High‐density lipoprotein and extracellular matrix promotes growth and plating efficiency of normal human mammary epithelial cells in serum‐free medium

Synthesis of the Epstein-Barr virus (EBV) associated early antigen (EA) can be induced by a variety of agents in Raji cells, a latently EBV-infected Burkitt lymphoma line. We investigated the role of lipoproteins in this EA induction system. Cell growth was not affected by lipoprotein-deficiency, but EA induction by most combinations of the inducers TPA (tetradecanoyl-phorbol-acetate), IdU (iododeoxyuridine), n-BA (n-butyric acid), anti-IgM and EA inducing factor (EIF), was greatly reduced. Only the inducer combination TPA/n-BA was completely independent of the presence of lipoproteins, indicating a different induction pathway. Removing the lipid moieties of the culturing serum did not result in reduced EA induction. Thus, the lowered EA inducibility in lipoprotein-deficiency is due to the absence of the protein moiety (apolipoprotein). Addition of HDL or VLDL partially reconstituted the original EA inducibility, whereas LDL had no effect. Lipoproteins were particularly important during the first 4 hours of induction, the phase where inducers may act on cell membrane structures (e.g., receptors). But lipoproteins were also required throughout the incubation period, even in a late and inducer independent phase.

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“…This agrees with other regulative qualities of HDL, e.g. growth factor activities [6,24,29] or monocyte activation [19].…”

Section: Discussionsupporting

confidence: 68%

The role of lipoproteins in EBV early antigen induction in Raji cells

Simon

Melzner

Bültmann

1989

Archives of Virology

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“…At present, the mitogen-stimulating function of HDL is widely discussed in the literature. The stimulating effect of HDL was demonstrated for the proliferation of lymphocytes [25], epithelial [26], endothelial [27], smooth muscle [28], and tumor [29] cells. It is known that in the G 1 -period of mitotic cycle the mRNA and protein synthesis increases, in the S-period the mRNA, rRNA and protein synthesis increases, in the G 2 -period the mRNA, rRNA and protein synthesis proceeds and proteins-initiators are formed, in the M-period proteins-initiators are activated and DNA is reduplicated.…”

Section: Discussionmentioning

confidence: 99%

Tetrahydrocortisol–apolipoprotein A-I complex specifically interacts with eukaryotic DNA and GCC elements of genes

Панин¹,

Тузиков²,

Gimautdinova³

2003

The Journal of Steroid Biochemistry and Molecular Biology

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“…Yang et al (25) obtained growth in a defined medium of primary cultures suspended in a collagen gel, and Biran et al (26) obtained growth of primary and secondary cultures on an extracellular matrix from bovine corneal en- Proc. NatL.…”

Section: Discussionmentioning

confidence: 99%

Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract.

Hammond¹,

Ham²,

Stampfer³

1984

Proc. Natl. Acad. Sci. U.S.A.

388

264

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A serum-free medium with bovine pituitary extract as the only undefined supplement has been developed for long-term culture of human mammary epithelial cells. This medium supports serial subculture of normal cells for 10-20 passages (1:10 splits) without conditioning or special substrates, and it supports rapid clonal growth with plating efficiencies up to 35%. It consists of an optimized basal nutrient medium, MCDB 170, supplemented with insulin, hydrocortisone, epidermal growth factor, ethanolamine, phosphoethanolamine, and bovine pituitary extract. Replacement of pituitary extract with prostaglandin E1 and ovine prolactin yields a defined medium that supports rapid clonal growth and serial subculture for three or four passages. Cultures initiated in these media from normal reduction mammoplasty tissue remain diploid and maintain normal epithelial morphology, distributiop of cell-associated fibronectin, expression of keratin fibrils, and a low level of expression of milk fat globule antigen. Large cell populations can now be generated and stored frozen, permitting multiple experiments over a period of time with cells from a single donor. These media greatly extend the range of experiments that can be performed both conveniently and reproducibly with cultured normal and tumor-derived human mammary epithelial cells.Growth of human mammary epithelial cells (HMEC) in medium MM, which contains hormones, fetal bovine serum, and conditioning factors from human epithelial and myoepithelial cells, has been reported previously (1,2). This medium supports rapid monolayer growth of normal HMEC for three or four passages with 1:10 splits. However, good clonal growth in MM requires cholera toxin and a fibroblast feeder layer (3). Tumor-derived cells respond similarly, but their doubling potential is more variable.Although medium MM makes possible many types of experiments with cultured HMEC (4-7), its usefulness is limited by lack of definition and by early cellular senescence. Other culture systems described for HMEC are generally even more restrictive (reviewed in ref. 8).This report describes long-term growth of HMEC with pituitary extract as the only undefined supplement and shorter-term monolayer and clonal growth in a defined medium. These results were achieved by applying to HMEC methods previously used in similar studies with other cell types (9-11). MATERIALS AND METHODSMedia and Solutions. Medium MM was prepared as previously described (2), except that HS578 Bst conditioned medium and cholera toxin were omitted. Medium MCDB 170 (Table 1) is an optimized formulation for HMEC. Medium MCDB 202a differs from MCDB 170 in concentrations of four components: cysteine HCl, 2.0 x 10-4 M; glutamine, 1.0 X 10-3 M, sodium pyruvate, 5.0 x 10-4 M, and zinc sulfate, 1.0 x 10-7 M. MCDB 202a is a minor modification of previously published medium MCDB 202 (13). MCDB 170 and MCDB 202a were both prepared according to the protocol for MCDB 104 (12), with appropriate adjustment of concentrations. These media were prepared withou...

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High‐density lipoprotein and extracellular matrix promotes growth and plating efficiency of normal human mammary epithelial cells in serum‐free medium

Cited by 37 publications

References 23 publications

The role of lipoproteins in EBV early antigen induction in Raji cells

The role of lipoproteins in EBV early antigen induction in Raji cells

Tetrahydrocortisol–apolipoprotein A-I complex specifically interacts with eukaryotic DNA and GCC elements of genes

Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract.

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