The method used in purification of nuclei from three variant cell lines of the Shionogi mouse mammary carcinoma alters their uptake of dihydrotestosterone.
Modifications of three isolation methods were used to purify nuclei from an androgen-dependent cell line (AD) and two androgen-independent cell lines (AI1 and AI2) of the Shionogi mouse mammary carcinoma. Yields of nuclei, contamination of the nuclei by whole cells, monitoring of cytoplasmic tags by phase-contrast microscopy, and biochemical analyses were used to compare the methods. Purification with the cationic detergent cetylpyridinium chloride (CPC) resulted in greater yields of nuclei than purification of nuclei using Triton N-101. Purification by glycerol loading followed by hypotonic shock, although resulting in somewhat less whole cell contamination of the nuclei, yielded fewer nuclei per gram wet weight starting tissue. Phase-contrast microscopy showed the relative absence of cytoplasmic tags when nuclei were prepared by either the Triton N-101 or CPC methods. However, the yield of protein per nucleus was less when nuclei were prepared using CPC. Androgen uptake by nuclei of three cell lines was markedly reduced in those nuclei prepared by the CPC method as compared with those prepared by the Triton N-101 method. In the case of the AD tumour cell line nuclei prepared by the CPC method, both the affinity of the nuclei for dihydrotestosterone and the number of uptake sites were reduced when compared with AD tumour cell line nuclei prepared by the modified Triton N-101 method.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D013196:Dihydrotestosterone / Q000378:metabolism*; D008297:Male; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism / Q000648:ultrastructure*; D051379:Mice; D013696:Temperature; D013997:Time Factors
D013196:Dihydrotestosterone