p21(WAF1/CIP1), a cyclin-dependent kinase inhibitor and a critical regulator of cell cycle, is controlled transcriptionally by p53-dependent and -independent mechanisms and posttranslationally by the proteasome. We have identified WISp39, a tetratricopeptide repeat (TPR) protein that binds p21. WISp39 stabilizes newly synthesized p21 protein by preventing its proteasomal degradation. WISp39, p21, and hsp90 form a trimeric complex in vivo. The interaction of WISp39 with Hsp90 is abolished by point mutations within the C-terminal TPR domain of WISp39. Although this WISp39 TPR mutant binds p21 in vivo, it fails to stabilize p21. Our results suggest that WISp39 recruits Hsp90 to regulate p21 protein stability. WISp39 downregulation by siRNA prevents the accumulation of p21 and cell cycle arrest after ionizing radiation. The results demonstrate the importance of posttranslational stabilization of p21 protein by WISp39 in regulating cellular p21 activity.

In MCF-7 breast cancer cells, estradiol (E P ) and pure antiestrogen RU 58668 down-regulate the estrogen receptor (ER). Interestingly, the protein synthesis inhibitor cycloheximide (CHX) abrogated solely the effect of E P suggesting a selective difference in the degradation of the receptor induced by estrogenic and antiestrogenic stimulations. A panel of lysosome inhibitors (i.e. bafilomycin, chloroquine, NH R Cl, and monensin), calpain inhibitors (calpastatin and PD 150606) and proteasome inhibitors (lactacystin and proteasome inhibitor I) were tested to assess this hypothesis. Among all inhibitors tested, lactacystin and proteasome inhibitor I were the sole inhibitors to abrogate the elimination of the receptor induced by both E P and RU 58668; this selective effect was also recorded in cells prelabeled with [Q H]tamoxifen aziridine before exposure to these ligands. Hence, differential sensitivity to CHX seems to be linked to the different mechanisms which target proteins for proteasomemediated destruction. Moreover, the two tested proteasome inhibitors produced a slight increase of ER concentration in cells not exposed to any ligand, suggesting also the involvement of proteasome in receptor turnover.z 1999 Federation of European Biochemical Societies.

No abstract

Data from immunocytochemical assessment of estrogen receptor (ER) regulation in MCF‐7 cells under estrogenic and anti‐estrogenic stimulation were compared with those obtained by enzyme immunoassay (Abbott ER‐EIA). Similar trends were observed, although ER level variations were less marked when assessed immunocytochemically. We confirmed reports of ER disappearance in the presence of estrogens (Es; E2 and DES) and pure anti‐estrogens (AEs; RU 58,668 and ICI 164,384) as well as its increase with partial AEs (4‐OH‐TAM and RU 39,119). E2‐induced ER down‐regulation was partly blocked by actinomycin D (AMD), okadaic acid (OK) and cycloheximide (CHX) when assessed by these 2 methods. Down‐regulation by pure AEs was not impeded by CHX, indicating that they operate differently from Es (i.e., transformation of ER to a form sensitive to constitutive degradation activity). In situ pre‐labeling of the cells with [3H]TAZ indicated that all investigated ligands eliminate pre‐existing ER through binding to newly synthetized receptors, since [3H]TAZ co‐valently associates with ER; E2 and RU 58,668 were more effective than 4‐OH‐TAM in this regard. CHX blocked ER disappearance even in the presence of pure AEs, which is in contrast to the data established with cells not pre‐exposed to [3H]TAZ. Nuclear location of [3H]TAZ‐ER complexes may explain this discrepancy, since pure AE‐ER complexes were reported to be incapable of nuclear translocation. Int. J. Cancer 78:760–765, 1998. © 1998 Wiley‐Liss, Inc.