Induction of antiestrogen resistance in human breast cancer cells by random insertional mutagenesis using defective retroviruses: identification of bcar-1, a common integration site.
Duration of response to antiestrogen therapy in metastatic breast cancer is limited due to the development of antiestrogen-resistant tumors. The mechanisms involved are not understood but could originate from (epi)genetic alterations within the tumor cells. We have applied in vitro random insertional mutagenesis with replication defective retroviruses to identify those genes playing a key role in development of antiestrogen resistance in human breast cancer cells. Eighty antiestrogen-resistant cell clones were isolated from 7 x 10(8) estrogen-dependent ZR-75-1 cells, mass-infected with defective retroviruses and subjected to 4-OH-tamoxifen selection. Integration site-specific DNA probes were made by inverse polymerase chain reaction techniques and used to search for common integration sites. Six cell clones were identified with retroviral genome integrations in the same orientation in a single locus, designated breast cancer antiestrogen resistance locus-1 (bcar-1). These bcar-1 cell clones had lost estrogen receptor expression and had become estrogen independent. Our results strongly suggest that alteration of the bcar-1 locus is responsible for development of antiestrogen resistance in human breast cancer cells in vitro. In addition, we have shown that in vitro insertional mutagenesis using defective retroviruses can be applied for gene tagging in human cells.
Dorssers
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Dr. Daniel den Hoed Cancer Center Department of Molecular Biology, Rotterdam, The Netherlands.
van Agthoven
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Dekker
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van Agthoven
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Kok
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D001345:Autoradiography; D001483:Base Sequence; D015139:Blotting, Southern; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology*; D002874:Chromosome Mapping; D015342:DNA Probes; D004251:DNA Transposable Elements / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D004351:Drug Resistance; D004965:Estrogen Antagonists / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics*; D013629:Tamoxifen / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D016662:Virus Integration / Q000235:genetics
D015342:DNA Probes; D004251:DNA Transposable Elements; D004273:DNA, Neoplasm; D004279:DNA, Viral; D004965:Estrogen Antagonists; D013629:Tamoxifen