Maria-Grazia Catelli

Association of the heat shock protein HSP90 with steroid hormone receptors and tyrosine kinase oncogene products

Monospecific, polyclonal rabbit antibody raised against the 90-kd non-hormone binding component of molybdate-stabilized steroid hormone receptor specifically recognises the 90-kd molecular weight heat shock protein (hsp 90) in mink cell extracts. Partial proteolytic digestion experiments indicate that this protein is identical to the 90-kd phosphoprotein found in a highly stable complex with the protein products of at least three members of the tyrosine kinase family of oncogenes (src, fes, fgr).

Antiproliferative and apoptotic activities of tosylcyclonovobiocic acids as potent heat shock protein 90 inhibitors in human cancer cells.

We evaluated whether inhibition of heat shock protein 90 (hsp90) function by novobiocin derivatives could induce the degradation of signal transducers that drive cancer cell growth and thereby promote apoptosis. Removal of the noviose moiety in novobiocin and introduction of a tosyl substituent at C-4 or C-7 coumarin nucleus provided derivatives 4TCNA and 7TCNA which compared favourably with novobiocin in MCF-7 breast cancer cells. Here we extend the antiproliferative and apoptotic properties of these analogues to a panel of cancer cell lines. Destabilization of hsp90 client proteins Raf-1, HER2, and cdk4 suggests inhibition of hsp90 chaperoning function. In HT29 colon and IGROV1 ovarian cancer cells, the growth inhibiting effect of 4TCNA and 7TCNA was consistent with the stimulation of cell death as assessed by the processing and activation of caspase 9, 8, 7 and 3 and the subsequent cleavage of poly(ADP-ribose) polymerase (PARP). In Ishikawa endometrial adenocarcinoma cells, 4TCNA also promoted apoptosis and the processing of PARP. These derivatives impacting multiple pathways involved in the neoplastic process may represent promising drugs for cancer therapy.

Monohydroxytamoxifen: An antioestrogen with high affinity for the chick oviduct oestrogen receptor

Abstract The monohydroxylated derivative of tamoxifen (a non-steroidal triaryl ethylene antioestrogen) shows an apparent affinity (Ki = 0.2 nM) for the chick oviduct oestrogen receptor which is higher than that of oestradiol itself, and ∼ 10 times higher than that of tamoxifen. Administered in vivo with oestradiol benzoate, it inhibited the increase of tissue growth, progesterone receptor content, ornithine decarboxylase activity (ODC), and ovalbumin and conalbumin synthesis, and also inhibited the oestradiol induced increase of ODC in vitro . It did not display any oestrogenic effect by itself. We conclude that antioestrogenic action may be exhibited by a molecule with higher affinity binding to the oestrogen receptor than oestradiol itself. Metabolic studies demonstrated that the antioestrogenic action of tamoxifen is not due to its prior conversion to monohydroxytamoxifen.

Do Receptor-Associated Nuclear Proteins Explain Earliest Steps of Steroid Hormone Function?

The functional importance of hsp 90 interaction with steroid hormone receptors in hormone action has been previously proposed. This hypothesis, although not yet proven, is supported by new data obtained in our and other laboratories, whereas no conflicting experimental result has been presented. Our recent studies deal with results of hsp 90 cloning, normal and mutated receptor transfection experiments, effects of antihormone RU 486 and immunohistochemical data.

Estrogen-like effects of combined dexamethasone and tamoxifen in the chick oviduct.

The effects of dexamethasone alone on withdrawn chick oviduct weight, DNA, protein content and progesterone receptor concentration were barely detectable, whereas ovalbumin and conalbumin synthesis were increased. When dexamethasone and tamoxifen were combined, a marked increase of total proteins, including egg white proteins, DNA and wet weight occurred. Progesterone receptor also was increased. The most striking result was the stimulation of DNA polymerase-alpha activity by combined dexamethasone and tamoxifen, whereas either compound was completely ineffective.

