Since the first two newsletters satisfied most of my needs to communicate how the cell cultures from my laboratory were derived and grown, this newsletter will serve as an exchange of information on studies being done with these cells, and provide some information characterizing the cell types growing in our culture systems.
Characterization of normal human mammary epithelial cells (HMEC) in culture:
The question of what HMEC cell types we have been growing in our different culture conditions has been lurking around the edges of all interpretations of data generated from these cell cultures. This issue is particularly significant in understanding studies done with the HMEC grown in the serum-free MCDB 170 medium since only a small subpopulation of the primary cell cultures survives "selection", maintaining the long term growth and contributing to the large pools of frozen cells at 5-8th passage which are used for most of our experiments. As was mentioned in the first newsletter, we have not found any indication of abnormal properties (i.e., properties associated with transformation) in these cells, but this still leaves open the question of the relationship of these cells to cell types found in vivo. In particular, normal breast epithelia is pseudostratified with a basal cell population adjacent to the basement membrane, covered by a polarized luminal cell population facing the lumen. Myoepithelial cells can be found in the basal cell layer, whereas secretion of milk components occurs in the luminal cell layer. Some of the studies briefly described below (and alluded to in the first newsletter) done in collaboration with Joyce Taylor and others at ICRF, and by Paul Yaswen, myself and others at LBL suggest that the HMEC grown in MCDB 170 initially exhibit a basal cell phenotype, and with age in vitro acquire some aspects of a luminal phenotype without losing expression of basal phenotype properties.
| passage | Keratin 8 | Keratin 18 | Keratin 19 | Keratin 14 | Keratin 5 | a-Smooth muscle actin |
|---|---|---|---|---|---|---|
| 1 | + (*2,*3) | - | - | +++ | +++ | ++ |
| 3 | ++ | ++ (*3) | - | + | +++ | + |
| 6 | + (*2,*3) | +++ (*3) | - | + to ++ | +++ | - |
| 13 | not done | +++ (*3) | - | ++ to +++ | not done | +- |
| Medium | Culture | Keratin 8 | Keratin 18 | Keratin 19 | Keratin 14 | PEM (*1) |
|---|---|---|---|---|---|---|
| MCDB 170 | RM184 Passage 2 | + (*2,*3) | - | - | + | - |
| MM | RM184 Passage 2; Open growth pattern | + (*2,*3) | - | - | + | - |
| MM | RM184 Passsage 2; Patchy, closed morphology | + | + | + (*2) | - | + |
Tables 1&2 shows some summary data from reference 7. Specifically, keratins 14/5 which in the mammary gland are normally found predominantly in basal cells (and in basal cells of stratified epithelium), are uniformly expressed by all the MCDB 170 cells at all passages. We have also looked at the RNA expression for keratin 5 and find it expressed in the normal HMEC at all passages examined. Keratins 8/18, which are expressed by mammary luminal cells in vivo (and in simple epithelial tissues), are weakly or not at all expressed in the growing pre-selection MCDB 170 cells*. Their expression increases in the post-selection cells, particularly those grown with a cAMP stimulator, so that cells near senescence are uniformly positive. Keratin 19 is predominantly found in a subpopulation of mammary luminal cells in vivo. It is also present in almost all breast tumor cells in vivo and in the tumor cell lines in vitro. We have been unable to detect expression of either the keratin 19 protein or keratin 19 mRNA in our MCDB 170 grown cells*. Thus, that particular luminal phenotype is absent. We are currently determining if changes in the cell culture conditions, or addition of specific factors to the medium, can induce expression of keratin 19. A subpopulation of cells grown in our MM medium do express keratins 19,8 and 18 and do not express keratin 14. Understanding the controls on keratin 19 expression is intriguing because of the possible relationship to malignant transformation. Our two immortally transformed cell lines, 184A1 and 184B5 both show decreased expression of keratins 5/14.
Polymorphic epithelial mucin/human milk fat globule antigens (HMFG)
These large glycoproteins on the cell surface are highly antigenic. A variety of poly- and monoclonal antibodies have been generated against these antigens. Specific monoclonal antibodies recognize specific epitopes, whose distribution (in a form recognizable by the antibody) may vary with the cells' state of differentiation or transformation. Monoclonal antibody HMFG-2, which recognizes tumor cells in vivo and in vitro, and to a lesser extent lactating and luminal cells in vivo, does not recognize our normal MCDB 170 HMEC. The immortally transformed line 184B5 is positive. Monoclonal antibody HMFG-1, which preferentially recognizes normal lactating cells in vivo and some resting luminal cells, does not recognize the pre-selection MCDB 170 cells. However, some expression is found in the post-selection cells, increasing with age in vitro. 184B5 is also recognized by HMFG-1.
