Effects of reduced BRCA1 protein on in vivo tumor cell growth
Because the AS-4 clone showed a growth advantage in soft agar, a phenotype that may be correlated with the ability to form tumors in vivo, the BRCA1 antisense sub-clone AS-4 was evaluated for its ability to form subcutaneous tumors in ovariectomized athymic mice in the presence or absence of an estrogen pellet. Mice were injected subcutaneously with AS-4 cells in matrigel on one side of each mouse and NEO cells in matrigel on the other side. Of the mice injected, 50% received an implanted estrogen pellet (0.18 mg estrogen) that was designed to release estrogen for 60 days. In the absence of estrogen, a significant difference was detected in tumorigenic growth between AS-4 and NEO cells (Fig. 5b). Almost twice as many sites were tumor positive for the AS-4 clones than for NEO injected sites. 100% (14/14) Tumor formation was reached for all AS-4 clones 42 days after injection, compared with 57% (eight out of 14) positive tumor formation of the NEO sites (Fig. 5b, upper panel). AS-4 cells also formed tumors that averaged twice the size of NEO control tumors (Fig. 5b, lower panel).
BG-1 cells without matrigel were nontumorigenic in athymic male or female mice (0 positive sites/20 sites injected at 5×106 cells per site), but these cells formed large, progressively growing tumors when injected with matrigel in the presence of estrogen (Fig. 5a). These tumors were very large (>1cm diameter) and did not regress even though the estrogen pellet was effective for only 60 days (Fig. 5a, lower panel). Similar to the agar experiments, both the BRCA1 antisense clone and LXSN control clone were positive for tumor formation in the presence of estrogen. AS-4 cells formed tumors with half of the latency of control cells in the presence of implanted estrogen (Fig. 5a, upper panel; 11 days versus 21 days until tumor formation). Neither AS-4 nor NEO cells formed progressively growing tumors in the absence of estrogen, however. All tumors in the mice without estrogen pellets had started to regress by 71 days after injection. The observed tumor regression was not surprising, because matrigel is not stable for longer than 14 days in culture, and probably not in vivo either (personal communication; Collaborative Biomedical Products, Bedford, MA, USA). By day 71, the matrigel would no longer confer an optimal growth environment for BG-1 cells.