Identification of a distinct side population of cancer cells in the Cal-51 human breast carcinoma cell line

Abstract

"Side population" (SP) cells, which pump out the fluorescent dye H33342 via the ABCG2 transporter, define a putative stem/progenitor cell population in the mammary gland. Breast cancer SP cells recently isolated from the MCF-7 cell line possess similar properties and may represent stem cell-like cancer cells. This study extends SP cell analysis to a broad panel of human breast cancer cell lines and investigates the expression of differentiation-associated markers in isolated cancer SP cells. Expression of ABCG2 was determined in 16 breast cancer cell lines by quantitative RT-PCR, Western blotting and immunohistochemistry. Subsequently, all cell lines were screened for the presence of SP cells. Human breast cancer cell lines commonly express ABCG2. ABCG2-immunoreactivity was clearly restricted to rare cancer cells in several cell lines including Cal-51. Analysis of H33342-labeled Cal-51 cells revealed a small fraction of putative SP cells accounting for one percent of all cells. The genuine nature of Cal-51 SP cells was unambiguously verified by demonstrating a 30-fold increased ABCG2-expression in isolated Cal-51 SP cells. During in vitro expansion, Cal-51 SP cells generated heterologous non-SP (NSP) cells and ABCG2-expression declined dramatically. In contrast, NSP cells failed to sustain proliferation. Freshly isolated Cal-51 SP cells also exhibited increased expression of Muc1 and CALLA. Noteworthy, non-malignant mammary epithelial SP cells lack these differentiation markers, highlighting fundamental differences between non-malignant and breast cancer-derived SP cells. In summary, we established Cal-51 SP cells as a novel in vitro model to study differential gene expression in breast cancer-derived SP and NSP cells.

Comprehensive genetic and functional characterization of IPH‐926: a novel CDH1‐null tumour cell line from human lobular breast cancer

“…The human ductal breast carcinoma cell line KPL-1 and the HL-60, IM-9, and RAJI cell lines were obtained from DSMZ (Braunschweig, Germany). Additional cell lines and their culture conditions have been described previously [26]. For functional analyses, cells were incubated in medium containing the FGFR1 inhibitor SU5402 (Calbiochem, Darmstadt, Germany) [27].…”

“…Standard end-point PCR was performed with 1 µl of cDNA as template in a 25 µl reaction volume using Platinum Taq DNA polymerase (Invitrogen) according to the manufacturer's recommendations. Quantitative assessment of gene expression normalized to the housekeeping gene β-GUS was performed with Sybr Green I (Invitrogen) on an ABI Prism 7700 system as described previously [26]. PCR primers are given in the Supporting information, Supplementary Methods.…”

“…For the detection of protein expression, cell lines were lysed in RIPA buffer and 30 µg of total cellular proteins was separated by 12% SDS-PAGE and transferred to nitrocellulose membranes [26]. Membranes were probed with antibodies against the following proteins: KRT18, KRT19, ErbB2, ER, E-cadherin, β-catenin, p120-catenin, FGFR1, PARP, and β-actin.…”

“…Immunohistochemical staining of FFPE tissue and cell lines was performed with the same antibodies as those used for western blots and the tyramine amplification technique as previously described [26].…”

KAI1/CD82 is a novel target of estrogen receptor‐mediated gene repression and downregulated in primary human breast cancer

“…21 For analysis of ER-dependent modulation of gene expression, cells were treated with the ER-antagonist fulvestrant (Tocris, Bristol, UK) at 1 lM for 48 hr. 22,23 RNA extraction from cell lines and primary breast cancer tissue Total RNA was extracted from cell lines using Trizol reagent (Invitrogen, Karlsruhe, Germany) and treated with RQ1 Dnase I (Promega, Mannheim, Germany) according to the manufacturer's recommendations.…”