Cloning and characterization of a 77-kDa oestrogen receptor isolated from a human breast cancer cell line

Abstract

Summary We have cloned and characterized a 77-kDa oestrogen receptor (ER) from an oestrogen-independent subclone of the MCF-7 human breast cancer cell line. This receptor contains an in-frame, tandem duplication of exons 6 and 7, located in the steroid-binding domain of the ER. This mutation has abrogated ligand binding, but not DNA binding, in this mutant ER. We previously described the partial structure of a unique oestrogen receptor (ER) that is expressed in an oestrogen-independent MCF-7:2A subclone of the breast cancer cell line MCF-7 (Pink JJ, Wu SQ, Wolf DM, Bilimoria MM, Jordan VC 1 996a, Nucleic Acids Res 24 962-969). Sequence analyses determined the molecular weight of this 80-kDa ER to be 77 kDa, and hereafter this protein will be designated as ER77. Examination of the entire coding sequence of the ER77 mRNA indicates that it contains a tandem duplication of exons 6 and 7. Using a coupled transcription/translation system, a 77-kDa ER, which corresponds to the protein observed in the MCF-7:2A cells, was expressed. The ER77 protein does not bind the ligands [3H] oestradiol or [3H]tamoxifen aziridine. In DNA binding gel shift assays, the in vitro synthesized ER77 binds to a consensus vitellogenin A2 oestrogenresponse element. In transient transfection experiments, the mutant ER, alone or in combination with the wild-type ER, does not induce expression of an oestrogen-responsive luciferase reporter construct. In fact, expression of the ER77 in the ER-positive T47D:A18 cell line inhibits E2-induced luciferase expression. Overexpression of wild-type ER in T47D:A1 8 cells leads to elevated constitutive expression of the luciferase reporter, which was inhibited by co-transfection with ERR7. These data suggest that the ER77 can interfere with normal ER activity and does not act as a constitutive activator of oestrogen-independent growth in MCF-7:2A cells. Consequently, the constitutive growth observed in MCF-7:2A cells is probably the result of other ER-mediated pathways.

Downregulation ofbeta-catenin and transdifferentiation of human osteoblasts to adipocytes under estrogen deficiency

“…Most interestingly, this study reveals expression of the 46-kDa and the 77-kDa ERa isoforms in the human hFOB 1.19 osteoblast cell line, but no expression of the 66-kDa isoform nor expression of the estrogen receptor-b (data not shown). Expression of the 46-kDa isoform in human osteoblasts has already been published, but so far the 77-kDa isoform has only been shown in MCF-7 breast cancer cells [22,23]. The 46-kDa isoform is an alternative splicing variant of the ERa gene product missing the activation domain AF-1, but still having the DNA-binding and ligand-binding domains and being able to dimerize after ligand binding [24].…”

Intergenically Spliced Chimeric RNAs in Cancer

“…There are two types of trans-splicing based on the source of primary RNA transcripts: intragenic trans-splicing and intergenic trans-splicing. Intragenic trans-splicing takes place between two copies of primary RNAs transcribed from the same genome locus, and can lead to exon-duplication and sense-antisense fusion [13, 36, 37] (Figure 2E). Intergenic trans-splicing takes place between two pre-mRNAs transcribed from different gene loci [10, 12, 38] (Figure 2F).…”

“…In humans, examples of intragenic trans-splicing, such as estrogen receptor alpha, have been reported [36, 37]. Four intragenically trans-spliced fusion RNAs were detected in human embryonic stem cells, one of which contributes to maintenance of pluripotency [13].…”

Chimeric RNAs as potential biomarkers for tumor diagnosis

“…Fusion RNAs that are generated from fusion genes can be also validated on genome levels by methods such as in situ hybridization. Alternatively, high sensitivity of RT-PCR has been proven useful for validation of many known chimeric RNAs (23,32). However RT-PCR has its own limitations and can sometimes introduce false results.…”

“…However, the exact mechanisms of this event for the majority of chimeric RNAs remain largely unconfirmed. In fact, in contrast to the popular and logical hypothesis that trans-splicing generates many chimeric RNAs, a recent paper by Li et al proposed a short transcriptional slippage model (32). The authors conducted a large-scale search for chimeric RNAs in yeast, fruit fly, mouse and human samples and identified 5 chimeric RNAs in yeast, 4,084 in fruit fly, 10,586 in mouse, and more than 30,000 putative chimeric RNAs from humans.…”