Oestetrol stimulates proliferation and oestrogen receptor expression in breast cancer cell lines: comparison of four oestrogens.

OBJECTIVES: Oestetrol (15-alpha-hydroxyoestriol, E4) is an endogenous oestradiol metabolite mainly produced at high concentrations in the fetal liver. In earlier studies E4 was investigated for its use as marker for pregnancies at risk, especially with vascular problems. Some current investigations suggest that the use of E4 in hormone therapy or contraception may have advantages in terms of breast cancer risk when compared to other oestrogens. METHODS: Proliferation of two oestrogen receptor-positive breast cancer cell lines (ZR 75-1 and HCC 1500) was investigated after incubation with oestrone (E1), oestradiol (E2), oestriol (E3), and oestetrol (E4). Receptor expression of oestrogen receptor-alpha (ERα) and -beta (ERß) was determined by Western-Blot. RESULTS: All four oestrogens elicited a significant proliferative stimulation at concentrations of 10(-10) und 10(-9) M as compared to controls. Oestrone displayed a significantly weaker effect than E2. Oestetrol was significantly less effective than E2 at the lower concentration. Expression of ERα and ERß was significantly upregulated by all oestrogens tested, without differences between the latter. CONCLUSIONS: Our results indicate a slightly lower proliferative effect of E4, but only at low concentrations. However, no difference was found regarding receptor expression. Breast cancer risk associated with use of oestetrol should be tested in clinical trials.

Nomegestrol acetate sequentially or continuously combined to estradiol did not negatively affect membrane-receptor associated progestogenic effects in human breast cancer cells.

OBJECTIVES: Recently the first monophasic contraceptive pill containing estradiol has been developed which is thought to be a milestone in contraception. Nomegestrol acetate (NOM) is the progestogenic component. Progesterone receptor membrane component 1 (PGRMC1) is highly expressed in the tissue of breast cancer patients, and can predict a progestogen dependent risk of breast cancer. METHODS: MCF-7 cells were transfected with PGRMC1 expression plasmid, and were stimulated with estradiol (E2, 10(-12) and 10(-10) M). NOM, progesterone (P), medroxyprogesterone acetate (MPA) and norethisterone (NET) (each 10(-7) M) were added sequentially or continuously. RESULTS: E2 at 10(-10) M elicited a significant increase of cell proliferation from 150 to 200%. No effect was seen at 10(-12) M. Addition of the progestogens to E2 at 10(-10) M had no significant effect. However, at an E2 10(-12) M, NET significantly stimulated cell proliferation more pronounced in the continuous combined model. No effect was seen for NOM, P and MPA. The E2/NET combined effect could be abrogated by the addition of an estrogen receptor (ER) antagonist. CONCLUSION: Since NOM did not increase proliferation it may be concluded that it will be neutral in terms of breast cancer risk when combined with E2 at least in women overexpressing PGRMC1.

Progression-specific genes identified in microdissected formalin-fixed and paraffin-embedded tissue containing matched ductal carcinoma in situ and invasive ductal breast cancers.

BACKGROUND: The transition from ductal carcinoma in situ (DCIS) to invasive breast carcinoma (IBC) is an important step during breast carcinogenesis. Understanding its molecular changes may help to identify high-risk DCIS that progress to IBC. Here, we describe a transcriptomic profiling analysis of matched formalin-fixed and paraffin-embedded (FFPE) DCIS and IBC components of individual breast tumours, containing both tumour compartments. The study was performed to validate progression-associated transcripts detected in an earlier gene profiling project using fresh frozen breast cancer tissue. In addition, FFPE tissues from patients with pure DCIS (pDCIS) were analysed to identify candidate transcripts characterizing DCIS with a high or low risk of progressing to IBC. METHODS: Fifteen laser microdissected pairs of DCIS and IBC were profiled by Illumina DASL technology and used for expression validation by qPCR. Differential expression was independently validated using further 25 laser microdissected DCIS/IBC sample pairs. Additionally, laser microdissected epithelial cells from 31 pDCIS were investigated for expression of candidate transcripts using qPCR. RESULTS: Multiple statistical calculation methods revealed 1784 mRNAs which are differentially expressed between DCIS and IBC (P < 0.05), of which 124 have also been identified in the gene profiling project using fresh frozen breast cancer tissue. Nine mRNAs that had been selected from the gene list obtained using fresh frozen tissues by applying pathway and network analysis (MMP11, GREM1, PLEKHC1, SULF1, THBS2, CSPG2, COL10A1, COL11A1, KRT14) were investigated in tissues from the same 15 microdissected specimens and the 25 independent tissue samples by qPCR. All selected transcripts were also detected in tumour cells from pDCIS. Expression of MMP11 and COL10A1 increased significantly from pDCIS to DCIS of DCIS/IBC mixed tumours. CONCLUSION: We confirm differential expression of progression-associated transcripts in FFPE breast cancer samples which might mediate the transition from DCIS to IBC. MMP11 and COL10A1 may characterize pure DCIS with a high risk developing IDC.

A bead-based western for high-throughput cellular signal transduction analyses.

Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. By combining gel-based protein separation with immobilization on microspheres, hundreds of replicas of the initial blot are created, thus enabling the comprehensive analysis of limited material, such as cells collected by laser capture microdissection, and extending traditional western blotting to reach proteomic scales. The combination of molecular weight resolution, sensitivity and signal linearity on an automated platform enables the rapid quantification of hundreds of specific proteins and protein modifications in complex samples. This high-throughput western blot approach allowed us to identify and characterize alterations in cellular signal transduction that occur during the development of resistance to the kinase inhibitor Lapatinib, revealing major changes in the activation state of Ephrin-mediated signalling and a central role for p53-controlled processes.

Correlation of the KISA% index and Scheimpflug tomography in 'normal', 'subclinical', 'keratoconus-suspect' and 'clinically manifest' keratoconus eyes.

PURPOSE: To analyse tomographic changes in eyes classified as 'normal', 'keratoconus-suspect' and 'clinically manifest keratoconus' based on the established KISA% definition of Rabinowitz and Rasheed and to develop the category of 'subclinical keratoconus eyes' to expand the classification into a 'subtopographic' range. METHODS: Tomographic and topographic analyses of 670 eyes performed with a rotating Scheimpflug imaging system (Pentacam(®), Oculus Inc., Wetzlar, Germany) were retrospectively analysed. Based on the KISA% keratoconus classification system, eyes were assigned to a 'normal', 'keratoconus-suspect' or 'manifest keratoconus' group. In addition, a new group of 'subclinical keratoconus eyes' was analysed, comprising unsuspicious fellow eyes of patients with keratoconus. T-tests, Wilcoxon rank-sum test, receiver operating characteristics (ROC) and robust regression analyses were performed to correlate tomographic parameters with the increasing KISA% index. RESULTS: KISA%-grouped keratoconus eyes showed robust tomographic changes. By adding the subclinical group, although the concurrent topography was unchanged, we were able to demonstrate statistically significant changes for almost all tomographic parameters (parameters with highest sensitivity/specificity: ART_max, [0.69/0.69], BAD_D [0.66/0.66]). The highest coefficient of determination (R(2)) with the KISA% index was demonstrated for Ele_f_max (R(2) = 0.70), Ele_f_TP (R(2) = 0.69), Ele_b_TP (R(2) = 0.69) and BAD_D (R(2) = 0.68). CONCLUSION: We recommend the use of the expanded KISA% index (eKISA% index) as the basis for the definition of keratoconus and normal groups in future keratoconus research projects.