Progesterone and estrogen receptors segregate into different cell subpopulations in the normal human breast.

Progesterone is critical in normal breast development and its synthetic derivatives are emerging as major drivers of breast cancer risk. The recent demonstration that progesterone regulates the stem cell compartment in the murine mammary gland, despite the absence of progesterone receptor (PR) in mammary stem cells, highlights the fact that PR distribution in progenitor cell subsets in the human breast remains to be conclusively shown. By utilising two independent cell sorting strategies to fractionate cells into distinct subpopulations enriched for different cell lineage characteristics, we have demonstrated a consistent enrichment of PR transcripts, relative to estrogen receptor transcripts, in the bipotent progenitor subfraction in the normal human breast. We have also shown co-expression of both steroid hormone receptors with basal markers in a subset of human breast cells, and finally we have demonstrated that PR+ bipotent progenitor cells are estrogen-insensitive, and that estrogen regulates PR in mature luminal cells only.

Progesterone receptor isoforms in normal and malignant breast.

Progesterone is an essential regulator of normal female reproductive function, yet recent studies on the use of progestins in hormone replacement therapy have clearly implicated progestins in breast cancer development, a disease initiated early in life at a time of frequent exposure to cycling ovarian hormones. The effects of progesterone are mediated by two distinct nuclear receptor proteins, PRA and PRB. In normal breast PRA and PRB are co-expressed at similar levels in luminal epithelial cells, suggesting that both proteins are required to mediate physiologically relevant progesterone signalling. However, early in breast carcinogenesis PRA:PRB expression is disrupted, resulting in frequent predominance of one isoform. Unbalanced expression of PRA and PRB results in altered hormonal response and aberrant targeting of genes that are not normally progestin-regulated, principally those involved in morphological changes and disruptions of the actin cytoskeleton, and in migration. Movement of PR into discrete nuclear domains, or foci, is a critical step in normal PR transcriptional activity that appears to be aberrant in cancers and likely related to alterations in nuclear morphology, gene expression and cell function associated with tumour cells. Given that exogenous progestins are consumed by millions of women worldwide in the form of hormone replacement therapy and oral contraceptives, it is vital to better understand the mechanisms of progesterone action in the breast.

Loss of co-ordinate expression of progesterone receptors A and B is an early event in breast carcinogenesis.

Progesterone receptor (PR) mediates the effects of progesterone in mammary tissues and plays a crucial role in normal breast development and in breast cancer. PR proteins are expressed as two isoforms, PRA and PRB, that have different capacities to activate target genes, yet it is unknown whether progesterone action in normal and malignant breast is mediated by PRA and/or PRB. This study determines the relative expression of PRA and PRB in normal breast and in benign, premalignant and malignant archival breast lesions by dual immunofluorescent histochemistry. In normal breast and in proliferative disease without atypia (PDWA) PRA and PRB were co-expressed within the same cells in comparable amounts, implicating both isoforms in progesterone action. In atypical lesions, however, there was a significant increase in predominant expression of PRA or PRB, with lesion progression from the normal state to malignancy. PR isoform predominance, especially PRA predominance, was evident in a high proportion of ductal carcinomas in situ (DCIS) and invasive breast lesions. In the normal breast and in PDWA, the relative expression of PRA and PRB in adjacent cells was homogenous. There was a significant increase in cell-to-cell heterogeneity of PR isoform expression in ADH and DCIS lesions and in the majority of breast cancers. Heterogeneous cell-to-cell expression of PR isoforms occurred prior to overall predominant expression of one isoform in premalignant breast lesions, demonstrating that loss of control of relative PRA:PRB expression is an early event in the development of breast cancer. PRA:PRB ratios within a breast lesion are likely to be important as both markers and effectors of tumor growth and development, and progressively aberrant PR isoform expression may play a role in the etiology of breast cancer.

Detection of progesterone receptor forms A and B by immunohistochemical analysis.