Steroid Receptor Associated Proteins: Heat Shock Protein 90 and P59 Immunophilin

Nearly ten years ago, hoping to obtain a monoclonal antibody against “native” 8S chick progesterone receptor prepared in the now classical way in the presence of molybdate, we obtained an antibody which displaced progesterone binding in sucrose gradients (Joab et al, 1984). It also displaced estrogen, androgen and glucocorticosteroid binding, and we now know that it can also displace mineralocorticosteroid receptors (Rafestin-Oblin,1989). In low salt gradients, where the 8S complex was conserved, the peak of bound steroid was shifted by the BF4 antibody, but in high salt, the complex dissociates and the 4S peak was not shifted, indicating that BF4 interacted not with the hormone-binding receptor molecule, but with another protein, which was part of the 8S, but not of the 4S peak. We had discovered a new receptor-associated protein, which turned out to be an abundant, cytosol, non-steroid-binding protein of 90 kDa. We soon found out that it was a well known heat shock protein (hsp 90) (Catelli et al, 1985), which could interact with other intracellular proteins such as oncogenic protein kinases (reviewed in Brugge, 1986, Zimiecki et al, 1986). Chick hsp 90 was cloned using BF4 antibody and then sequenced. Among other features it has a negatively charged region, which was called “zone A” (which is predicted to contain two α helices with a proline containing loop in the middle, and where the negative charges could be aligned by the computer on the phosphate backbone of the B-DNA helix)(Binart et al, 1989). At about the same time, the structure of steroid receptors had been described (Hollenberg et al, 1985, Walter et al, 1985, Gronemeyer et al, 1987), and we hypothesized that zone A could be engaged in an ionic interaction with the DNA binding domain (DBD) of steroid receptors, and that somehow hsp 90 could cap the DBD and maintain the receptor in an inactive conformation (Baulieu, 1987).

Indirect participation of Hsp90 in the regulation of the cyclin E turnover.

Cyclin E is the Cdk2-regulatory subunit required for the initiation of DNA replication at the G1/S transition. It accumulates in late G1 phase and gets rapidly degraded by the ubiquitin/proteasome pathway during S phase. The degradation of cyclin E is a consequence of its phosphorylation and subsequent isomerization by the peptidyl-prolyl isomerase Pin1. We show that in the colon cancer cells HT-29 the inhibition of the chaperone function of Hsp90 by geldanamycin (GA) enhances the ubiquitinylation of cyclin E and triggers active degradation via the proteasome pathway. As Hsp90 forms multiprotein complexes with and regulates the function and cell contents of numerous signaling proteins, this observation suggests a direct interaction between Hsp90 and cyclin E. However, experiments using cell lysate fractionation did not reveal the presence of complexes containing both Hsp90 and cyclin E. Coupled transcription/translation experiments also failed to detect the formation of complexes between newly synthesized cyclin E and Hsp90. We conclude that Hsp90 can regulate the degradation of cellular proteins without binding to them, by an indirect mechanism. This conclusion postulates a new category of proteins that are affected by the inactivation of Hsp90. Our observations do not support the possible involvement of a PPIase in this indirect mechanism. Besides, we did not observe active geldanamycin-dependent degradation of cyclin E in the prostate cancer-derived cell line DU-145, indicating that the Hsp90-dependent stabilization of cyclin E requires specific regulatory mechanism which may be lost in certain types of cancer cells.

Are receptor-associated nuclear proteins associated with the earliest effects of steroid hormones?

The functional importance of the interaction of hsp90 with receptors for steroid hormones in the action of these hormones has been suggested. This hypothesis, although not yet proven, is supported by new data obtained in our laboratory and in those of others, whereas no conflicting experimental results have been presented. Our recent studies have dealt with the cloning of hsp90, transfection of normal and mutated receptors, the effects of the antihormone RU486 and immunohistochemistry.

Possible Functional Interaction Between Steroid Hormone Receptors and Heat Shock Protein Mr 90.000 (hsp 90)

In the native, heterooligomeric, non active 8S-receptor, hsp 90 caps the receptor DNA-binding site. Hormone (agonist) “transforms” the 8S-form, releasing hsp 90 and active receptor (4S-form) which can bind to DNA and trigger the hormonal response. An antihormone (antagonist) as RU 486 stabilizes the 8S-form, and thus decreases availability of the DNA-binding site of the receptor. Hsp 90 may be prototype of “antireceptors” which do not bind to DNA but interact with the DNA-binding site of regulatory proteins thus modulating their function.