% cytotoxicity in rats fed NF diet to 21% cytotoxicity in rats fed HF diet, but was not affected by LF feeding. No difference in NK activity was observed among the groups at the later time periods. DMBA had no effect on NK activity at 20 or 55 days after its administration. In a second experiment, where DMBA (15 mg/rat) was given to half of the rats at 50 days of age and NF or HF diets were started 3 days later, NK activity was 35% in rats fed NF diet and 21% in rats fed HF diet, 5 days after the diets were started. No difference in NK activity in rats fed either diet was observed at later time periods. DMBA decreased both NK activity and spleen cellularity transiently. In both experiments, PGE synthesis by spleen cells cultured for 18 h was not affected by dietary fat intake, but was slightly increased 3 days after DMBA administration. Results from these experiments suggest that the stimulation of DMBA-induced mammary tumorigenesis by polyunsaturated fat and by DMBA itself may possibly be mediated by a transient decrease in splenic NK cell activity.

Isolation and characterization of cortisol-sensitive and -resistant P1798 mouse lymphosarcoma cell lines

Three cell lines have been isolated and characterized from the P1798 mouse lymphosarcoma. One line, derived from a glucocorticoid-resistant tumor, was glucocorticoid-resistant in vitro. The other two cell lines, derived from glucocorticoid-sensitive and -resistant parental tumors, respectively, were shown to be glucocorticoid-sensitive in vitro. The glucocorticoid receptor from all three cell lines bound glucocorticoid with similar affinity and capacity. However, based on Sephacryl S-300 gel filtration, the glucocorticoid receptor from the resistant cell line was smaller than that of the two sensitive cell lines. Moreover, the glucocorticoid receptor from the resistant cell line accumulated to a greater extent in the nucleus. This resistant cell line thus resembles the nti variant of the S49 lymphoma cell line. All three cell lines were tumorigenic and metastatic when reimplanted into mice, contained the normal mouse diploid complement of 40 chromosomes and exhibited the same responsiveness to cortisol in vivo as they did in vitro. It is concluded that the ready passage of these cell lines in vitro or in vivo and the presence of the small receptor in the resistant line should make them excellent model systems for the study of glucocorticoid resistance.

Binding of the glucocorticoid receptor complex to the nucleosomal core in the P1798 mouse lymphosarcoma.

Binding of the glucocorticoid receptor complex to nucleosomes has been studied using the mouse P1798 lymphosarcoma. Cells were incubated with [3H]triamcinolone acetonide (TA), and nuclei prepared and digested with 3 different concentrations of micrococcal nuclease. After fractionation with EDTA and NaCl, it was observed that [3H]TA bound with similar specific radioactivity to mononucleosomes containing both core and linker DNA, of 183 +/- 5, and 168 +/- 4 base pair lengths, respectively, as well as to core size DNA, of 148 +/- 3 base pair length, suggesting that the glucocorticoid receptor bound to the core portion of the nucleosome. Steroid binding was found to be associated with regions of the nucleosome that were depleted in histone H1 and enriched in high mobility group (HMG) proteins 1 and 2; only negligible binding was noted in nucleosomes enriched in histone H1 and depleted in HMG proteins. In addition to binding to core nucleosomes, the glucocorticoid receptor complex was also shown to bind to a fraction sedimenting at 5-6 S on sucrose gradients characterized by subnucleosome and mononucleosome size DNA, as well as by core histones. While binding of the steroid receptor complex to linker regions of the nucleosome cannot be ruled out, this data would appear to present the first concrete evidence that glucocorticoid binding, at least in the P1798 lymphosarcoma, is to core nucleosomes. Some caution in interpretation of the results is indicated, however, on 2 points: (1) receptor redistribution during nuclease digestion cannot be ruled out; (2) only the binding of a small proportion of the steroid receptor complex may be physiologically relevant.

Regulation of rat natural killing—I. Inhibition of cytolysis and activation by protein synthesis inhibitors☆

Rat splenic natural killer (NK) cell activity against 51Cr-labeled YAC-1 or TMT-081 tumor cells can be augmented by culturing at 37 degrees C for 18 h. The protein synthesis inhibitors, cycloheximide and emetine, inhibited such NK activation and also inhibited NK lysis when added directly to the NK assay. Both drugs also inhibited conjugation of effector cells to target cells. The inhibitory effect of cycloheximide on both the NK lysis and NK activation was reversible while that of emetine was irreversible. Both agents were able to inhibit 3H-amino acid incorporation at the concentrations that inhibited NK activity and activation. Culture-activated NK cells were found to be less susceptible to inhibition by cycloheximide and emetine.