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1.
Proc Natl Acad Sci U S A ; 105(44): 16940-5, 2008 Nov 04.
Article in English | MEDLINE | ID: mdl-18957543

ABSTRACT

Tumors derived from rat LA7 cancer stem cells (CSCs) contain a hierarchy of cells with different capacities to generate self-renewing spheres and tubules serially ex vivo and to evoke tumors in vivo. We isolated two morphologically distinct cell types with distinct tumorigenic potential from LA7-evoked tumors: cells with polygonal morphology that are characterized by expression of p21/(WAF1) and p63 and display hallmarks of CSCs and elongated epithelial cells, which generate tumors with far less heterogeneity than LA7 CSCs. Serial transplantation of elongated epithelial cells results in progressive loss of tumorigenic potential; tumor heterogeneity; CD44, E-cadherin, and epithelial cytokeratin expression and increased alpha-smooth muscle actin I and vimentin expression. In contrast, serial transplantation of LA7 CSCs can be performed indefinitely and results in tumors that maintain their heterogeneity, consistent with self-renewal and multilineage differentiation potential. Collectively, our data show that polygonal cells are CSCs, whereas epithelial elongated cells are lineage-committed progenitors with tumorigenic potential, and suggest that tumor progenitors, although lacking indefinite self-renewal potential, nevertheless may make a substantial contribution to tumor development. Because LA7 cells can switch between conditions that favor maintenance of pure CSCs vs. differentiation into other tumor cell types, this cell system provides the opportunity to study factors that influence CSC self-renewal and differentiation. One factor, p63, was identified as a key gene regulating the transition between CSCs and early progenitor cells.


Subject(s)
Mammary Glands, Animal/cytology , Mammary Neoplasms, Experimental/pathology , Neoplastic Stem Cells/cytology , Animals , Cell Differentiation , Cell Line, Tumor , Cell Lineage , Clone Cells , Disease Models, Animal , Female , Immunohistochemistry , Mammary Glands, Animal/metabolism , Mice , Mice, SCID , Neoplasms/metabolism , Neoplasms/pathology , Neoplastic Stem Cells/metabolism , Rats , Stem Cells/cytology
2.
Proc Natl Acad Sci U S A ; 104(25): 10476-81, 2007 Jun 19.
Article in English | MEDLINE | ID: mdl-17566110

ABSTRACT

The cancer stem cell hypothesis posits that tumors are derived from a single cancer-initiating cell with stem cell properties. The task of identifying and characterizing a single cancer-initiating cell with stem cell properties has proven technically difficult because of the scarcity of the cancer stem cells in the tissue of origin and the lack of specific markers for cancer stem cells. Here we show that a single LA7 cell derived from rat mammary adenocarcinoma has the following properties: the differentiation potential to generate all of the cell lineages of the mammary gland; the ability to generate branched duct-like structures that recapitulate morphologically and functionally the ductal-alveolar-like architecture of the mammary tree; and the capacity to initiate heterogeneous tumors in nonobese diabetic-SCID mice. In addition, we show that cultured cells derived from tumors generated by a single LA7 cell-injection have properties similar to LA7 cells, can generate all of the cell lineages of the mammary gland, and recapitulate the ductal-alveolar-like architecture of the mammary tree. The properties of self-renewal, extensive capacity for proliferation, multilineage differentiation potential, and single-cell tumor-initiation potential suggest that LA7 cells are cancer stem cells and can be used as a model system to study the dynamics of tumor formation at the single-cell level.


