Mechanism of collagen biosynthesis up-regulation in cultured leiomyoma cells.

Uterine leiomyoma is the most common tumour in women with a reported incidence of 25-30%. The tumors are benign, composed of smooth muscle cells with variable amount of collagen - rich fibrous tissue. It is well established that accumulation of extracellular matrix in leiomyoma is key feature of tissue fibrosis. However, the pathogenesis of leiomyoma is still unclear. The aim of this study was to evaluate the metabolism of collagen in cultured leiomyoma cells and in control myometrium cells. The effect of estradiol, selective modulators of estrogen receptors (raloxifene, tamoxifen) and estrogen receptor down regulator (ICI 182.780) on collagen biosynthesis (measured by 5-[3H]-proline incorporation assay and measurement of prolidase activity) and collagen degradation (measured by metalloproteinase activity assay) was studied. It was found that collagen biosynthesis is strongly stimulated by low doses of estradiol (5 nM) in leiomyoma cells while it is not changed in control myometrium cells. An increase in estradiol concentration to 10 nM results in drastic decrease of this process both in leiomyoma as well as control cells. Although raloxifene and tamoxifen only slightly affected collagen biosynthesis in control myometrium cells, they significantly inhibited the process in leiomyoma cells. There was no coordinate correlation between collagen biosysignificantly inhibited the process in leiomyoma cells. There was no coordinate correlation between collagen biosynthesis and prolidase activity suggesting that regulation of this process may take place at transcriptional level. Both estrogen and SERMs were found to inhibit MMP-2 in leiomyoma as well as in control myometrium cells. The data suggest that stimulatory action of estrogen on collagen biosynthesis and inhibitory effect on MMP-2 activity in uterine leiomyoma may contribute to accumulation of this protein in ECM of this tissue

The High-Acceptance Dielectron Spectrometer HADES

HADES is a versatile magnetic spectrometer aimed at studying dielectron production in pion, proton and heavy-ion induced collisions. Its main features include a ring imaging gas Cherenkov detector for electron-hadron discrimination, a tracking system consisting of a set of 6 superconducting coils producing a toroidal field and drift chambers and a multiplicity and electron trigger array for additional electron-hadron discrimination and event characterization. A two-stage trigger system enhances events containing electrons. The physics program is focused on the investigation of hadron properties in nuclei and in the hot and dense hadronic matter. The detector system is characterized by an 85% azimuthal coverage over a polar angle interval from 18 to 85 degree, a single electron efficiency of 50% and a vector meson mass resolution of 2.5%. Identification of pions, kaons and protons is achieved combining time-of-flight and energy loss measurements over a large momentum range. This paper describes the main features and the performance of the detector system

Understanding the Role of Estrogen Receptor Status in PRODH/POX-Dependent Apoptosis/Survival in Breast Cancer Cells

It has been suggested that activation of estrogen receptor α (ER α) stimulates cell proliferation. In contrast, estrogen receptor β (ER β) has anti-proliferative and pro-apoptotic activity. Although the role of estrogens in estrogen receptor-positive breast cancer progression has been well established, the mechanism of their effect on apoptosis is not fully understood. It has been considered that ER status of breast cancer cells and estrogen availability might determine proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis. PRODH/POX is a mitochondrial enzyme that converts proline into pyrroline-5-carboxylate (P5C). During this process, ATP (adenosine triphosphate) or ROS (reactive oxygen species) are produced, facilitating cell survival or death, respectively. However, the critical factor in driving PRODH/POX-dependent functions is proline availability. The amount of this amino acid is regulated at the level of prolidase (proline releasing enzyme), collagen biosynthesis (proline utilizing process), and glutamine, glutamate, α-ketoglutarate, and ornithine metabolism. Estrogens were found to upregulate prolidase activity and collagen biosynthesis. It seems that in estrogen receptor-positive breast cancer cells, prolidase supports proline for collagen biosynthesis, limiting its availability for PRODH/POX-dependent apoptosis. Moreover, lack of free proline (known to upregulate the transcriptional activity of hypoxia-inducible factor 1, HIF-1) contributes to downregulation of HIF-1-dependent pro-survival activity. The complex regulatory mechanism also involves PRODH/POX expression and activity. It is induced transcriptionally by p53 and post-transcriptionally by AMPK (AMP-activated protein kinase), which is regulated by ERs. The review also discusses the role of interconversion of proline/glutamate/ornithine in supporting proline to PRODH/POX-dependent functions. The data suggest that PRODH/POX-induced apoptosis is dependent on ER status in breast cancer cells

