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 canc er 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 vit ro random insertional mutagenesis with replication defective retroviru ses to identify those genes playing a key role in development of antie strogen resistance in human breast cancer cells. Eighty antiestrogen-r esistant cell clones were isolated f rom 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 wer e made by inverse polymerase chain reaction techniques and used to sea rch for common integration sites. Six cell clones were identified with retroviral genome integrations in the same orientation in a single lo cus, designated breast cancer antiestrogen resistance locus-1 (bcar-1) . These bcar-1 cell clones had lost estrogen receptor expression and h ad become estrogen independent. Our results strongly suggest that alte ration of the bcar-1 locus is responsible for development of antiestro gen resistance in human breast cancer cells in vitro. In addition, we have shown that in vitro insertional mutagenesis using defective retro viruses can be applied for gene tagging in human cells.