AIM: The measurement of progesterone receptors (PR) is recommended as part of the clinical management of breast and endometrial cancers, and immunohistochemistry on formalin fixed tissue is now the method of choice. PR is expressed as two isoforms, PRA and PRB, and although both these proteins are expressed in hormone dependent cancers, there is evidence that a large proportion of tumours express a predominance of one isoform. Therefore, it is essential to document the individual detection of PRA and PRB by the presently available anti-PR antibodies. The aim of this study is to investigate the detection of PR isoforms A and B in formalin fixed, paraffin wax embedded cell lines and tissue sections by immunohistochemistry, using a panel of commercial and in house antibodies to human PR. METHODS: PR negative cell lines stably transfected to express only PRA (MCF-7Mll/PRA) or PRB (MDA-MB-231/PRB), and tissue sections of human breast carcinoma and normal endometrium were stained using an immunoperoxidase method. A panel of primary PR specific antibodies was evaluated for ability to detect both PRA and PRB proteins, and for intensity and distribution of positive staining under optimal conditions. RESULTS: Of the 11 antibodies assessed, only four recognised PRA and PRB similarly. Six recognised PRA proteins but were unable to detect PRB expression in the cell lines expressing only PRA or PRB. In tissues expressing high amounts of PRA and PRB, all antibodies tested demonstrated positive PR staining. However, in tissues expressing a predominance of PRB, differential staining patterns were observed, with variations in staining intensity and in the proportion of cells positive for PR. CONCLUSIONS: Most PR specific antibodies tested failed to detect PRB in formalin fixed tissue by immunohistochemical techniques, despite their ability to do so by immunoblot analysis. These observations suggest that there are conformational differences between PRA and PRB that mask epitopes on the PRB protein recognised by most anti-PR antibodies. The selection of antibodies that recognise both PRB and PRA in formalin fixed tissue is essential for the accurate evaluation of PR positivity in clinical specimens.

Relative expression of progesterone receptors A and B in endometrioid cancers of the endometrium.

The nuclear receptor for the female hormone progesterone (PR) is widely expressed in uterine cancer. PR is expressed as two proteins (PRA and PRB) with different functions, and in vitro evidence reveals PRA to inhibit PRB function, so the cellular ratio of PRA:PRB is likely to be an important determinant of progesterone action. The relative expression of PRA and B and their involvement in the pathogenesis of endometrial cancer is not known. The aims of this study were to determine PRA and B expression by dual immunofluorescent histochemistry in endometrial adenocarcinomas compared with expression in normal and hyperplastic glands, and to correlate expression in tumors with clinical features including grade. Significantly lower PR levels were found in tumors compared with normal glands and areas of complex atypical hyperplasia within the same specimen. The normal glands expressed both of the isoforms at similar levels, whereas there was increased predominance of one isoform in hyperplastic areas and in tumors, which suggested that the loss of coordinated expression of PR isoforms was an early event in tumor progression. The majority of tumors [27 (58%) of 46] expressed only one PR isoform, and the proportion expressing either PRA or B was the same [14 (30%) of 46, and 13 (28%) of 46, respectively]. One-half of all tumors ([23 (50%) of 46] expressed either PRA only or a predominance of PRA, and a few tumors [10 (22%) of 46] expressed comparable levels of PRA and B. Similar levels of PRA and B were noted only in FIGO grade 1 tumors, whereas higher grades (2 and 3) were associated with a predominance of one isoform. In summary, expression of only one PR isoform was common in endometrial cancers, which indicates that the decreased PR levels observed in these cancers arise from the loss of one PR isoform. Expression of a single PR isoform was associated with higher clinical grade, which suggests a relationship between the loss of PR isoform expression and features of poorer prognosis. Disruption of relative PR isoform expression was observed in complex atypical hyperplasia, which suggests that early alterations in the ratio of PRA:PRB may precede and/or be implicated in the development of endometrial adenocarcinoma. Alterations in the ratio of PR isoform expression are likely to cause disordered regulation of target genes, resulting in altered progestin action in the uterus, and this may be involved in the pathogenesis of endometrial cancer.

Heterogeneity of progesterone receptors A and B expression in human endometrial glands and stroma.

The human progesterone receptor (PR) is expressed as two isoforms, PRA and PRB, which function as ligand-activated transcription factors. In-vitro studies suggest that the isoforms differ functionally and that their relative expression in a target cell may determine the nature and magnitude of response to progesterone. We have shown recently that PRA and PRB are co-expressed in target cells of the human endometrium. The purpose of this study was to investigate the homogeneity of expression of PRA and PRB in target cells of the human uterus throughout the menstrual cycle. In the functionalis, PRA and PRB were expressed in comparable levels in glandular epithelium during the proliferative phase of the cycle, whereas there was persistence of PRB but not PRA in the glands during mid-secretory phase. In the stroma, there was predominance of the PRA isoform throughout the cycle. There was remarkable homogeneity in the relative expression of PRA and PRB in adjacent cells within the same tissue compartment, suggesting that the mechanisms regulating relative PR isoform expression are similarly active in these cells. By contrast, heterogeneity between glands was observed under some circumstances in the functionalis of the endometrium, suggesting PR isoform down-regulation by progesterone to be asynchronous. Heterogeneity was also seen between the glands of the basalis and functionalis of the endometrium implying region-specific responses to hormonal stimuli. This study demonstrates adjacent cell homogeneity in the relative expression of PRA and PRB in normal human endometrial tissue and a differential response to ovarian steroid hormones between cell types and between different regions within the same tissue.

