Factors affecting the loss of MED12-mutated leiomyoma cells during in vitro growth.

Uterine leiomyomas (UL) are the most prevalent symptomatic human tumors at all and somatic mutations of the gene encoding mediator subcomplex 12 (MED12) constitute the most frequent driver mutations in UL. Recently, a rapid loss of mutated cells during in vitro growth of UL-derived cell cultures was reported, resulting in doubts about the benefits of UL-derived cell cultures. To evaluate if the rapid loss of MED12-mutated cells in UL cell cultures depends on in vitro passaging, we set up cell cultures from nine UL from 40-50 year old Caucasian patients with at least one UL. Cultured UL cells were investigated for loss of MED12-mutated cells. Genetic characterization of native tumor samples and adjacent myometrium was done by array analysis. "Aged" primary cultures without passaging were compared to cells of three subsequent passages. Comparative analyses of the mutated/non-mutated ratios between native tissue, primary cells, and cultured tumor cells revealed a clear decrease of MED12-mutated cells. None of the tumors showed gross alterations of the array profiles, excluding the presence of gross genomic imbalances besides the MED12 mutations as a reason for the intertumoral variation in the loss of MED12-mutated cells. Albeit at a lesser rate, loss of MED12-mutated cells from cell cultures of UL occurs even without passaging thus indicating the requirement of soluble factors or matrix components lacking in vitro. Identification of these factors can help to understand the mechanisms of the growth of the most frequent type of uterine leiomyomas and to decipher novel drug targets.

The biochemical properties of the Arabidopsis ecto-nucleoside triphosphate diphosphohydrolase AtAPY1 contradict a direct role in purinergic signaling.

The Arabidopsis E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) AtAPY1 was previously shown to be involved in growth and development, pollen germination and stress responses. It was proposed to perform these functions through regulation of extracellular ATP signals. However, a GFP-tagged version was localized exclusively in the Golgi and did not hydrolyze ATP. In this study, AtAPY1 without the bulky GFP-tag was biochemically characterized with regard to its suggested role in purinergic signaling. Both the full-length protein and a soluble form without the transmembrane domain near the N-terminus were produced in HEK293 cells. Of the twelve nucleotide substrates tested, only three--GDP, IDP and UDP--were hydrolyzed, confirming that ATP was not a substrate of AtAPY1. In addition, the effects of pH, divalent metal ions, known E-NTPDase inhibitors and calmodulin on AtAPY1 activity were analyzed. AtAPY1-GFP extracted from transgenic Arabidopsis seedlings was included in the analyses. All three AtAPY1 versions exhibited very similar biochemical properties. Activity was detectable in a broad pH range, and Ca(2+), Mg(2+) and Mn(2+) were the three most efficient cofactors. Of the inhibitors tested, vanadate was the most potent one. Surprisingly, sulfonamide-based inhibitors shown to inhibit other E-NTPDases and presumed to inhibit AtAPY1 as well were not effective. Calmodulin stimulated the activity of the GFP-tagless membranous and soluble AtAPY1 forms about five-fold, but did not alter their substrate specificities. The apparent Km values obtained with AtAPY1-GFP indicate that AtAPY1 is primarily a GDPase. A putative three-dimensional structural model of the ecto-domain is presented, explaining the potent inhibitory potential of vanadate and predicting the binding mode of GDP. The found substrate specificity classifies AtAPY1 as a nucleoside diphosphatase typical of N-terminally anchored Golgi E-NTPDases and negates a direct function in purinergic signaling.