Although vimentin has been described as the intermediate filament found in connective tissue cells, some epithelial cells in culture have been shown to express vimentin. Normal HMEC grown in MM have been previously shown to be vimentin positive. We have now seen that the MCDB 170 cells also express both the vimentin protein and mRNA at all passages examined. There are mixed reports about vimentin expession in human breast cells in vivo. Some studies have not detected it, whereas others report vimentin expression in some basal cells. Both 184A1 and 184B5 have little or no vimentin expression at the mRNA or protein level. A1N4-TH, the malignantly transformed derivative of 184A1, does express vimentin but B5KTu, the malignant derivative of 184B5 does not [6,12].
This actin is seen in mammary basal cells in vivo. It is found in the pre-selection MCDB 170 grown cells, and absent in the post-selection cells except at senescence.
* Primary cultures of HMEC derived from organoids placed in MCDB 170 medium show two main phenotypes. The top cell layer is keratin 8/18/19 and HMFG 1&2 positive. While this luminal-like cell population may show migration in culture, its does not appear to proliferate. Secondary passage cell populations show only the basal-like cell phenotype.
What can be deduced from this information?
(1) Breast cells in vivo are not known for their co-expression of keratins 5/14 with 8/18 (or 5/14 with the
HMFGs). That HMEC in culture do express all 4 of these keratins suggests that the culture conditions used
may alter the normal regulation of keratin expression. The most obvious difference between in vivo and in vitro
is the absence of the pseudostratified, polarized cellular orientation in vitro. The cultured cells are a monolayer
with impermeable plastic on one side, rather than a basement membrane. Such a situation may provide a
"neither fish-nor-fowl" environment where the cells express both basal and luminal properties. These abnormal
positional effects may also account for the high levels of expression of the normally basal associated
intermediate filaments keratins 5/14 and vimentin. We have tried to test this possibility by placing the normal
HMEC on reconstituted basement membrane material (EHS/Matrigel) in MCDB 170 culture [12]. Under
these conditions, the HMEC may form tubular and globular strucutres. Preliminary studies have indicated some
decrease in the level of keratin 5 mRNA and some increase in that of keratin 18; vimentin levels were
unchanged, and keratin 19 mRNA was not detected. Clearly, there is plenty of room for further optimization
of the culture conditions to make the cells behave more like they would in vivo.
(2) The above results are consistent with the interpretation that MCDB 170 supports the proliferation of
basal cells for the 2-3 passages until "selection", at which point most of these cells senesce and a cell type
expressing some properties of both basal and luminal cells is selected and continues to proliferate. Since our
post-selection cells express the luminal markers of keratins 8/18 and HMFG as well as keratins 5/14, and are
derived from a population that is 5/14, a-smooth muscle actin positive but 8/18, HMFG negative, it would seem
inappropriate to automatically assume that any keratin 5/14 expressing cells (in vivo or in vitro) are
myoepithelial. These data suggest that the human mammary gland may resemble the rat mammary gland, where
it has been postulated that a stem cell capable of developing into the myoepithelial and luminal epithelial
pathways is present in the basal cell layer [14].
1. Band, V., Zajchowski, D., Stenman, G., Morton, C.C., Kulesa, V., Connolly, J., and Sager, R., A newly established metastatic breast tumor cell line with integrated amplified copies of c-erbB-2 and double minute chromosomes. Genes, Chromosomes and Cancer 1:48-58, 1989.
2. Bates, S.E., Valverius, E., Ennis, B.W., Bronzert, D.A., Sheridan, J.P., Stampfer, M., Lippman, M.E., and Dickson, R.B., Expression of the TGFa/EGF Receptor Pathway in Normal Human Breast Epithelial Cells. Endocrin., in press, 1990.
3. Eldridge, S.R., Martens, T.W., Sattler, C.A., and Gould, M., Association of decreased intercellular communication with the immortal but not the tumorigenic phenotype in human mammary epithelial cells. Cancer Res. 49:4326-4331, 1989.
4. Hosobuchi, M., and Stampfer, M., Effects of Transforming Growth Factor b on Growth of Human Mammary Epithelial Cells in Culture, In Vitro 25:705-713, 1989.
5. Salomon, D.S., Kidwell, W.R., Kim, N., Ciardiello, F., Bates, S.E., Valverius, E.M., Lippman, M.E., Dickson, R.B., and Stampfer, M., Modulation by Estrogen and Growth Factors of Transforming Growth Factor-Alpha and Epidermal Growth Factor Receptor Expression in Normal and Malignant Human Mammary Epithelial Cells, Recent Results in Cancer Research 113:57-69, 1989.