Subject(s)
Cell Differentiation , Cell Proliferation , Neoplastic Stem Cells/pathology , Adenocarcinoma/pathology , Animals , Benzimidazoles/metabolism , Breast Neoplasms/pathology , Carbazoles/metabolism , Cell Line, Tumor , Cell Lineage , Cells, Cultured , Clone Cells , Female , Fluorescent Antibody Technique, Indirect , Fluorescent Dyes/metabolism , Immunohistochemistry , Keratin-14/metabolism , Keratin-18/metabolism , Mammary Glands, Animal/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Neoplastic Stem Cells/metabolism , Organ Culture Techniques , Rats , Stem Cell Transplantation , Transplantation, Heterologous
3.
Proc Natl Acad Sci U S A ; 101(52): 18147-52, 2004 Dec 28.
Article in English | MEDLINE | ID: mdl-15608061

ABSTRACT

Expression profiles of breast carcinomas are difficult to interpret when they are obtained from tissue in toto, which may contain a large proportion of non-cancer cells. To avoid this problem, we microscopically isolated cells from a primary invasive ductal carcinoma of the breast and from an axillary node harboring a metastatic breast carcinoma, to obtain pure populations of carcinoma cells ( approximately 500) and used them for serial analysis of gene expression. The expression profiles generated from both populations of cells were compared with the profile of a disease-free mammary epithelium. We showed that the expression profiles obtained are exclusive of carcinoma cells with no contribution of non-epithelial cells. From a total of 16,939 unique tags analyzed, we detected 559 statistically significant changes in gene expression; some of these genes have not been previously associated with breast cancer. We observed that many of the down-regulated genes are the same in both cancers, whereas the up-regulated genes are completely different, suggesting that the down-regulation of a set of genes may be the basic mechanism of cancer formation, while the up-regulation may characterize and possibly control the state of evolution of individual cancers. The results obtained may help in characterizing the neoplastic process of breast cancer.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Carcinoma/pathology , Epithelial Cells/metabolism , Gene Expression Regulation, Neoplastic , Lymphatic Metastasis , Breast/metabolism , Carcinoma/genetics , DNA, Complementary/metabolism , Down-Regulation , Epithelium/metabolism , Gene Library , Humans , In Situ Hybridization , Up-Regulation
4.
Proc Natl Acad Sci U S A ; 101(7): 1880-5, 2004 Feb 17.
Article in English | MEDLINE | ID: mdl-14766990

ABSTRACT

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.


Subject(s)
Cell Differentiation , Mammary Glands, Animal/cytology , Mammary Glands, Animal/metabolism , Membrane Microdomains/metabolism , Membrane Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Cell Line , Precipitin Tests , Protein Binding , Protein Transport , Proto-Oncogene Proteins c-fyn , Rats , src-Family Kinases/metabolism
6.
Proc Natl Acad Sci U S A ; 99(13): 8660-5, 2002 Jun 25.
Article in English | MEDLINE | ID: mdl-12077301

ABSTRACT

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 beta-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-beta 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.


Subject(s)
Cell Differentiation , Mammary Glands, Animal/cytology , Animals , Annexin A1/physiology , Base Sequence , Cell Differentiation/drug effects , Cell Line , DNA Primers , Dimethyl Sulfoxide/pharmacology , Electrophoresis, Gel, Two-Dimensional , HSP90 Heat-Shock Proteins/physiology , Hydrocortisone/pharmacology , Milk Proteins/genetics , Prolactin/pharmacology , Proteome , Rats , Reverse Transcriptase Polymerase Chain Reaction
7.
J Mammary Gland Biol Neoplasia ; 7(4): 373-84, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12882522

ABSTRACT

The study of the development of the mammary gland at the molecular level in animals is difficult because of the complex tissue organization. This review introduces a proteomic approach to investigate mammary gland development in a cell culture system that we have previously developed as an in vitro model for studying mammary cell differentiation. The model is based on two cell lines, one of which is able to differentiate spontaneously and produce hemispherical blisters, called domes, when confluent. Through proteomic dissection of dome-forming cells, two types of key regulatory genes have been identified: genes inducing cellular structural modifications and genes related to functional modifications. We identified several genes in the pathway leading to dome formation in vitro and showed that the functional and structural changes taking place in dome-forming cells correspond to cellular changes occurring in vivo when tubules and alveoli are developed in the mammary gland at pregnancy.