Recombinant Human Prolidase (rhPEPD) Induces Wound Healing in Experimental Model of Inflammation through Activation of EGFR Signalling in Fibroblasts

The potential of recombinant human prolidase (rhPEPD) to induce wound healing in an experimental model of IL-1β-induced inflammation in human fibroblasts was studied. It was found that rhPEPD significantly increased cell proliferation and viability, as well as the expression of the epidermal growth factor receptor (EGFR) and downstream signaling proteins, such as phosphorylated PI3K, AKT, and mTOR, in the studied model. Moreover, rhPEPD upregulated the expression of the β1 integrin receptor and its downstream signaling proteins, such as p-FAK, Grb2 and p-ERK 1/2. The inhibition of EGFR signaling by gefitinib abolished rhPEPD-dependent functions in an experimental model of inflammation. Subsequent studies showed that rhPEPD augmented collagen biosynthesis in IL-1β-treated fibroblasts as well as in a wound healing model (wound closure/scratch test). Although IL-1β treatment of fibroblasts increased cell migration, rhPEPD significantly enhanced this process. This effect was accompanied by an increase in the activity of MMP-2 and MMP-9, suggesting extracellular matrix (ECM) remodeling during the inflammatory process. The data suggest that rhPEPD may play an important role in EGFR-dependent cell growth in an experimental model of inflammation in human fibroblasts, and this knowledge may be useful for further approaches to the treatment of abnormalities of wound healing and other skin diseases

Functional Consequences of Intracellular Proline Levels Manipulation Affecting PRODH/POX–Dependent Pro-Apoptotic Pathways in a Novel in Vitro Cell Culture Model

Background/Aims: The effect of impaired intracellular proline availability for proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis was studied. Methods: We generated a constitutively knocked-down PRODH/POX MCF-7 breast cancer cell line (MCF-7shPRODH/POX) as a model to analyze the functional consequences of impaired intracellular proline levels. We have used inhibitor of proline utilization in collagen biosynthesis, 2-metoxyestradiol (MOE), inhibitor of prolidase that generate proline, rapamycin (Rap) and glycyl-proline (GlyPro), substrate for prolidase. Collagen and DNA biosynthesis were evaluated by radiometric assays. Cell viability was determined using Nucleo-Counter NC-3000. The activity of prolidase was determined by colorimetric assay. Expression of proteins was assessed by Western blot and immunofluorescence bioimaging. Concentration of proline was analyzed by liquid chromatography with mass spectrometry. Results: PRODH/POX knockdown decreased DNA and collagen biosynthesis, whereas increased prolidase activity and intracellular proline level in MCF-7shPRODH/POX cells. All studied compounds decreased cell viability in MCF-7 and MCF-7shPRODH/POX cells. DNA biosynthesis was similarly inhibited by Rap and MOE in both cell lines, but GlyPro inhibited the process only in MCF-7shPRODH/POX and MOE+GlyPro only in MCF-7 cells. All the compounds inhibited collagen biosynthesis, increased prolidase activity and cytoplasmic proline level in MCF-7shPRODH/POX cells and contributed to the induction of pro-survival mode only in MCF-7shPRODH/POX cells. In contrast, all studied compounds upregulated expression of pro-apoptotic protein only in MCF-7 cells. Conclusion: PRODH/POX was confirmed as a driver of apoptosis and proved the eligibility of MCF-7shPRODH/POX cell line as a highly effective model to elucidate the different mechanisms underlying proline utilization or generation in PRODH/POX-dependent pro-apoptotic pathways