Bettina Hanstein

Molecular mechanism of estrogen receptor (ER)α-specific, estradiol-dependent expression of the progesterone receptor (PR) B-isoform

The physiological effects of progesterone are mediated by the progesterone receptor (PR) isoforms PRA and PRB, transcribed from a single gene, under control of two distinct promoters. Both the isoforms display different, promoter- and cell line-specific transactivation properties. Upregulation of both isoforms in response to estradiol stimulation has been described, although the two promoters contain no classical estrogen response element (ERE). Therefore, we decided to investigate the regulation of PRB-expression through distinct estrogen receptor (ER)-isoforms: ERalpha and ERbeta We demonstrate, that in HeLa cells treated with E2, PRB promoter activity was enhanced (five-fold) by ERalpha, but not by ERbeta. ERbeta was also unable to stimulate activity of the PRB promoter in BT20 and Ishikawa cells, where ERalpha induced reporter activity by two-fold. Deletion of the AF1-but not AF2 domain from ERalpha resulted in loss of the transactivation potential in all cell lines tested. Furthermore, in BT20 cells deletion of the AF2 domain of ERalpha resulted in stronger transcriptional activation than that mediated through wild-type ERalpha. In SK-BR-3 cells both ERs repressed PRB promoter activity and this repression was enhanced by co-transfection of SRC1. However, strong estrogen-dependent stimulation was observed after deletion of AF2. We conclude that PRB expression is stimulated by ERalpha but not ERbeta in an unique, AF1-dependent but AF2-independent mechanism.

In vivo cervical cancer growth inhibition by genetically engineered cytotoxic T cells

Purpose: The CD44 v7/8 splice variant that is frequently expressed in cervical carcinoma and rarely expressed in normal tissues displays promising properties as a target antigen for cancer immune therapy. In this study, cytotoxic T lymphocytes (CTLs) were genetically engineered to gain CD44v7/8 target specificity. Methods: Clone 96 (CI96), an established murine cytotoxic T-cell line, and naïve murine T cells were retrovirally transduced with a fusion gene construct encoding for the single chain fragment scFv of the monoclonal antibody VFF17 and for the ζ chain of the T-cell receptor (TCR). The therapeutic potential of genetically engineered T cells was tested in vitro and in vivo. Results: Surface expression of the chimeric TCR on infected Cl96 and naïve T cells was shown by FACS analysis. CD44v7/8-positive target cells were efficiently lysed by transduced Cl96 and naïve T cells, demonstrating the functionality and specificity of the chimeric TCR. In a xenograft BALB/c mouse model, efficient growth retardation of CD44v7/8-positive tumours was mediated by genetically engineered Cl96(VFF17)cyYZ cells. Conclusions: We were able to reprogramme the target specificity of recombinant Cl96 and naïve CTLs resulting in efficient cytolysis of CD44v7/8-positive cervical cancer cells. High transduction rates and the specific cytolysis of CD44v7/8-redirected CTLs are promising tools for an immune gene therapy approach for advanced cervical cancer.

Novel cGMP liposomal vectors mediate efficient gene transfer

Viral vector systems are the most commonly used gene transfer tools for clinical gene therapy. However, lipofection systems are potential alternatives because of lower immunogenicity and easier cGMP production, but in vivo stability and transduction efficacy need to be improved. Therefore, we investigated gene transduction efficiency of our novel cGMP cationic lipids, CCQ22 and CCQ32, by FACS analysis. Toxicity analysis was performed to determine the cytotoxic side effects of the novel lipids. To evaluate the stability of the compounds in the context of local delivery to patients with intraperitoneally metastatic ovarian cancer, gene transfer was also tested in the presence of malignant ascites. Our novel cGMP standard lipids mediated gene transfer rates of more than 50%. However, for most cell lines cytotoxic side effects were similar to our reference lipofection system. In general, ascites had no major influence on gene transduction rates with the novel lipids. Our results suggest that CCQs may compare favorably with commercially available lipofection systems. These promising results facilitate further analysis of the compounds.

