Lipids covalently bound to keratins of mouse mammary epithelial cells.

Keratin polypeptides obtained from mouse mammary epithelial cells (MMEC) were found to be modified by covalent attachment of lipids. MMEC in primary culture were incubated in 3H-palmitate and treated with 1.5M KCl/1% Triton X-100 to obtain a cytoskeletal (CS) fraction containing primarily keratin and actin filaments. After exhaustive extraction to remove labeled lipids, the CS proteins were separated by gel electrophoresis, and the labeled 46 kD (K18) and 55 kD (K8) keratin polypeptides were excised, subjected to acid hydrolysis and the chloroform-soluble products were resolved on thin layer chromatography. For both keratins, covalently bound lipid included major peaks which co-chromatographed with fatty acid standards. Also, unlabeled lipid resolving with fatty acid standards was found covalently bound to both keratins. The results are discussed in terms of keratin-lipid-membrane interactions.

Covalent binding of lipids to cytoskeletal proteins of mouse mammary epithelial cells.

Cytoskeletal proteins obtained from mouse mammary epithelial cells (MMEC) were found to be modified by covalent attachment of lipids. Primary cultures of MMEC were incubated in the presence of 3H-palmitate for 4 h. A cytoskeletal (CS) fraction was prepared by treatment of the cells with 1.5M KCl and 1% Triton X-100. The residual material, consisting primarily of keratin and actin filaments was exhaustively (10-20 rounds, including sonications) extracted with chloroform/methanol to remove non-covalently bound labeled lipids. The CS protein was then acid-hydrolyzed and the chloroform-soluble products subjected to thin layer chromatography (TLC). Two-thirds of the covalently bound radiolabel appeared as a very hydrophobic peak on a TLC system optimized for separation of neutral lipids. Ten percent separated into 4-5 peaks on a polar lipid TLC system. A small amount of label was traced to fatty acid-like components. Autoradiography of two-dimensional gels indicated that all the CS proteins resolvable by Coomassie blue staining were also radiolabeled. The results are discussed in terms of CS-lipid-membrane interactions.

Comparative growth of normal and malignant mouse mammary epithelium cultured serum-free on a biomatrix from preadipocytes.

Growth of normal and malignant mouse mammary epithelial cells (MMEC) on a biomatrix of substrate-attached material from 3T3-L1 preadipocytes was evaluated to devise culture conditions that are suitable for transformation studies but do not involve embedding cells in a gel. The biomatrix was prepared as described by Levine and Stockdale, and serum-free medium contained bovine serum albumin, insulin, progesterone, prolactin, and linoleic acid. Each cell type produced a distinctive pattern of colony architecture in this culture system. Cells from virgin mice (vMMEC) usually formed elaborate, three-dimensional structures resembling ducts and alveoli; cells from pregnant mice (pMMEC) grew as flat monolayers; and tumor cells grew in multilayered clusters. Cell growth was monitored by an assay for succinate dehydrogenase. Similar growth rates were found through Day 8 in cultures of vMMEC and D2 carcinoma cells. Growth of vMMEC slowed thereafter, whereas tumor cells typically continued growing through Day 14 to 18. Increase in cell number during 18 days in culture was 3-, 7-, 9-, and 11-fold for cells from pregnant and virgin mice, BALB/cfC3H and D2 carcinomas, respectively. The percent cells in S phase on Day 2 of culture was 9% for pMMEC, 4 to 11% for BALB/cfC3H tumor cells, 20% for vMMEC, and 24% for D2 tumor cells. Thus, this culture system promotes extended growth of MMEC and offers several advantages over embedding cells in a collagen gel. It may therefore be applicable to in vitro transformation studies with MMEC.

Lipids noncovalently associated with keratins and other cytoskeletal proteins of mouse mammary epithelial cells in primary culture.

Lipids noncovalently associated with cytoskeletal (CS) proteins of mouse mammary epithelial cells (MMEC) grown in primary culture were analyzed. A CS fraction, prepared by subjecting MMEC to 1.5 M KCl and 1% Triton X-100 in phosphate buffered saline (pH 7.4), was extracted 4-6 times with chloroform/methanol. Thin-layer chromatography (TLC) indicated that in comparison to whole cell lipid extracts, CS lipids consisted mostly of neutral lipids, especially triacylglycerols and, possibly cholesteryl esters. TLC analysis of chloroform/methanol CS extracts prepared from MMEC that had been incubated 4 h in [3H]palmitate revealed similar results, with the majority of label appearing in triacylglycerols and other neutral lipids. By autoradiography of sodium dodecyl sulfate polyacrylamide gels, all of the major CS proteins appeared labelled. The major regions of autoradiographic density of the gel were excised, the protein solubilized, and the lipids extracted and subjected to TLC. Most of the radiolabel appeared at the origin and ion front and resolved as neutral lipids. In contrast, keratins of 54-55 kDa and 46 kDa appeared to be associated noncovalently with a higher ratio of polar lipids (possibly phospholipids) to nonpolar (neutral lipids). Very little radioactivity, mostly neutral lipid, was associated with actin. A previously unidentified CS component of 30 kDa had primarily noncovalently bound neutral lipid. The results are discussed in terms of the apparent interactions of keratin filaments with the plasma membrane, nuclear envelope and cytoplasmic organelles.