Rical order (cf. Table 1). Ordinates gives relative expression of p14Arf
Rical order (cf. Table 1). Ordinates gives relative expression of p14Arf and MDM2, respectively.analyses showed an increased number of p53-positive cells as well when comparing UL with matching myometrium. In some of the samples IHC using Ki-67 was performed as well. Whereas, corresponding to the literature [13], 1 – 2 positive cells were seen in the controls, they were absent from any of the nutlin-3-treated samples.Uterine leiomyoma cells do not recover during long-term inhibition of MDMFigure 4 Highly significant (p < 0.001) correlation between the MDM2 expression (x-axis) and the p19Arf expression (y-axis) in myometrium (white boxes) and leiomyoma tissues (violet circles). One myometrial tissue served as calibrator (expression: 1).To see how the gene expression patterns for p21, BAX, and Ki-67 change after six days of MDM2 inhibition and if the cells become resistant we have additionally performed gene expression analyses for these genes on a series of three UL along with matching myometrium (Figure 7A-C). Akin to a shorter exposure to nutlin-3 the long term experiment did also significantly increase the expression of p21 and BAX and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27107493 decreased that of Ki-67 in a dose-dependent manner. Furthermore, clearMarkowski et al. BMC Women?’?s Health 2012, 12:2 http://www.biomedcentral.com/1472-6874/12/Page 8 ofFigure 5 As a rule fibroids display a higher nutlin-3 sensitivity than matching myometrium. Explants from five fibroids from three patients were UNC0642 site checked for their nutlin-3 sensitivity after incubation with 3 M or 10 M nutlin-3 respectively, for 72 hours. As an indicator for sensitivity the expression of BAX (A, B) and p21 (C, D) mRNA was determined by qRT-PCR. Myometrium (violet columns) was always set 100 and the expression of the corresponding fibroids (light blue columns) refers to that value. Numbers below each row indicate the patient’s lab no. (cf. Table 1). The corresponding leiomyomas are depicted in numerical order. Statistically significances are given by asterisks (*, p < 0.05; PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27484364 **, p < 0.01; ***, p < 0.001).differences between the myometrium and any of the matching fibroids became apparent that again point to a reduced sensitivity of the myometrium against the MDM2 inhibition.Discussion Recently, we have shown that compared to myometrium leiomyomas express higher levels of p14Arf, an important upstream regulator of p53 [7]. Herein, this observation has been confirmed on a much larger series of cases. In addition, a marked heterogeneity of the p14Arf expression was noted when comparing UL from one patient. Most likely due to the well-documented positive feedback-loop that exists between p53 and MDM2 [14,15], the fibroids do also express higher levels of MDM2 leading to a positive correlation between p14Arf and MDM2 expression [7] that may keep fibroids in a delicate balance between growth and senescence. This finding has been confirmed here based on a larger series and makes MDM2 an interesting target molecule for the growth control of leiomyomas. Accordingly, we were able to demonstrate that in leiomyoma cells in vitro treatment with nutlin-3, a smallmolecule inhibitor of MDM2, activates the expression of canonical groups of genes associated with senescenceand apoptosis downstream of p53 [9]. Herein, we were able to show that as a rule tissue explants taken from leiomyomas display a higher sensitivity after treatment with nutlin-3 than do those from matching myometrium. Of note, nutlin-3 increased the expression of p53-depe.