In rat uterus and human breast cancer MCF-7 cell cytosol, the antiestrogens tamoxifen (Tam) and 4-hydroxytamoxifen (OH-Tam) bind to "antiestrogen binding sites" (ABS), which do not bind estradiol (E). Demonstrated in total cytosol by binding studies with radioactive antiestrogens in the presence of a large concentrationlof E, ABS can be physically separated from E-binding estrogen receptor (ER) by removing the latter with an E-containing bioaffinity adsorbent or with heparin-Sepharose gel. ABS concentration is 10-20% gland Nuclear; 17,21-dimethyl-19-nor-4,9-pregna-4,9-diene-3,20-dione) were used. Unlabeled steroids were from Steraloids and Roussel-Uclaf. Tam and OH-Tam were gifts from Imperial Chemical Industries, and CI 628 and nafoxidine-a-(4-pyrrolidinoethoxy)phenyl-4-methoxy-a-nitrostilbene] and 1-{2-[p-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]-ethyl}pyrrolidine-were gifts from Parke, Davis and Upjohn, respectively.[3H]Thymidine (28 Ci/mmol) was from Amersham; protease (bovine pancreas), a-chymotrypsin, and trypsin were from Worthington; DNase and RNase II were from Sigma, and heparin-Sepharose CL-6B was from Pharmacia. Rat Uterus. Sprague-Dawley rats (3 mo old) were used during the estrus period. After they were killed, their uteri were homogenized at 0-40C in 4 vol of 20 mM Tris HCl, pH 7.4/1 mM dithiothreitol (TD buffer). The homogenate was centrifuged at 800 x g for 15 min, and the supernatant was recentrifuged at 105,000 x g for 1 hr. The resulting supernatant was referred to as cytosol and contained 6-15 mg of protein per ml. The pellets obtained at 800 X g and 105,000 X g were washed twice with 1 ml of TD buffer and resuspended in 1-3 ml of the same buffer containing 20% calf serum stripped of endogenous hormones by a 30-min incubation at 370C with 5 mg of charcoal per ml.MCF-7 Cells. Gift of M. Rich (Michigan Cancer Foundation, Detroit), MCF-7 cells were grown in an incubator in humidified 5% C02/95% air at 370C in 75-cm2 Nunclon plastic flasks (Nunc) in RPMI 1640 medium supplemented with 2 g of sodium bicarbonate per liter, 2 mM glutamine (pH 7.4 at 230C), and 4% fetal calf serum stripped of endogenous hormones.Tam-Resistant Cells RTx6. Clone RTx6 was isolated from cells growing in the presence of 10 ,uM Tam for 15 days by us-
The growth-inhibitory protein p21WAF1/CIP1 is a potent inhibitor of various cyclin-dependent kinases, the expression of which is regulated at the transcriptional level by p53-dependent and -independent mechanisms. We examined p21WAF1/CIP1 mRNA and protein expression in 5 human ovarian-adenocarcinoma cell lines, 1 primary culture of normal surface epithelium and 17 human ovarian-tumor specimens. In culture cells, the p21WAF1/CIP1 protein was expressed in normal ovarian epithelial cells and at a high level in the adenocarcinoma 2008 and IGROV-1 cell lines. p21 WAF1/CIP1 expression was undetectable at the mRNA and protein levels in the NIH-OVCAR-3 and SKOV-3 ovarian-adenocarcinoma cell lines which are respectively mutated and deleted in the p53 gene. Heterogeneous expression of p21WAF1/CIP1 observed in ovarian-cancer cell lines in culture was also found in vivo on tumor specimens. p21WAF1/CIP1 expression is undetectable in 25% of the ovarian biopsies examined. Since it has been found that the p53 gene is mutated in 79% of ovarian cancer, the absence of p21WAF1/CIP1 expression in 25% of these ovarian cancer could not be correlated with p53 mutation. The proliferation index of the 17 tumors showed great variation from one tumor to another. However, no significant correlation was found between p21WAF1/CIP1 expression and the proliferation rate of the tumors.
Significantly increased numbers of ORO-PM were associated with various lung pathologies. However, the higher numbers observed in HIV patients require further investigations.
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