Progesterone receptor isoform identification and subcellular localization in endometrial cancer.
ABSTRACT These studies were undertaken to characterize the subcellular localization of the two major isoforms of progesterone receptors (PR), PRA and PRB, in endometrial cancer.
Immunohistochemistry, immunoprecipitation, and confocal microscopy were performed using Hec50co and KLE endometrial cancer cell models expressing PRA or PRB as a consequence of transduction. The location of PRB compared to PRA was determined, and antibodies were tested for specificity with respect to PR isoform recognition. Immunohistochemical analyses of PR expression and subcellular compartmentalization were also performed on 20 formalin-fixed endometrial cancer tumors.
Morphological and biochemical evaluations demonstrated that PRA is localized to the nucleus, even in the absence of progesterone. In contrast, a large proportion of PRB is cytoplasmic in the absence of ligand, but is rapidly translocated to the nucleus in the presence of progesterone. The differential distribution of PRA and PRB proved to be a hallmark of malignant and nonmalignant epithelia in 20 samples of archival endometrial tissue from women with the pre-operative diagnosis of endometrial cancer. All endometrial cancer specimens demonstrated cytoplasmic PRB in 50% or more of the cells, and five of the seven tumors that were moderately to poorly differentiated demonstrated no PRB staining in the nuclei. Nuclear PRB was significantly associated with increasing tumor differentiation (P = 0.031).
In the absence of ligand, PRA is nuclear and PRB is largely cytoplasmic. This suggests that PRA may exert ligand-independent nuclear effects, while PRB may have nongenomic cytoplasmic actions in endometrial cancer cells.
Article: In situ photolinked nuclear progesterone receptors of human breast cancer cells: subunit molecular weights after transformation and translocation.[show abstract] [hide abstract]
ABSTRACT: The subunit structure of mammalian cytoplasmic progesterone receptors (PR) has been difficult to study because these proteins are subject to in vitro proteolysis; the structure of nuclear PR is unknown. We have now developed an in situ photoaffinity labeling method for PR that permits study of their subunits with minimal in vitro incubations. The strategy is to use [3H]R5020, a synthetic photoactive progestin, and suitable incubation temperatures to place receptors into their precise intracellular sites in intact cells. The cells, still intact, are then irradiated with UV at 300 nm for 2 min. This irradiation efficiently (approximately 15%) yields covalently linked hormone-receptor complexes at any intracellular location. Cells are than rapidly ruptured, nuclei are separated, and receptors are extracted with salt and/or directly solubilized with detergents before the subunits are displayed on denaturing polyacrylamide gels. With this as well as with modified in vitro methods, we show here that untransformed human breast cancer PR have two dissimilar subunits (mol wt, 115,000 and 81,000) present in equimolar amounts. The same subunits, with apparently unmodified mass, can be demonstrated in nuclei after they have been translocated by progestin treatment. Therefore, PR transformation and acquisition of nuclear binding capacity does not require prior proteolytic processing of subunits or other major structural modifications that are detectable on single dimension gels.Endocrinology 01/1984; 113(6):2195-201. · 4.46 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: The nuclear localization of the progesterone receptor is mediated by two signal sequences: one is constitutive and lies in the hinge region (between the DNA and steroid binding domains), the other is hormone dependent and is localized in the second zinc finger of the DNA binding domain. The use of various inhibitors of energy synthesis in cells expressing permanently or transiently the wild-type receptor or a receptor mutated within the nuclear localization signals, demonstrated that the nuclear residency of the receptor reflects a dynamic situation: the receptor diffusing into the cytoplasm and being constantly and actively transported back into the nucleus. The existence of this nucleo-cytoplasmic shuttle mechanism was confirmed by receptor transfer from one nucleus to the other in heterokaryons. Preliminary evidence was obtained, using oestrogen receptor, that this phenomenon may be of general significance for steroid receptors.The EMBO Journal 01/1992; 10(12):3851-9. · 9.20 Impact Factor