Cells obtained from N-nitrosomethylurea-induced rat mammary tumors were grown in vitro using the soft-agar clonogenic assay technique. Their growth was studied in regular media containing serum as well as in media lacking serum, but to which insulin was added. Deletion of serum from the media resulted in a mean decrease of 49% in the number of colonies formed in vitro in 13 of 18 tumors and was without effect in the remaining 5 tumors. The addition of either 17β-estradiol (10-8m) or ovine prolactin (OPRL, 100 ng/ml) to the defined media resulted in an increase in the number of colonies formed in 12 of 18 tumors. The mean numbers of colonies per Petri dish in 17β-estradiol- and OPRL-treated Petri dishes were 95 ± 5.4 (S.E.) and 92 ± 6.2% of the values seen in serum-containing media. Simultaneous addition of both hormones to the defined media resulted in a significant increase in the number of colonies formed which was greater than that seen when either hormone was added separately. Of four tumors where neither hormone influenced colony formation, the addition of both 17β-estradiol and OPRL resulted in an increase in the number of colonies formed in three tumors.

We conclude that the N-nitrosomethylurea-induced mammary tumors can be grown in soft agar using defined media and that their growth can be enhanced by either 17β-estradiol or OPRL. These hormones have a synergistic effect on the growth of some of these tumors in vitro. These data are consistent with the known in vivo effects of these hormones on the N-nitrosomethylurea-induced rat mammary tumors.

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Supported in part by a grant from the Cleveland Foundation.

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