Growth and differentiation of progenitor/stem cells derived from the human mammary gland.

Estrogen is necessary for the full development of the mammary gland and it is also involved in breast cancer development. We set out to identify and characterise progenitor/stem cells in the human mammary gland and to explore the role of estrogen in their proliferation and differentiation. Three candidate stem cell populations were isolated: double positive (DP) cells co-expressed the luminal and myoepithelial markers, EMA and CALLA, respectively, whereas double negative (DN) cells did not express these cell surface markers; side population (SP) cells were characterised by their differential ability to efflux the dye Hoechst 33342. The ABC transporter, breast cancer resistance protein (BCRP) was more highly expressed in SP cells than in non-SP cells and a specific BCRP inhibitor, Ko143, reduced SP formation, suggesting that BCRP confers the SP phenotype in mammary epithelial cells, as has been demonstrated in other tissues. Interestingly, SP cells were double negative for the EMA and CALLA antigens and therefore represent a separate and distinct population to DP cells. Single cell multiplex RT-PCR indicated that the SP and DN cells do not express detectable levels of ERalpha or ERbeta, suggesting that estrogen is not involved in their proliferation. DP cells expressed ERalpha but at a lower level than differentiated luminal cells. These findings invoke a potential strategy for the breast stem/progenitor cells to ignore the mitogenic effects of estrogen. All three cell populations generated mixed colonies containing both luminal and myoepithelial cells from a single cell and therefore represent candidate multipotent stem cells. However, DN cells predominately generated luminal colonies and exhibited a much higher cloning efficiency than differentiated luminal cells. Further characterisation of these candidate progenitor/stem cells should contribute to a better understanding of normal mammary gland development and breast tumorigenesis.

[1]  S. Ho,et al.  Expression of estrogen receptor (ER)-alpha and ER-beta in normal and malignant prostatic epithelial cells: regulation by methylation and involvement in growth regulation. , 2000, Cancer research.

[2]  S. Shousha,et al.  Purified malignant mammary epithelial cells maintain hormone responsiveness in culture , 2003, British Journal of Cancer.

[3]  Elaine Fuchs,et al.  Stem Cells A New Lease on Life , 2000, Cell.

[4]  B. Gusterson,et al.  Identification of myoepithelial cells in human and rat breasts by anti-common acute lymphoblastic leukemia antigen antibody A12. , 1986, Journal of the National Cancer Institute.

[5]  R. Weigel,et al.  GATA‐3 is expressed in association with estrogen receptor in breast cancer , 1999, International journal of cancer.

[6]  J. Sloane,et al.  Immunodetectable cyclin D 1 is associated with oestrogen receptor but not Ki67 in normal, cancerous and precancerous breast lesions , 2001, British Journal of Cancer.

[7]  J. Gustafsson,et al.  Cloning of a novel receptor expressed in rat prostate and ovary. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[8]  R. McKay,et al.  A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. , 2002, Genes & development.

[9]  S. Morrison,et al.  Prospective identification of tumorigenic breast cancer cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[10]  P. Argos,et al.  Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A , 1986, Nature.

[11]  H. Nakauchi,et al.  The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype , 2001, Nature Medicine.

[12]  A. Howell,et al.  The effect of age and menstrual cycle upon proliferative activity of the normal human breast. , 1988, British Journal of Cancer.

[13]  W. Wahli,et al.  Steroid and nuclear receptors Villefranche‐sur‐Mer, France, May 25–27, 1999 , 1999, The EMBO journal.

[14]  J. Marx.  Mutant Stem Cells May Seed Cancer , 2003, Science.

[15]  J. Russo,et al.  Pattern of distribution of cells positive for estrogen receptor α and progesterone receptor in relation to proliferating cells in the mammary gland , 1999, Breast Cancer Research and Treatment.

[16]  G. Koretzky,et al.  CD45 and the immune response. , 1993, Journal of the American Society of Nephrology : JASN.

[17]  R C Coombes,et al.  Isolation of pure populations of epithelial and myoepithelial cells from the normal human mammary gland using immunomagnetic separation with Dynabeads. , 1995, Analytical biochemistry.