Subject(s)
Breast Neoplasms/metabolism , Breast/metabolism , Cell Culture Techniques/methods , Proteome , Animals , Breast/cytology , Cell Differentiation , Electrophoresis, Gel, Two-Dimensional , Humans , Mammary Glands, Animal/cytology , Mammary Glands, Animal/metabolism , Oligonucleotide Array Sequence Analysis , RNA, Messenger/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
8.
Proc Natl Acad Sci U S A ; 98(10): 5608-13, 2001 May 08.
Article in English | MEDLINE | ID: mdl-11331746

ABSTRACT

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 beta-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 beta-subunit expression in the absence of DMSO. Therefore, dome formation requires the expression of tropomyosin-5b, in addition to the ENaC beta-subunit and the rat8 proteins, and is under the negative control of maspin.


Subject(s)
Mammary Glands, Animal/metabolism , Proteins/physiology , Proteome , Serpins/physiology , Tropomyosin/physiology , Animals , Base Sequence , Blotting, Northern , DNA Primers , Epithelial Sodium Channels , Genes, Tumor Suppressor , Mammary Glands, Animal/cytology , Proteins/antagonists & inhibitors , Proteins/genetics , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Serpins/genetics , Sodium Channels/metabolism , Tropomyosin/genetics , Tumor Cells, Cultured
10.
Proc Natl Acad Sci U S A ; 96(24): 13766-70, 1999 Nov 23.
Article in English | MEDLINE | ID: mdl-10570147

ABSTRACT

In this work, we extend the study of the genes controlling the formation of domes in the rat mammary cell line LA7 under the influence of DMSO. The role of the rat8 gene has already been demonstrated. We have now studied two additional genes. The first, called 133, is the rat ortholog of the human epithelial membrane protein 3 (EMP3), a member of the peripheral myelin protein 22 (PMP22)/EMP/lens-specific membrane protein 20 (MP20) gene family that encodes for tetratransmembrane proteins; it is expressed in the LA7 line in the absence of DMSO but not in its presence. The second gene is the beta subunit of the amiloride-sensitive Na(+) channel. Studies with antisense oligonucleotides show that the formation of domes is under the control of all three genes: the expression of rat8 is required for both their formation and their persistence; the expression of the Na(+) channel beta subunit is required for their formation; and the expression of gene 133 blocks the expression of the Na(+) channel genes, thus preventing formation of the domes. The formation of these structures is also accompanied by the expression of alpha(6)beta(1) integrin, followed by that of E-cadherin and cytokeratin 8. It appears, therefore, that dome formation requires the activity of the Na(+) channel and the rat8-encoded protein and is under the negative control of gene 133. DMSO induces dome formation by blocking this control.


Subject(s)
Membrane Glycoproteins/genetics , Sodium Channels/genetics , Animals , Base Sequence , Biomarkers , Cadherins/genetics , Cell Line , Cloning, Molecular , Epithelial Sodium Channels , Gene Expression Regulation , Humans , Integrin alpha6beta1 , Integrins/biosynthesis , Keratins/genetics , Mammals , Molecular Sequence Data , Rats
12.
Proc Natl Acad Sci U S A ; 95(3): 1079-84, 1998 Feb 03.
Article in English | MEDLINE | ID: mdl-9448288

ABSTRACT

We have developed a model system for studying differentiation in the mammary gland, by using two clonal cultures deriving from a rat breast adenocarcinoma. They differ in the ability to form domes, structures the significance of which is unknown. By using the subtractive cDNA library approach, we isolated a cDNA that is highly expressed in the dome-forming cells, and identical to the rat8 gene and highly homologous to the human 9-27 gene. Antisense treatment of the dome-forming cells specifically and reproducibly abolishes dome formation, while forced expression of the gene in non-dome-forming cells causes morphological changes suggestive of "flat" domes. In situ hybridization on rat tissues shows that the gene is expressed in epithelia, especially in those forming tubular structures, suggesting a relatedness between these structures and domes. Cytokeratin 8 and E cadherin are strongly expressed in the domes but not outside them, suggesting that the rat8 gene triggers the cells to express molecules that tighten the lateral connections between the cells; the process is likely to parallel that occurring during the differentiation of the mammary gland.


