Differentiation and morphogenesis of mammary cells in vitro

Prinetti

Scotti

et al. 2004

We previously identified rat8 in the pathway involved in epithelial cell differentiation that occurs in the rat mammary gland at pregnancy when tubules and alveoli are formed. rat8, which encodes an IFN-inducible membrane protein, is the rat homologue of the mouse gene fragilis. By differential detergent extraction and isopycnic sucrose density gradients, we show that rat8 protein is associated to lipid membrane domains together with Lyn and Fyn, members of the Src tyrosine kinase family. We also show that recruitment of rat8 to lipid membrane domains is a necessary step in mammary epithelial cell differentiation. Immunoprecipitation analysis, performed with an anti-Fyn protein antibody, shows that rat8 was present in the Fyn immunoprecipitate. Antisense oligonucleotides, used to inhibit Fyn protein expression, block mammary cell differentiation. Taken together, these results suggest that the functional interaction, via lipid membrane domains, of rat8 and Fyn proteins is required for mammary cell differentiation. Therefore, rat8, like fragilis, may be involved in developmental decisions and the demarcation of a subset of cells in the mammary gland that cause epithelial cells to develop into a network of tubuloalveolar structures involved in secretion.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Association of rat8 with Fyn protein kinase via lipid rafts is required for rat mammary cell differentiationin vitro

Prinetti

Scotti

et al. 2004

“…For proteomic analysis, cells were cultured in triplicate, each of them derived from a different frozen culture. The LA7 cells, after having grown to confluence, were exposed to 1.5% DMSO as inducer of cell differentiation (13). Cells were harvested by scraping to avoid using trypsin to prevent extracellular protein digestion.…”

Section: Methodsmentioning

confidence: 99%

Proteomic dissection of dome formation in a mammary cell line: Role of tropomyosin-5b and maspin

Bini

Valaperta

et al. 2001

In this work we extended the study of genes controlling the formation of specific differentiation structures called ''domes'' formed by the rat mammary adenocarcinoma cell line LA7 under the influence of DMSO. We have reported previously that an interferon-inducible gene, rat-8, and the ␤-subunit of the epithelial sodium channel (ENaC) play a fundamental role in this process. Now, we used a proteomic approach to identify proteins differentially expressed either in DMSO-induced LA7 or in 106A10 cells. Two differentially expressed proteins were investigated. The first, tropomyosin-5b, strongly expressed in DMSO-induced LA7 cells, is needed for dome formation because its synthesis inhibition by the antisense RNA technology abolished domes. The second protein, maspin, strongly expressed in the uninduced 106A10 cell line, inhibits dome formation because 106A10 cells, transfected with rat8 cDNA (the function of which is required for the organization of these structures), acquired the ability to develop domes when cultured in presence of an antimaspin antibody. Dome formation in these cultures are accompanied by ENaC ␤-subunit expression in the absence of DMSO. Therefore, dome formation requires the expression of tropomyosin-5b, in addition to the ENaC ␤-subunit and the rat8 proteins, and is under the negative control of maspin.

“…The derived LA7 cell line spontaneously undergoes differentiation in vitro by forming domes, and this differentiation can be strongly promoted by differentiation inducers, such as DMSO. In contrast, the 106 cell line does not undergo this differentiation either spontaneously or under the action of inducers (14)(15)(16). The two cell lines also differ in their ability to produce tumors in nude mice, which is reduced for the LA7 cells (I.Z.…”

mentioning

confidence: 99%

“…WDMN1, originally identified as a gene whose expression is diminished in metastatic rat mammary adenocarcinomas, is a member of a family of protease inhibitors and encodes a secreted protein that may inhibit metastases by inhibiting proteases (19)(20)(21). WDNM1 and ␤-casein genes appear to be specific markers of mammary gland functional differentiation because in vivo the expression of these two genes occurs earlier in pregnancy (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), suggesting that their expression in LA7 cells may be indicative of a possible relationship between the biological processes resulting in dome formation in vitro and the processes resulting in the alveoli differentiation of the mammary gland at pregnancy.…”

mentioning

confidence: 99%

Dome formation in cell cultures as expression of an early stage of lactogenic differentiation of the mammary gland

Bini

Albani

et al. 2002

The study of the development of the mammary gland at the molecular level in the animal is difficult because of the complex tissue organization of the gland. We have previously developed an in vitro system for genetic analysis of mammary cell differentiation, based on the cell line LA7 clonally derived from a rat mammary adenocarcinoma. This cell line, after induction with DMSO, differentiates forming structures called domes. This process is under strict gene regulation, and we have previously identified several of the genes involved. In the present paper, we have defined the meaning of dome formation in relation to mammary development, by showing that treatment of LA7 cells with the lactogenic hormones hydrocortisone and prolactin induces dome formation; in the animal, these hormones precede and accompany milk production. Moreover, dome formation is accompanied by expression within the cells of the milk protein genes WDMN1 and ␤-casein, which are differentiation markers for the gland during pregnancy and lactation. We also show that two proteins, highly expressed in the mammary gland during lactation, HSP90-␤ and annexin I, are strongly expressed in DMSO-induced LA7 cells. Both proteins are essential in the formation of domes because when their synthesis is blocked by antisense RNA oligonucleotides, dome formation is abolished. Thus our in vitro system is a model for lobulo-alveolar development, and the genes identified in the pathway of dome formation are likely to be involved in the early differentiation steps occurring in the rat mammary gland during pregnancy and lactation.