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Immunorecognition of the active form of the oestrogen receptor by using a monoclonal antibody.

ABSTRACT: In previous studies, two forms (alpha and beta) of the oestrogen receptor, with different immunological characteristics, were observed in the cytosol fraction of fetal guinea-pig uterus, by using a monoclonal antibody to the human oestrogen receptor (D547Sp gamma). Only the alpha form was recognized by the antibody, shifting its sedimentation coefficient in high-salt sucrose gradients. The present work investigated the effect of several factors (time, temperature, high salt concentrations and Na2MoO4) on the interconversion of these two forms. Only the beta form was observed when cytosol was incubated with oestradiol for only 2-3 h, but 20 h later, 40-60% of the total oestradiol-receptor complexes were found as the alpha form. The transformation from the beta to the alpha form was accelerated by temperature (25 degrees C, 15 min) and exposure to high salt concentrations (0.4 M-KCl). On the other hand, Na2MoO4 completely blocked the transformation induced by time and temperature, but had little effect on that induced by KCl. The appearance of the alpha form always correlated with an increase in receptor binding to nuclei and DNA-cellulose. Finally, it was found that the isolated beta form, recovered from the gradient, was transformed into the alpha form after overnight dialysis under reduced pressure. The present data suggest that the alpha form, which is recognized by the monoclonal antibody, is the activated form of the oestrogen receptor.

SUBMITTER: Giambiagi N

PROVIDER: S-EPMC1152603 | biostudies-other | 1985 Aug

REPOSITORIES: biostudies-other

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Project description:Patents of some therapeutic monoclonal antibodies (mAb) used for cancer treatment are ending soon, allowing for the entry of similar analogs (biosimilars) onto the market. To have a biosimilar approved by health agencies, the manufacturer must typically demonstrate functional and physicochemical similarity between the biosimilar and the approved reference product. Functional similarity is often validated with cell-based assays, but the information gained is limited. In contrast, RNA-seq methods enable a sensitive transcriptomic analysis, providing detailed information on pathways and cellular responses. Trastuzumab inhibits signaling of the cell-surface receptor HER2, which is overexpressed in approximately 30% of all breast cancer patients. Here, we compare the functional effect of the mAb Trastuzumab and a corresponding biosimilar. The BT-474 breast cancer cell line was treated with the reference product, Trastuzumab (Herceptin®), or a proposed biosimilar (ApoTras), and the cellular transcriptomes were analyzed by RNA-seq. Functional similarity was assessed using two statistical contrasts. One contrast evaluated the mechanism of action by comparing treated samples (Her and ApoTras, n = 19) vs. untreated controls (n = 10), which identified 2623 differentially expressed genes (DEG) at a minimum fold change of 1.25. The other contrast directly compared ApoTras (n = 9) vs. Her (n = 10) to determine differences in expression between treatments and identified 24 DEG using a 1.25 fold- change threshold. This low DEG number reveals a very high similarity of effect of both mAb treatments on the cancer cells. A gene set overrepresentation analysis of DEG for mAb treatments revealed mechanisms of action, which were consistent with known trastuzumab effects. Supporting the small number of changes between both treatments, a post-hoc power analysis for the between-treatment comparison estimated power of 0.88 to detect a gene expression fold-changes of 2.0. To summarize these data, we introduce an overall similarity index (SI) to quantify treatment similarity based on changes in gene expression. Using statistical contrasts with RNA-seq analysis provides a powerful tool for the comparison of biological effects of biosimilar and originator compounds and can be broadly used for functional comparisons of drug treatments.

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Immunorecognition of the active form of the oestrogen receptor by using a monoclonal antibody.

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