Subject(s)
Mammary Glands, Animal/growth & development , Multigene Family , Animals , Biomarkers , Cadherins/analysis , Cell Differentiation/drug effects , Clone Cells , DNA, Complementary/chemistry , Databases, Factual , Female , Gene Library , Genes , Humans , In Situ Hybridization , Keratins/analysis , Mammary Glands, Animal/drug effects , Oligonucleotides, Antisense/pharmacology , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Rats , Tumor Cells, Cultured
15.
Nature ; 376(6537): 216, 1995 Jul 20.
Article in English | MEDLINE | ID: mdl-7617027
17.
Gene ; 135(1-2): 259-60, 1993 Dec 15.
Article in English | MEDLINE | ID: mdl-8276266
18.
Genomics ; 7(2): 294-7, 1990 Jun.
Article in English | MEDLINE | ID: mdl-2347595
19.
Int J Cancer Suppl ; 4: 6-9, 1989.
Article in English | MEDLINE | ID: mdl-2681012

ABSTRACT

I present a new hypothesis for cancer progression, based on observations with experimental tumors and on our growing understanding of the regulation of gene expression in mammalian cells. The experimental observations demonstrate that progression has a stochastic course and is associated with profound perturbations of cell differentiation. The hypothesis proposes that an initial event (such as the activation of an oncogene) alters the state of the regulatory network that controls the expression of cellular genes, directing it to evolve in a direction not consonant with the developmental program of the genome. Possible consequences of this epigenetic hypothesis for cancer research are discussed.


Subject(s)
Gene Expression Regulation, Neoplastic , Neoplasms/etiology , Animals , Humans , Models, Biological , Neoplasms/genetics , Neoplasms/pathology , Oncogenes
20.
Mol Cell Endocrinol ; 60(2-3): 177-87, 1988 Dec.
Article in English | MEDLINE | ID: mdl-3063568

ABSTRACT

A major problem in ovarian physiology is the lack of conveniently quantifiable markers of atresia. Towards this end, we identified a monoclonal antibody (anti-OA-2) that selectively recognizes granulosa cells in atretic follicles. When cryostat sections of rat ovaries were incubated with anti-OA-2, granulosa cells in atretic follicles showed intense immunofluorescent labeling. In contrast, no anti-OA-2 immunoreactivity was observed in the granulosa of the healthy follicles. The amount of anti-OA-2 binding was significantly enhanced when atresia was stimulated by treatment with human chorionic gonadotropin, testosterone, or estrogen withdrawal. The results of immunoprecipitation and Western blot analyses indicated that the OA-2 antigen is a 39 kDa protein which is actively synthesized by the granulosa during atresia. The 39 kDa protein is localized at or near the inner surface of the plasma membrane. We conclude that the anti-OA-2 monoclonal will prove useful as a convenient analytical tool to study the regulation of granulosa atresia.


Subject(s)
Antibodies, Monoclonal , Follicular Atresia , Follicular Phase , Granulosa Cells/analysis , Proteins/analysis , Animals , Antigens/analysis , Antigens/immunology , Blotting, Western , Chorionic Gonadotropin/pharmacology , Diethylstilbestrol/pharmacology , Female , Fluorescent Antibody Technique , Follicular Atresia/drug effects , Follicular Phase/drug effects , Histocytochemistry , Immunosorbent Techniques , Ovary/analysis , Proteins/immunology , Rats , Rats, Inbred Strains , Testosterone/pharmacology
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