Synthetic Modifications of Estrogens and Androgens

Estrogens the female sex hormones have numerous biological actions. Estradiol is the most abundant estrogen in women before menopause. It influences the development, maturation and function of the female reproductive tract. It also plays a role in mammary cancer. Accordingly determinations of estradiol level in body fluids assist in the evaluation of ovarian function and diagnosis for malignancies. Estriol is the primary estrogen in pregnant women and secreted from the fetoplacental unit. Measurement of estriol in maternal body fluids is the basis of fetoplacental monitoring test. Concentration of estrogens in body fluids is determined by immunoassay. Accuracy of this measurement depends on the availability of a specific antibody. As estrogens are not antigenic, their derivatives (haptens) are coupled with a carrier and this hapten-protein conjugate is used to generate antibodies. Specificity of the generated antibody largely depends on the structure of hapten. Therefore the synthesis of a hapten with a right structure is crucial for the accurate measurement of a steroid. We have synthesised new haptens for estradiol and estriol by adding an alkyl or alkoxy side chain at the C-7 of estrane skeleton. The side chains carry a terminal amino group, which can be used for conjugation with a carrier molecule. Estrogens and their biosynthetic precursor androgens both exist as fatty acid esters. They are known to act as hormone storage but their physiological role is not completely known yet. Our collaborator is studying their effect in cardiovascular diseases. We synthesised fatty acid ester derivatives of several steroids in high yield by a very rapid procedure (in 1 min) under microwave irradiation in an ionic liquid (IL). An expedient regioselective hydrolysis at C-3 of estradiol diesters is also reported. 8-Isoestrogens are compounds of pharmaceutical interests, their synthesis, structure, conformation and biological activity studies are ongoing. 7-Hydroxy-8-isoestradiol and 7-alkyl ether of it were synthesised as well. During this study we have developed a selective O-debenzylation method. A mild route for selective removal of benzylic protection on phenol in presence of benzyl protected alcohol was explored.

DNA damage recognition in the normal epithelium of human prostate and seminal vesicles

Prostate cancer is one of the most prevalent cancer types in men. The development of prostate tumors is known to require androgen exposure, and several pathways governing cell growth are deregulated in prostate tumorigenesis. Recent genetic studies have revealed that complex gene fusions and copy - number alterations are frequent in prostate cancer, a unique feature among solid tumors. These chromosomal aberrations are though to arise as a consequence of faulty repair of DNA double strand breaks (DSB). Most repair mechanisms have been studied in detail in cancer cell lines, but how DNA damage is detected and repaired in normal differentiated human cells has not been widely addressed. The events leading to the gene fusions in prostate cancer are under rigorous studies, as they not only shed light on the basic pathobiologic mechanisms but may also produce molecular targets for prostate cancer treatment and prevention. Prostate and seminal vesicles are part of the male reproductive system. They share similar structure and function but differ dramatically in their cancer incidence. Approximately fifty primary seminal vesicle carcinomas have been reported worldwide. Surprisingly, only little is known on why seminal vesicles are resistant to neoplastic changes. As both tissues are androgen dependent, it is a mystery that androgen signaling would only lead to tumors in prostate tissue. In this work, we set up novel ex vivo human tissue culture models of prostate and seminal vesicles, and used them to study how DNA damage is recognized in normal epithelium. One of the major DNA - damage inducible pathways, mediated by the ATM kinase, was robustly activated in all main cell types of both tissues. Interestingly, we discovered that secretory epithelial cells had less histone variant H2A.X and after DNA damage lower levels of H2AX were phosphorylated on serine 139 (γH2AX) than in basal or stromal cells. γH2AX has been considered essential for efficient DSB repair, but as there were no significant differences in the γH2AX levels between the two tissues, it seems more likely that the role of γH2AX is less important in postmitotic cells. We also gained insight into the regulation of p53, an important transcription factor that protects genomic integrity via multiple mechanisms, in human tissues. DSBs did not lead to a pronounced activation of p53, but treatments causing transcriptional stress, on the other hand, were able to launch a notable p53 response in both tissue types. In general, ex vivo culturing of human tissues provided unique means to study differentiated cells in their relevant tissue context, and is suited for testing novel therapeutic drugs before clinical trials. In order to study how prostate and seminal vesicle epithelial cells are able to activate DNA damage induced cell cycle checkpoints, we used primary cultures of prostate and seminal vesicle epithelial cells. To our knowledge, we are the first to report isolation of human primary seminal vesicle cells. Surprisingly, human prostate epithelial cells did not activate cell cycle checkpoints after DSBs in part due to low levels of Wee1A, a kinase regulating CDK activity, while primary seminal vesicle epithelial cells possessed proficient cell cycle checkpoints and expressed high levels of Wee1A. Similarly, seminal vesicle cells showed a distinct activation of the p53 - pathway after DSBs that did not occur in prostate epithelial cells. This indicates that p53 protein function is under different control mechanisms in the two cell types, which together with proficient cell cycle checkpoints may be crucial in protecting seminal vesicles from endogenous and exogenous DNA damaging factors and, as a consequence, from carcinogenesis. These data indicate that two very similar organs of male reproductive system do not respond to DNA damage similarly. The differentiated, non - replicating cells of both tissues were able to recognize DSBs, but under proliferation human prostate epithelial cells had deficient activation of the DNA damage response. This suggests that prostate epithelium is most vulnerable to accumulating genomic aberrations under conditions where it needs to proliferate, for example after inflammatory cellular damage.