[18]  M. J. van de Vijver,et al.  Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues. , 2001, Cancer research.

[19]  J. Russo,et al.  Role of Hormones in Mammary Cancer Initiation and Progression , 2004, Journal of Mammary Gland Biology and Neoplasia.

[20]  Fiona M. Watt,et al.  Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression , 1993, Cell.

[21]  G. Dontu,et al.  In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. , 2003, Genes & development.

[22]  M. Bissell,et al.  Human mammary luminal epithelial cells contain progenitors to myoepithelial cells. , 1999, Developmental biology.

[23]  R. Coombes,et al.  Breast Estrogen and Progesterone Receptors in the Normal Female Updated Version , 2006 .

[24]  M. Dean,et al.  The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[25]  G. Smith,et al.  An entire functional mammary gland may comprise the progeny from a single cell. , 1998, Development.

[26]  J. Craft,et al.  Clonal origins of human breast cancer , 2001, The Journal of pathology.

[27]  J. Schellens,et al.  Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C. , 2002, Molecular cancer therapeutics.

[28]  G H Smith,et al.  Mammary epithelial stem cells , 2001, Microscopy research and technique.

[29]  A. S. Conner,et al.  Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo , 1996, The Journal of experimental medicine.

[30]  M. J. van de Vijver,et al.  Expression of the breast cancer resistance protein in breast cancer. , 2002, Clinical cancer research : an official journal of the American Association for Cancer Research.

[31]  S. Rafii,et al.  VEGFR-3 and CD133 identify a population of CD34+ lymphatic/vascular endothelial precursor cells. , 2003, Blood.

[32]  John T. Dimos,et al.  A Stem Cell Molecular Signature , 2002, Science.

[33]  I. Weissman,et al.  Stem cells, cancer, and cancer stem cells , 2001, Nature.

[34]  J. Dick,et al.  A newly discovered class of human hematopoietic cells with SCID-repopulating activity , 1998, Nature Medicine.

[35]  C. Eaves,et al.  Characterization of bipotent mammary epithelial progenitor cells in normal adult human breast tissue , 2001, Breast Cancer Research and Treatment.

[36]  B. Gusterson,et al.  Distribution of myoepithelial cells and basement membrane proteins in the normal breast and in benign and malignant breast diseases. , 1982, Cancer research.

[37]  E. Lane,et al.  Keratin expression in human mammary epithelial cells cultured from normal and malignant tissue: relation to in vivo phenotypes and influence of medium. , 1989, Journal of cell science.

[38]  S. Lakhani,et al.  Comparative Genomic Hybridization Analysis of Myoepithelial Carcinoma of the Breast , 2000, Laboratory Investigation.

[39]  I. Weissman,et al.  Direct isolation of human central nervous system stem cells. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[40]  C. Potten,et al.  The intestinal epithelial stem cell: the mucosal governor , 1997, International journal of experimental pathology.

[41]  J. Dick,et al.  Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell , 1997, Nature Medicine.

[42]  K. Schaefer,et al.  Monoclonality in normal epithelium and in hyperplastic and neoplastic lesions of the breast , 2001, The Journal of pathology.

[43]  J. Smith,et al.  Cell proliferation in the human mammary epithelium. Differential contribution by epithelial and myoepithelial cells. , 1986, The American journal of pathology.

[44]  A. Sapino,et al.  Specific demonstration of myoepithelial cells by anti-alpha smooth muscle actin antibody. , 1988, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[45]  James A. Clark,et al.  Induction of Mammary Gland Development in Estrogen Receptor-α Knockout Mice. , 2000, Endocrinology.

[46]  I. Weissman,et al.  AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[47]  A. S. Conner,et al.  Isolation and Functional Properties of Murine Hematopoietic Stem Cells that are Replicating In Vivo By Margaret A. Goodell, Katja Brose, Glenn Paradis,* , 1996 .

[48]  M. Alison.  Tissue‐based stem cells: ABC transporter proteins take centre stage , 2003, The Journal of pathology.

