蔗糖密度勾配遠心法によるヒト乳癌estrogen receptorおよびprogesterone receptorに関する研究

抄録

The steroid-receptor assays in advanced or recurrent human breast cancer have recently become important as a method for the predictions of a therapeutic response to endocrine therapy. Estrogen receptor (ER) and progesterone receptor (PgR) were measured by sucrose gradient centrifugation. Breast cancers (109), benign mammary tumors (22) and normal mammary tissues (10) were examined.
The tissue powders were homogenized with a motor-driven, teflon-coated glass homogenizer in a 2 ml buffer of 0.01 M Tris-HCl containing 0.0015 M EDTA pH 7.4 per gram of tissue. The homogenate was then centrifuged at 105,000g for 60 minutes to obtain the supernatant. The protein concentration of the supernatant had to be adjusted about 1.2%. One-half ml of the supernatant was incubated with 1 pM ³H-Estradiol (98.5 Ci/mmol) or 1 pM ³H-R5020 (70-80 Ci/mmol) at 0°-4°C for 3 hrs, and then the free steroids and nonbound protein were removed by dextran-coated charcoal. Two hundred fifty of the cytosol was layered on a 5% to 20% sucrose density gradient solution and centrifuged at 40,000 rpm for 18 hrs.
After centrifugation, fractions were collected in 40 test tubes at the rate of five drops per tube.
One ml of Tris-HCl buffer was added to each fraction, and the protein density at 278 nm was measured with a spectrophotometer. Ten ml of scintillator (Nonione-Toluene-PPOPOPOP) was added to each vial, and the radioactivity was counted with a liquid scintillation spectrometer.
The results obtained were as follows :
1) Specific binding of ER was observed at the 8S and one of PgR was in the 8S region.
2) About 45% of human breast cancers were ER (+), and about 20% were PgR (+). The positive rate of PgR was lower than that of ER. As for benign mammary tumors, one out of 10 fibroadenomas and one out of 3 giant fibroadenomas were ER (+), and all of the normal mammary tissues were ER (-).
3) There was no difference between premenopausal females and postmenopausal females in the positive rate of ER and PgR, and the percentage was not related to clinical stage or status of lymphnode metastasis. As for blood type, there was no difference in the positive rates of ER and PgR among A, B and 0 types. In the AB type, ER and PgR were negative in all the examined cases.
4) In the positive rate of ER, papillotubular carcinomas tended to be a little lower than the other histological types, while in the binding sites of ER, medullary tubular carcinomas were higher than scirrhous carcinomas. As for PgR, medullary tubular carcinomas tended to be higher than the others.
5) In 10 cases, the occurrence of ER in primary tumors and metastatic or recurrent lesions was almost identical, and their binding sites were at almost the same level.
6) In 39 cases which measured both ER and PgR, all 8 cases of PgR (+) showed ER (+), and there was a close relationship between ER and PgR.
7) The relationship between the occurrence of ER and the clinical response to endocrine therapy was examined in 21 cases. Six out of 12 cases of ER (+) (50%) and one out of 4 cases of ER (±) showed a response, but 5 cases of ER (-) showed no response.
8) Endocrine therapy was carried out in 11 cases out of the 39 cases in which both ER and PgR were measured. The cases of both ER (+) and PgR (+) seemed to respond better than those of ER (+) only.