Article

High susceptibility of a human breast epithelial cell type with stem cell characteristics to telomerase activation and immortalization

Department of Pediatrics and Human Development, College of Human Medicine, Michigan State University, East Lansing 48824, USA.
Cancer Research (Impact Factor: 9.28). 01/2000; 59(24):6118-23.
Source: PubMed

ABSTRACT We have recently characterized two types of normal human breast epithelial cells (HBECs) from reduction mammoplasty. Type I cells express estrogen receptor, luminal epithelial cell markers, and stem cell characteristics (i.e., the ability to differentiate into other cell types and to form budding/ductal structures on Matrigel), whereas Type II cells show basal epithelial cell phenotypes. In this study, we have examined whether Type I HBECs are more susceptible to telomerase activation and immortalization after transfection with SV40 large T-antigen. The results show that both types of cells acquire extended life span [(EL); i.e., bypassing senescence] at a comparable frequency. However, they differ significantly in the ability to become immortal in continuous culture, ie., 11 of 11 Type I EL clones became immortal compared with 1 of 10 Type II EL clones. Both parental Type I and Type II cells as well as their transformed EL clones at early passages [approximately 30 cumulative population doubling level (cpdl)] showed a low level of telomerase activity as measured by the telomeric repeat amplification protocol assay. For all 11 of the Type I EL clones and the single Type II EL clone that became immortal, telomerase activities were invariably activated at middle passages (approximately 60 cpdl) or late passages (approximately 100 cpdl). For the four Type II EL clones randomly selected from the nine Type II clones that did not become immortal, the telomerase activities were found to be further diminished at mid-passage, before the end of the life span. Thus, normal HBECs do have a low level of telomerase activity, and Type I HBECs with stem cell characteristics are more susceptible to telomerase activation and immortalization, a basis on which they may be major target cells for breast carcinogenesis.

Download full-text

Full-text

Available from: James E Trosko, Jun 19, 2014
0 Followers
 · 
48 Views
 · 
12 Downloads
  • Source
    • "The differentiation of human breast cells obtained from outgrowth of organoids into squames is well described [48]. The ability of these basal cells to form “relatively large spherical structures with a central core of squamous metaplasia” on basement membrane has also been noted [49,50]. Squamous differentiation of cells isolated from reduction mammoplasty has more recently been reported [51,52]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background Normal, healthy human breast tissue from a variety of volunteer donors has become available for research thanks to the establishment of the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB). Multiple epithelial (K-HME) and stromal cells (K-HMS) were established from the donated tissue. Explant culture was utilized to isolate the cells from pieces of breast tissue. Selective media and trypsinization were employed to select either epithelial cells or stromal cells. The primary, non-transformed epithelial cells, the focus of this study, were characterized by immunohistochemistry, flow cytometry, and in vitro cell culture. Results All of the primary, non-transformed epithelial cells tested have the ability to differentiate in vitro into a variety of cell types when plated in or on biologic matrices. Cells identified include stratified squamous epithelial, osteoclasts, chondrocytes, adipocytes, neural progenitors/neurons, immature muscle and melanocytes. The cells also express markers of embryonic stem cells. Conclusions The cell culture conditions employed select an epithelial cell that is pluri/multipotent. The plasticity of the epithelial cells developed mimics that seen in metaplastic carcinoma of the breast (MCB), a subtype of triple negative breast cancer; and may provide clues to the origin of this particularly aggressive type of breast cancer. The KTB is a unique biorepository, and the normal breast epithelial cells isolated from donated tissue have significant potential as new research tools.
    BMC Cell Biology 06/2014; 15(1):20. DOI:10.1186/1471-2121-15-20 · 2.84 Impact Factor
  • Source
    • "Adult stem-like cells derived from human breast tissues have been isolated and characterized for stem cell properties23242527. Here we employed human breast epithelial stem-like cells for ORA to evaluate their NER activities, in comparison with those of breast epithelial non-stem cells derived from the same individuals. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nucleotide excision repair (NER) excises bulky DNA lesions induced by mutagens and carcinogens. The repair process includes recognition of DNA damage, excision of a short patch of nucleotides containing the damaged base, re-synthesis of a new DNA strand and ligation of the nicks to restore the sequence integrity. Mutation or aberrant transcription of NER genes reduces repair efficiency and results in the accumulation of mutations that is associated with the development of cancer. Here we present a rapid, sensitive and quantitative assay to measure NER activity in human cells, which we term the Oligonucleotide Retrieval Assay (ORA). We used oligonucleotide constructs containing the UV-damaged adduct, cyclobutane pyrimidine dimer (CPD), to transfect human cells, and retrieved the oligonucleotides for quantification of the repaired, CPD-free DNA by real-time quantitative PCR. We demonstrate that ORA can quantify the extent of NER in diverse cell types, including immortalized, primary and stem-like cells.
    Scientific Reports 05/2014; 4:4894. DOI:10.1038/srep04894 · 5.58 Impact Factor
  • Source
    • "Previously, we have reported the development of immortal (M13SV1), weakly tumorigenic (M13SV1R2) and highly tumorigenic (M13SV1R2N1) cell lines from a human breast epithelial cell type with stem cell characteristics after successive SV40 large T-antigen transfection, X-ray irradiation and ectopic expression of C-erbB2/neu oncogene [25,28]. These M13SV1R2 cells lost their tumorigenicity concomitant with the expression of two tumor suppressor genes, maspin and alpha-6 integrin, after culturing in a growth factor/hormone-deprived medium for >10 passages (referred to as R2d) [16]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cancer cells are believed to arise primarily from stem cells. CD44+/CD24(-) have been identified as markers for human breast cancer stem cells. Although, HER2 is a well known breast cancer oncogene, the mechanisms of action of this gene are not completely understood. Previously, we have derived immortal (M13SV1), weakly tumorigenic (M13SV1R2) and highly tumorigenic (M13SV1R2N1) cell lines from a breast epithelial cell type with stem cell phenotypes after successive SV40 large T-antigen transfection, X-ray irradiation and ectopic expression of HER2/C-erbB2/neu. Recently, we found that M13SV1R2 cells became non-tumorigenic after growing in a growth factor/hormone-deprived medium (R2d cells). In this study, we developed M13SV1R2N1 under the same growth factor/hormone-deprived condition (R2N1d cells). This provides an opportunity to analyze HER2 effect on gene expression associated with tumorigenesis by comparative study of R2d and R2N1d cells with homogeneous genetic background except HER2 expression. The results reveal distinct characters of R2N1d cells that can be ascribed to HER2: 1) development of fast-growing tumors; 2) high frequency of CD44+/CD24(-) cells (~50% for R2N1d vs. ~10% for R2d); 3) enhanced expression of COX-2, HDAC6 mediated, respectively, by MAPK and PI3K/Akt pathways, and many genes associated with inflammation, metastasis, and angiogenesis. Furthermore, HER2 expression can be down regulated in non-adhering R2N1d cells. These cells showed longer latent period and lower rate of tumor development compared with adhering cells. HER2 may induce breast cancer by increasing the frequency of tumor stem cells and upregulating the expression of COX-2 and HDAC6 that play pivotal roles in tumor progression.
    Molecular Cancer 11/2010; 9(1):288. DOI:10.1186/1476-4598-9-288 · 5.40 Impact Factor
Show more