pactamycin and Breast-Neoplasms

Previous studies suggest that post-transcriptional events play an important role in estrogen-induced loss of estrogen receptor expression. The present study shows that treatment of MCF-7 cells with estradiol resulted in a six-fold decrease in estrogen receptor mRNA half-life from 4 h in control cells to 40 min in estradiol treated cells. To determine the role of protein synthesis in the regulation of estrogen receptor mRNA stability, several translational inhibitors were utilized. Pactamycin and puromycin, which prevent ribosome association with mRNA, inhibited the effect of estradiol on receptor mRNA stability, whereas cycloheximide, which has no effect on ribosome association with mRNA, had no effect on estradiol regulation of estrogen receptor mRNA stability. In control cells, the total cellular content of estrogen receptor mRNA was associated with high molecular weight polyribosomes. Treatment with estradiol resulted in a 70% decrease in estrogen receptor mRNA associated with polyribosomes but had no effect on the polyribosome distribution of estrogen receptor mRNA. In an in vitro degradation assay, polyribosomes isolated from estradiol-treated cells degraded ER mRNA faster than polyribosomes isolated from control cells. The nuclease activity associated with the polysome fraction appeared to be Mg2+ independent and inhibited by RNasin. Freeze-thawing and heating at 90 degrees C for 10 min resulted in the loss of nuclease activity. These studies suggest that an estrogen-regulated nuclease activity associated with ribosomes alters the stability of estrogen receptor mRNA.

Topics: Breast Neoplasms; Centrifugation, Density Gradient; Cycloheximide; Drug Stability; Enzyme Inhibitors; Estradiol; Half-Life; Humans; Kinetics; Magnesium; Pactamycin; Placental Hormones; Protein Synthesis Inhibitors; Puromycin; Receptors, Estrogen; Ribonucleases; Ribosomes; RNA, Messenger; Tumor Cells, Cultured

The c-fms proto-oncogene encodes the receptor for a hematopoietic growth factor, CSF-1. Recently, the importance of c-fms and its ligand CSF-1 in malignancies of non-hematopoietic origin, such as breast, ovarian, endometrial, pulmonary, and trophoblastic cancers has been recognized. We have previously shown that glucocorticoids induce a large increase in c-fms mRNA and protein levels in breast carcinoma cell lines. In this report, we investigate the mechanism underlying such c-fms overexpression by dexamethasone. We show that dexamethasone treatment of two breast carcinoma cell lines (BT20-c-fms expressor, and SKBR3-co-expressor of both c-fms and CSF-1) does not increase the rate of c-fms gene transcription, suggesting a post-transcriptional mechanism of regulation of c-fms expression by dexamethasone. The effect of protein synthesis inhibition was studied to help determine whether there was a role for intermediary regulatory proteins in the regulation of c-fms expression. We find that several protein synthesis inhibitors interfere with dexamethasone induction of c-fms transcripts, suggesting the existence of regulatory proteins. These regulatory proteins do not appear to be constitutively expressed, as we show no effect of protein synthesis inhibition on c-fms transcript expression in resting BT20 cells. These findings suggest that the putative regulatory proteins are induced by dexamethasone. Furthermore, the addition of a protein synthesis inhibitor, pactamycin, to dexamethasone-treated BT20 cells results in a decrease in c-fms mRNA stability.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amidohydrolases; Aminopeptidases; Breast Neoplasms; Dexamethasone; Humans; Macrophage Colony-Stimulating Factor; Pactamycin; Proto-Oncogene Mas; RNA Processing, Post-Transcriptional; RNA, Messenger; Tumor Cells, Cultured