6. Sommers, C.L., Walker-Jones, D., Heckford S.E., Worland, P., Valverius, E., Clark, R., McCormick, F., Stampfer, M., Abularach, S., and Gelmann, E., Vimentin Rather Than Keratin Expression in Some Hormone-independent Breast Cancer Cell Lines and in Oncogene-transformed Mammary Epithelial Cells. Cancer Res. 49:4258-4263, 1989.
7. Taylor-Papadimitriou, J., Stampfer, M., Bartek, J., Lewis, A., Boshell, M., Lane, E.B., and Leigh, I.M., Keratin Expression in Human Mammary Epithelial Cells Cultured from Normal and Malignant Tissue: Relation to in vivo Phenotypes and Influence of Medium. J. Cell Sci., in press 1989.
8. Valverius, E., Walker-Jones, D., Bates, S.E., Stampfer, M.R., Clark, R., McCormick, F., Dickson, R.B., and Lippman, M.E., Production of and Responsiveness to Transforming Growth Factor b in Normal and Oncogene Transformed Human Mammary Epithelial Cells. Cancer Res. 49:6269-6274, 1989.
9. Valverius, E., Bates, S.E., Stampfer, M., Clark, R., McCormick, F., Salomon, D.S., Lippman, M.E., and Dickson, R.B., Transforming Growth Factor Alpha and its Receptor in Human Mammary Epithelial Cells: Modulation of Epidermal Growth Factor Receptor Function with Oncogenic Transformation, Mol. Endocr. 3:203-214, 1989.
10. Walen, K.H., and Stampfer, M.R., Chromosome Analyses of Human Mammary Epithelial Cells (HMEC) at Stages of Chemically-induced Transformation Progression to Immortality, Cancer Genet. Cytogen., 37:249-261, 1989.
11. Walker-Jones, D., Valverius, E.M., Stampfer, M.R., Lippman, M.E., and Dickson, R.B., Transforming Growth Factor Beta (TGFb) Stimulates Epithelial Membrane Antigen Expression in Normal and Oncogene-Transformed Human Mammary Epithelial Cells, Cancer Res. 49:6407-6411, 1989.
12. Yaswen, P., Peehl, D., and Stampfer, M.R., Changes in Regulated mRNA Expression Associated with Immortalization and Oncogenic Transformation of Human Mammary Epithelial Cells, submitted for publication.
13. Zajchowski, D., Pauzie, N., Tager, A., Band, V., Stampfer, M., and Sager, R., Expression of Growth Factors and Oncogenes in Normal and Tumor-derived Human Mammary Epithelial Cells, Cancer Res., 48:7041-7047, 1988.
14. Dulbecco, R., Allen, W.R., Bologna, M., and Bowman, M., Marker evolution during the development of the rat mammary gland: stem cells identified by markers and the role of myoepithelial cells, Cancer Res. 46:2449-2456, 1986.
Sandra Eldridge, CIIT Currently, I am developing the unscheduled DNA synthesis (UDS) assay in human mammary epithelial cells for investigating tissue-specificity of chemically-induced DNA repair. I am looking at your 184B5 cell line to optimize experimental conditions and will eventually look at the potential of a wide variety of different classes of chemicals to induce DNA repair in secondary cultures of normal human mammary epithelial cells.
Betsey Porter, Olivia Periera-Smith, Baylor
The normal and immortally transformed HMEC were scored for reactivity with an antibody that specifically recognizes fibronectin (FN) secreted from senescent human fibroblasts. The antibodies recognize an amino acid sequence in FN that is present in FN from young cells as well as senescent, but is only expressed in senescent cells in culture. If cells are capable of division (young, quiescent, immortal) they interact with young FN in such a way that the sequence is not available to react with the MAbs. The MAbs appear to recognize the FN secreted from the senescent cells of the 184 epithelial strain specifically. However, the pattern of expression is different than in fibroblasts. The staining on fibroblasts is cell associated; the epithelial cells stain completely extracellular. The cells themselves are not stained, but the coverslip is dotted with brown stain. This brown stain can be anywhere from light and powdery to completely covered with dark brown stain. Human keratinocytes stain similar to fibroblasts in that they stain cell-associated, but endothelial cells stain the surface of the plate similar to the mammary epithelial cells.
Young and immortal lines secrete FN but do not react with the MAbs. TGFb appears to induce some staining in these dividing populations. The FN is secreted and remains entirely extracellular.
Clonetics Corp. (Jeffrey Janus, Technical Marketing) Clonetics is currently testing its mammary epithelial cell growth medium MEGM for future commercial release to the research community. MEGM is a fully supplemented serum free growth medium (fully supplemented MCDB 170 with minor modification). Basal, unsupplemented MCDB 170 will also be available. For more information, call Clonetics Technical Sales (see listings).