Nongenomic effects of estrogens on signal transduction pathways in estrogen-sensitive carcinoma cell lines

Research thus far has concentrated mainly on the classical steroid hormone (SH) receptors and on the underlying mechanisms of antihormone interactions with these receptors. This is mainly because, in the conventional view, estrogen (E) and progesterone produce most of their effects through interaction with cellular/nuclear receptors with subsequent alteration of the gene-regulating machinery. However, emerging data suggest that these lipophilic hormones are also able to produce rapid effects within several seconds, which cannot be adequately explained through the classical mechanism. Further investigation has recently led to the discovery of membrane-bound forms of E-receptors, which are coupled to cytosolic signal transduction proteins. These rapid responses have been observed in several tissues such as myometrial cells, neurons, endothelium, osteoblasts, granulosa cells and some breast cancer cell lines. The binding of E to these cell-surface forms of E-receptors is thought to activate several second messenger systems via the activation of G-proteins, resulting in the activation of different protein kinases. One such kinase is the mitogen-activated protein (MAP) kinase, which may serve as a stimulus for cell proliferation. We report preliminary results showing the functional existence of such receptors in the human breast carcinoma cell line MCF-7. Using spectrofluorometry to measure the intracellular calcium concentration, evidence has been collected that the addition of E to these cells causes a rapid rise in the intracellular calcium concentration. This mechanism may prove to be an important initial signaling pathway, leading to the activation of specific protein kinases and subsequent proliferation.

Körpergewichtsregulation und Knochenstoffwechsel

Die frauenärztliche Betreuung umfasst viele Gesichtspunkte, die eine Beratung in grundsätzlichen Fragen der Lebensführung darstellen. Diese Tatsache ist wiederholt mit dem Hinweis beschrieben worden, dass Gynäkologinnen und Gynäkologen für viele Frauen die Rolle des Hausarztes erfüllen. Dies bestätigen viele Patientinnen in der Sprechstunde auch mit dem Hinweis, dass sie „ja zu keinem anderen Arzt gingen“ und Fragen in der gynäkologischen Sprechstunde klären möchten.

Transcriptional regulation through the estrogen receptor (ER)-α and splice variants of ER-β via classical and nonclassical signal transduction pathways

The aim of this study was to analyze the transcriptional regulation of different ERs via alternative cis-elements. For this purpose we performed transient transfections with a luciferase reporter plasmid for estrogen-responsive elements (ERE) and AP-1 elements, and expression plasmids for ER-α, ER-β1 and ER-β2. Cells were left unstimulated or stimulated with E2, tamoxifen or raloxifen. We found that, in the breast cancer cell line SKBR3, ER-β1 lead to a significant inhibition of AP-1 activity by E2, whereas through ER-β2 E2 lead to a stimulation of transcriptional activity. Antiestrogens inhibited transcription through ER-β1 but did not exhibit an effect through ER-β2. When transfecting ER-β1 in SKBR3 cells the basal transcriptional activity increased, in contrast to the results obtained when transfecting the human osteosarcoma cell line U2OS with the same receptor. E2 in SKBR3 cells leads to a significant transcriptional inhibition, whereas this effect is not seen in U2OS cells. Also the antiestrogens tamoxifen and raloxifen exhibit in SKBR3 cells via the same receptor a transcriptional inhibition, but in contrast in U2OS cells they lead to a stimulation of transcription. In summary, splice variants of ER-β are able to regulate the actvity of the AP-1 complex differentially, indicating that the relative expression of these variants in a tumor could modulate its hormonal sensitvity. Sponsored by DFG Ha 2404/2-1.

Molekularbiologie des Östrogenrezeptors und seine Regulation durch Östrogen und Antiöstrogene

Zum ThemaHinweise auf eine zentrale Rolle von Östrogen für das Wachstum metastasierender Mammakarzinome wurden zum ersten Mal bereits vor über 100 Jahren beschrieben. Heute sind Antiöstrogene zentraler Bestandteil der Therapie von Mammakarzinomen aller Stadien. Mittlerweile ist bekannt, daß die Wirkung von Östrogen über den Östrogenrezeptor (ER), ein Mitglied der Familie der nukleären Rezeptoren vermittelt wird. In diesem Artikel sollen die molekularen Mechanismen beschrieben werden, über die der Östrogenrezeptor die Transkription von Zielgenen aktiviert und wie derzeit verfügbare Antiöstrogene ihre therapeutischen Effekte vermitteln. Es werden mögliche Mechanismen des Therapieversagens von Antiöstrogenen diskutiert sowie die Wirkung in der Entwicklung befindlicher Antiöstrogene dargestellt.