Nuclease sensitivity of estradiol-charged estrogen receptor binding sites in nuclei isolated from normal and neoplastic rat mammary tissues.
The interaction of partially purified calf uterine estradiol-charged estrogen receptor ([3H]ER) with rat nuclei was studied in vitro. We previously observed a significantly greater number of [3H]ER binding sites (at saturation) in nuclei of R3230AC mammary tumors from intact vs ovariectomized (ovex) rats with no difference in the affinity of [3H]ER binding for these nuclei. We now report on the nuclease sensitivity of [3H]ER binding sites in nuclei from these tumors and from normal rat tissues. Digestion of tumor nuclei with deoxyribonuclease I (DNase I) prior to incubation with [3H]ER in vitro resulted in a progressive loss of [3H]ER binding capacity, which was not accompanied by alterations in the affinity of [3H]ER for the nuclei (Kd = 1-3 nM). A significantly lower concentration (P less than 0.005) of DNase I eliminated 50% of the [3H]ER binding sites in nuclei of tumors from intact hosts (8 unit.min/ml) compared to tumors from ovex hosts (22 unit.min/ml). These results indicate that DNA regions capable of binding ER are more susceptible to DNase I digestion in tumors from intact rats than those from ovex hosts, suggesting that the endogenous hormonal milieu is responsible, at least in part, for maintenance of nuclease-sensitive DNA conformations in this hormone-responsive mammary tumor. The amount of DNase I required to eliminate 50% of [3H]ER binding to nuclei from lactating mammary gland, liver, and kidney ranged from 14 to 56 unit.min/ml. Therefore, accessibility of [3H]ER binding sites to nuclease digestion in normal rat tissue is generally less than that of R3230AC tumors.
Klinge
C M
CM
Department of Biochemistry, University of Rochester Medical Center, NY 14642.
Bambara
R A
RA
Zain
S
S
Hilf
R
R
D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000032:analysis; D003850:Deoxyribonuclease I / Q000494:pharmacology*; D005260:Female; D066298:In Vitro Techniques; D008321:Mammary Glands, Animal / Q000187:drug effects / Q000378:metabolism* / Q000637:transplantation; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism*; D010052:Ovariectomy; D051381:Rats; D011916:Rats, Inbred F344; D011960:Receptors, Estrogen / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity
D011960:Receptors, Estrogen; D004247:DNA; D003850:Deoxyribonuclease I