Adenomyosis is a fairly frequent disorder in adult women characterized by the haphazard location of endometrial glands and stroma deep within the myometrium of the uterus. This study compared the effects on uterine development of the selective estrogen receptor modulators, tamoxifen, toremifene, and raloxifene with estradiol when given orally to female mice on days 2 to 5 after birth. Uterine adenomyosis was found in all (14 of 14) mice dosed with tamoxifen and most mice (12 of 14) treated with toremifene, but in none of the vehicle-dosed controls, in only one animal treated with raloxifene at 42 and 90 days after dosing and in none of the mice treated with estradiol at 42 days. At 6 days, the uterus in the groups that developed a high incidence of adenomyosis showed histological evidence of disturbed differentiation of the myometrium. Gene-expression XY-scatterplots using Clontech mouse 1.2 Atlas mouse cDNA expression arrays analyzing total uterine RNA showed nerve growth factor-alpha, preadipocyte factor-1, and insulin-like growth factor-2 were key genes differentially modified by tamoxifen or toremifene treatment, relative to the controls. As these genes may play an important role in regulating differentiation and development of the myometrium, these data suggest that adenomyosis may be caused primarily by defects in the formation of the myometrium.

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Tamoxifen acts as a strong estrogen antagonist in human breast but as an estrogen agonist in the uterus. The action of tamoxifen is mediated through estrogen receptors (ER␣ and ER␤), which bind to a variety of responsive elements, to activate transcription. To examine the role of these varied elements in the response to antiestrogens, we studied the activation of a panel of differing promoters, by these compounds, in human breast, bone, and endometrial derived cell lines. No agonistic activity was observed in breast cells, whereas all antiestrogens, particularly tamoxifen, exhibited agonistic effects in uterine cell lines. All antiestrogens studied were agonistic in co-transfections of a collagenase reporter gene and ER␤, but tamoxifen alone was agonistic with ER␣ in (uterine) HEC-1-A cells. The ER␣ mediated, agonism of tamoxifen was not observed in primary cultures of human uterine stromal cells, whereas the ER␤-mediated agonism of all selective estrogen receptor modulators was present. This suggests that the two receptors operate by distinct pathways and that the response of cells to antiestrogens is dependent on the ER subtypes expressed.Selective estrogen receptor modulators (SERMs), 1 such as tamoxifen, act through interaction with the estrogen receptor (ER) (1). Estrogens and SERMs bind to the ligand binding domain of the ER, allowing dimerization and binding to a palindromic estrogen response element (ERE) upstream of estrogen-sensitive genes. The bound dimer then acts to transactivate transcription (2). SERMs act by competing with estrogen for ER binding. However, estrogenic activity is inhibited in some tissues but unaffected in others (3-5). The cloning of a second estrogen receptor (6) (ER␤) raised the possibility of two different ER homodimers together with a heterodimer of the two ERs (7,8). The cloning of an N-terminal extended (9) and ligand binding domain insertion splice variants (10, 11) of the human ER␤ suggests a wide range of possible homo-and heterodimers, each of which may possess differing ligand sensitivities. Many promoters have been identified that are estrogensensitive but lack an ERE. These include 1) the activation of genes possessing AP-1 elements, including collagenase (12-14), 2) the activation of expression of genes possessing Sp1 binding sites, which are often associated ERE half-sites (15-20), 3) the activation of the TGF␤3 gene through a novel sequence termed the raloxifene response element (21, 22), and more recently 4) the activation of genes possessing the antioxidant response element (23, 24). The estrogen receptor has also been found to down-regulate the interleukin-6 gene by preventing the binding of .The conformation of the ER allosterically varies depending on the DNA sequence bound (28) and thus will differ between response elements. Conformational changes may well affect both the ligand binding domain and its interaction with other proteins such as coactivators and corepressors (29 -32). Therefore, alternative estrogen signaling pathways allow for a broad range of a...

Research into the metabolism of the non-essential amino acid (NEAA) proline in cancer has gained traction in recent years. The last step in the proline biosynthesis pathway is catalyzed by pyrroline-5-carboxylate reductase (PYCR) enzymes. There are three PYCR enzymes: mitochondrial PYCR1 and 2 and cytosolic PYCR3 encoded by separate genes. The expression of the PYCR1 gene is increased in numerous malignancies and correlates with poor prognosis. PYCR1 expression sustains cancer cells’ proliferation and survival and several mechanisms have been implicated to explain its oncogenic role. It has been suggested that the biosynthesis of proline is key to sustain protein synthesis, support mitochondrial function and nucleotide biosynthesis. However, the links between proline metabolism and cancer remain ill-defined and are likely to be tissue specific. Here we use a combination of human dataset, human tissue and mouse models to show that the expression levels of the proline biosynthesis enzymes are significantly increased during colorectal tumorigenesis. Functionally, the expression of mitochondrial PYCRs is necessary for cancer cells’ survival and proliferation. However, the phenotypic consequences of PYCRs depletion could not be rescued by external supplementation with either proline or nucleotides. Overall, our data suggest that, despite the mechanisms underlying the role of proline metabolism in colorectal tumorigenesis remain elusive, targeting the proline biosynthesis pathway is a suitable approach for the development of novel anti-cancer therapies.

We show using Molecular imprinted Polymers (MIPs) and LC-MS/SRM that we can identify the KRAS mutation in cancer patients plasma as well as carry out epitope discovery for drug target evaluation.