Rebecca C. Osborn

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.

Transforming c-Ki-ras mutation is a preneoplastic event in mouse mammary carcinogenesis induced in vitro by N-methyl-N-nitrosourea.

Mouse mammary epithelial cells can be transformed in primary cultures to preneoplastic and neoplastic states when treated with N-methyl-N-nitrosourea (MNU). Mammary carcinomas arising from MNU-induced hyperplastic alveolar nodules (a type of mouse mammary preneoplastic lesion) contained transforming c-Ki-ras genes when examined by the NIH 3T3 focus assay. Hybridization of allele-specific oligonucleotides to c-Ki-ras sequences amplified by the polymerase chain reaction demonstrated the presence of a specific G-35----A-35 point mutation in codon 12 in each of the NIH 3T3 foci as well as the mammary carcinomas. This mutation resulted in the substitution of the normal glycine with an aspartic acid. Furthermore, this mutation in the c-Ki-ras proto-oncogenes was also detected in 9 of 10 hyperplastic alveolar nodules. These results demonstrate that the specific c-Ki-ras mutation is a preneoplastic event in MNU-induced mouse mammary carcinogenesis.

CULTURED PROLIFERATING RAT MAMMARY EPITHELIAL CELLS

SummaryNormal epithelial cells from the rat mammary gland proliferated in culture when plated with lethally irradiated cells of the LA7 rat mammary tumor line. Proliferation of the normal rat cells occured as the LA7 cells slowly died from the radiation. By labeling the cultures with3H-thymidine it was determined that most of the proliferating rat cells were those adjacent to the LA7 feeder cells. The epithelial cells from the primary culture proliferated after subsequent passages if the cells were plated at each subculture with newly irradiated LA7 cells. If the cells were plated at a ratio of ∼1:8 rat:LA7 a confluent layer of normal rat cells covered the plastic substrate after 6 to 7 wk. The cells have so far been carried up through Passage 7, which amounted to ∼19 doublings in cell number, and still proliferate vigorously. The growth medium for this culture system was Dulbecco’s modified Eagle’s medium:Ham’s F12 1:1 supplemented with fetal bovine serum, insulin, and antibiotics. The presence in the cells of keratin, desmosomes, and cell junctions attested to their epithelial origin. The cultures were composed of cells with diploid or near diploid chromosome numbers. Samples of the cultured cells were implanted into the cleared fat pads of nude mice. Most of the implants from Passage 2 formed normal mammary ductal structures, but the incidence of outgrowths decreased significantly with later passages until no out-growths resulted from the implantation of cells from Passage 5. The one unusual, feeder-independent cell line that arose from a primary culture seemed to be immortal in culture, contained a hyperdiploid chromosome complement, and formed abnormal structures when implanted into cleared fat pads.

Metabolism of 7,12-dimethylbenz[a]anthracene by mouse mammary epithelial cells cultured serum-free inside collagen gels

Mammary epithelial cells from 3-4-month-old BALB/c virgin mice were cultured inside collagen gels in the following serum-free media: Dulbeccos Modified Eagles Medium/Hams F-12 (1:1) supplemented with (A) insulin, bovine serum albumin, epidermal growth factor; (B) insulin, bovine serum albumin, progesterone, prolactin; (C) insulin, bovine serum albumin, progesterone, prolactin, linoleic acid. Cell number increased with all media used. At day 7 of culture, [3H]dimethylbenz[a]anthracene (DMBA) was added to the cultures and its metabolism to water soluble and organic soluble compounds was determined. Mouse mammary epithelial cells were able to metabolize [3H]DMBA to water and organosoluble metabolites. By 72 h, 77-94% of the added DMBA had been metabolized by the epithelial cells in the three media to water and organosoluble metabolites in equivalent amounts. The distribution between water soluble and organosoluble metabolites was approximately equivalent. The high pressure liquid chromatography profiles of organosoluble fractions from the media indicated that the major products appeared to be the phenols, 2-,3-, or 4-hydroxydimethylbenz[a]anthracene, the hydroxymethyl derivatives, 7-methylbenz[a]anthracene and 7-hydroxymethylbenz[a]anthracene, trans-3,4-dihydro-3,4-dihydroxydimethylbenz[a]anthracene and one or two major fractions eluting just behind the marker cis-5,6-dihydro-5,6-dihydroxydimethylbenz[a]anthracene. The major fraction eluting just ahead of the cis-5,6-dihydro-5,6-dihydroxydimethylbenz[a]anthracene was most likely trans-8,9-dihydro 8,9-dihydroxydimethylbenz[a]anthracene. The profiles were similar for the cells cultured in all three serum-free media. The results demonstrate that mouse mammary epithelial cells cultured inside collagen gels with serum-free media can metabolize DMBA to putative carcinogenic forms.

Hormone-Dependent Activation of c-Ki-ras in N-Methyl-N-Nitrosourea (MNU)-Induced Mouse Mammary Tumors

Mouse mammary epithelial cells (MMEC) cultured in the presence of mammogenic hormones (progesterone and prolactin), treated with MNU, and transplanted to syngeneic mice developed a high frequency of hyperplastic alveolar nodules (HAN) and carcinomas. The majority of these transformants had activated c-Ki-ras with a specific point mutation in the twelfth codon (G35–A35). In contrast, MMEC cultured with EGF developed a low frequency of ductal hyperplasias. MMEC cultured with lithium developed a high frequency of HAN and carcinomas, but no c-Ki-ras has been detected. To determine whether these findings parallel mammary carcinogenesis in vivo, mice were pituitary isografted to increase their levels of progesterone and prolactin and injected with MNU. Greater than 90% of the treated mice developed mammary carcinomas and the majority of these cancers had an identical phenotype to those induced in vitro in the presence of progesterone and prolactin. The majority (75%) of the cancers induced in vivo also had the identical point mutation in the c-Ki-ras protooncogene to those found in the in vitro studies. These studies suggest that the hormonal milieu at the time of carcinogen exposure affects not only the incidence and phenotype of the mammary lesions, but also the molecular events associated with mammary carcinogenesis.