[49]  I. Weissman,et al.  Self-renewal, differentiation or death: regulation and manipulation of hematopoietic stem cell fate. , 1999, Molecular medicine today.

[50]  V. Quaranta,et al.  Immunohistochemical localization of integrins in the normal, hyperplastic, and neoplastic breast. Correlations with their functions as receptors and cell adhesion molecules. , 1991, The American journal of pathology.

[51]  F. Watt,et al.  Stem cell patterning and fate in human epidermis , 1995, Cell.

[52]  A. Howell,et al.  Dissociation between steroid receptor expression and cell proliferation in the human breast. , 1997, Cancer research.

[53]  C. Eaves,et al.  Phenotypic and functional characterization in vitro of a multipotent epithelial cell present in the normal adult human breast. , 1998, Differentiation; research in biological diversity.

[54]  Min Ye,et al.  Myeloid or lymphoid promiscuity as a critical step in hematopoietic lineage commitment. , 2002, Developmental cell.

[55]  Valerie Speirs,et al.  Coexpression of Estrogen Receptor α and β: Poor Prognostic Factors in Human Breast Cancer? , 1999 .

[56]  J. Smith,et al.  Growth factors and their receptors in neoplastic mammary glands. , 1995, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie.

[57]  T. Enver,et al.  Stromal expression of Jagged 1 promotes colony formation by fetal hematopoietic progenitor cells. , 1998, Blood.

[58]  E. Lane,et al.  Characterization in vitro of luminal and myoepithelial cells isolated from the human mammary gland by cell sorting. , 1991, Differentiation; research in biological diversity.

[59]  J. Kearney,et al.  AC133, a novel marker for human hematopoietic stem and progenitor cells. , 1997, Blood.

[60]  Mina J Bissell,et al.  Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties. , 2002, Genes & development.

[61]  A. Ashworth,et al.  Correction: Functional and molecular characterisation of mammary side population cells , 2002, Breast Cancer Research.

[62]  D. Melton,et al.  "Stemness": Transcriptional Profiling of Embryonic and Adult Stem Cells , 2002, Science.

[63]  M J Bissell,et al.  Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones. , 2000, Endocrine-related cancer.

[64]  M. Roussel,et al.  Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. , 1998, Genes & development.

[65]  P. Bøhler,et al.  Cytogenetic analysis shows that carcinosarcomas of the breast are of monoclonal origin , 1998, Genes, chromosomes & cancer.

[66]  G. Bannikov,et al.  Monoclonal antibody mapping of keratins 8 and 17 and of vimentin in normal human mammary gland, benign tumors, dysplasias and breast cancer , 1988, International journal of cancer.

[67]  M. Fortini,et al.  An activated Notch receptor blocks cell-fate commitment in the developing Drosophila eye , 1993, Nature.

[68]  R. Nagle,et al.  Differential diagnosis of benign epithelial proliferations and carcinomas of the breast using antibodies to cytokeratins. , 1988, Human pathology.

[69]  J. Schuetz,et al.  Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[70]  B. Torok-Storb,et al.  The ABCG2 transporter is an efficient Hoechst 33342 efflux pump and is preferentially expressed by immature human hematopoietic progenitors. , 2002, Blood.

[71]  F. Kiechle,et al.  Hoechst 33342-induced apoptosis is associated with decreased immunoreactive topoisomerase I and topoisomerase I-DNA complex formation. , 2001, Annals of clinical and laboratory science.

[72]  A. Ashworth,et al.  Functional and molecular characterisation of mammary side population cells , 2002, Breast Cancer Research.

[73]  V. Speirs,et al.  Distinct expression patterns of ERα and ERβ in normal human mammary gland , 2002 .

[74]  S. Noguchi,et al.  Clonal analysis of predominantly intraductal carcinoma and precancerous lesions of the breast by means of polymerase chain reaction. , 1994, Cancer research.

[75]  M. Greaves,et al.  Multilineage gene expression precedes commitment in the hemopoietic system. , 1997, Genes & development.