Colocalization of progesterone receptors A and B by dual immunofluorescent histochemistry in human endometrium during the menstrual cycle.

The human progesterone receptor (PR) is expressed as two isoforms, PRA and PRB, that function as ligand-activated transcription factors. In vitro studies suggest that the isoforms differ functionally and that the relative levels in a target cell may determine the nature and magnitude of response to progesterone. However, it is not known whether the two isoforms are normally coexpressed in vivo. To understand the functional significance of relative PR isoform expression in normal physiology, it is essential to determine whether PRA and PRB are coexpressed in the same cell. This study reports the development of a dual immunofluorescent staining technique to demonstrate PRA and PRB proteins by single cell analysis in the same tissue section of human endometrium during the menstrual cycle. PRA and PRB are coexpressed in target cells of the human uterus. In the glands, PRA and PRB were expressed before subnuclear vacuole formation and glycogenolysis, implicating both isoforms in this process, whereas persistence of PRB during the midsecretory phase suggested its significance in glandular secretion. In the stroma, the predominance of PRA throughout the cycle implicates this isoform in post-ovulatory progesterone-mediated events. These results support the view that PRA and PRB mediate distinct pathways of progesterone action in the glandular epithelium and stroma of the human uterus throughout the menstrual cycle.

Immunohistochemical detection of progesterone receptors in archival breast cancer.

The progesterone receptor (PR) is an important marker of response to endocrine agents in breast cancer. Immunohistochemical demonstration of PR in formalin fixed tissue has previously proved difficult, and heat pretreatment is considered necessary to retrieve the antigen. There are few data on the effectiveness of autoclaving in unmasking PR, however, and it is not known whether all PR epitopes are equally unmasked. The objectives of this study were to compare the efficacy of autoclaving and microwaving to retrieve PR antigen in archival breast tumors, to determine whether there is an epitope-dependent variability in the pretreatment required, and to examine different slide types and adhesives to reduce the problem of section loss frequently associated with these procedures. Paraffin embedded sections were cut at 2 or 4 microm, mounted onto various slide types with or without the addition of adhesive, and heat pretreated prior to immunoperoxidase staining. Whereas PR immunoreactivity was clearly demonstrated in tissue after both autoclaving and microwaving, autoclaving produced a significantly stronger staining intensity under the conditions used in this study. The duration of autoclaving required to reveal PR fully differed for different epitopes examined. In the absence of heat pretreatment, PR was not detected. Section retention was improved by the use of adhesives and by cutting tissue at 2 microm. Maximum retention was obtained using positively charged slides coated with Mayer albumen adhesive. We conclude that for maximal tissue preservation autoclave pretreatment is the preferred method of PR antigen retrieval from archival breast tumors, that there is epitope-dependent variability in pretreatment required, and that section loss during this procedure can be minimized by choice of slide type, the use of adhesive, and by cutting sections at 2 microm.

Paclitaxel sensitizes multidrug resistant cells to radiation.

The unique action of paclitaxel, to stabilize microtubules and block cells at the radiosensitive G2M phase of the cell cycle, suggests, it may sensitize tumors to radiotherapy. We have investigated the potential of this interaction to overcome multidrug resistance in vitro using the HL60 cell line and its P-glycoprotein expressing, multidrug resistant H/E8 subline. HL60 cells showed a modest 1.4-fold (p < 0.01) increase in sensitivity to 2 Gy radiation given 24 h after a 1 h treatment with paclitaxel. The H/E8 subline, which has increased radiation resistance and expresses an extended multidrug resistance phenotype, showed significant sensitization to radiation (up to 2.3-fold sensitization; p < 0.01) even with doses of paclitaxel which had no effect on cell viability or were associated with any G2/M block in the cell cycle. In the presence of verapamil, an inhibitor of P-glycoprotein mediated efflux, drug resistant cells could be sensitized to 2 Gy radiation by similar paclitaxel doses as the parental cell (> or = 30 nM; p < 0.01). These results indicate a therapeutic advantage may be possible in the treatment of resistant tumors by the combined use of paclitaxel with radiation.