Selection and Characterization of a Breast Cancer Cell Line Resistant to the Antiestrogen LY 117018

Abstract

We have selected and cloned a stable variant of the MCF-7 human breast cancer cell line (LY 2) that is resistant to LY 117018 (LY), a potent antiestrogen that inhibits cell growth at concentrations as low as 10(-10) M. The cell line was selected by increasing the concentration of LY in the growth medium in a stepwise manner from 10(-8) to 10(-6) M as the cells become resistant. LY2 has been cloned in soft agar and carried for over 50 passages with no change in resistance. Other antiestrogens, such as tamoxifen and 40-hydroxytamoxifen no longer inhibit cell proliferation of LY 2. The cell line is still responsive to estrogen in a cell proliferation assay, but contains somewhat less estrogen receptors than MCF-7. The cytosolic estrogen receptor sediments to a 4S position on high salt sucrose density gradient centrifugation and is completely shifted to a denser gradient region when the receptor is incubated with a monoclonal antiestrophilin. The nuclear estrogen receptor when covalently labeled with [3H]tamoxifen aziridine has the same mol wt (62,000) in both MCF-7 and LY2 cells, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a competitive binding assay, LY 117018 competes for [3H]estradiol binding to its cytosol receptor with the same Ki in both MCF-7 and LY2 cells. When the induction of estrogen-specific proteins was examined, no detectable progesterone receptor could be detected in either estrogen-induced or control LY2 cells, in contrast to MCF-7 cells. However, both 52,000- and 160,000-dalton proteins were estrogen inducible in the medium of LY2 and MCF-7 cells, as measured by labeling with [35S]methionine. The phenotypic stability of the antiestrogen resistance in LY2 cells coupled with the cross-resistance the antiestrogens of widely different structures make this cell line an ideal model system for the study of hormone resistance in human breast cancer. In addition, while the mechanism of resistance is currently not elucidated, the selective loss of estrogen-inducible functions in this cell line may provide powerful clues for future study.

NF-κB Signaling Is Required for XBP1 (Unspliced and Spliced)-Mediated Effects on Antiestrogen Responsiveness and Cell Fate Decisions in Breast Cancer

“…MCF-7 (ER␣ ϩ , estrogen dependent, and antiestrogen sensitive), MDA-MB-231 (ER␣ negative), and MDA-MB-231-ER␣ (ER␣ ϩ ) human breast cancer cells were maintained in improved minimal essential medium (IMEM) with 5% fetal bovine serum (FBS). MCF-7-derived LCC1 (ER␣ ϩ , estrogen independent, and antiestrogen sensitive) (23), LCC9 (ER␣ ϩ , estrogen independent, and TAM and fulvestrant resistant) (24), LY2 (ER␣ ϩ , estrogen independent, and LY117018, TAM, and fulvestrant resistant) (25,26), and MCF-7/RR (ER␣ ϩ , estrogen independent, and TAM and fulvestrant resistant) (27) cells were maintained in IMEM without phenol red and supplemented with 5% charcoal-stripped calf serum (CCS). COS-1 cells were maintained in Dulbecco's modified Eagle medium (DMEM) with 10% FBS.…”

Global Gene Repression by the Steroid Receptor Coactivator SRC-1 Promotes Oncogenesis

“…Cell culture, treatments, and transfections Endocrine-sensitive MCF7 cells were obtained from American Type Culture Collection and endocrine-resistant LY2 cells were a kind gift from R. Clarke, Georgetown University, Washington DC (15) and have been classified as luminal B (16). Cells were grown as previously described (17).…”

Ets-2 and p160 proteins collaborate to regulate c-Myc in endocrine resistant breast cancer

“…Cell culture and cell treatments Breast cancer cell lines-endocrine-sensitive MCF7 (ATCC, LGC Promochem, Teddington, Middlesex, UK), endocrine independent/endocrine sensitive, MCF-7 derived LCC1 cells (Brunner et al, 1993) and endocrine-resistant, MCF7-derived LY2 (Bronzert et al, 1985) (kind gifts from Robert Clarke, Georgetown University, Washington DC)-were maintained in phenol red-free Eagle's minimum essential medium with 2 mM L-glutamine and supplemented with charcoal dextran stripped fetal bovine serum, 10% (Invitrogen, Paisley, UK). Endocrine-insensitive SKBR3 (ATCC) were maintained in RPMI medium with 2 mM L-glutamine and supplemented with fetal bovine serum, 10% (Invitrogen).…”