PARENT SESSION

(T200) REACTIVE OXYGEN SPECIES (ROS) ACT AS SIGNALING MOLECULES IN THE MITOGENIC PATHWAY OF MYOMETRIAL AND LEIOMYOMA SMOOTH MUSCLE CELLS.

Dyer, Summer¹, Nowak, Romana¹, ¹ University of Illinois, Urbana, IL

ABSTRACT- Uterine leiomyomas, or fibroids, are benign tumors of the myometrium affecting more than half of women during their reproductive years. Fibroids are the leading indication for hysterectomy and result in more than $2 billion in hospital charges each year. These tumors are characterized by the proliferation of smooth muscle cells (SMCs) and increased production of collagen. We hypothesize that leiomyomas develop as a response to injury in a manner similar to the development of vascular SMC lesions occurring after angioplasty or during atherosclerosis. Reactive oxygen species (ROS) at low concentrations have been shown to behave as second messenger signaling molecules in vascular SMCs and renal mesangial cells. The goal of our study was to determine whether ROS are necessary components of the signaling pathway for platelet derived growth factor (PDGF) and epidermal growth factor (EGF) in uterine SMCs. These growth factors are known to be strong stimulators of mitogenesis for SMCs. Primary cultures of leiomyoma and myometrial SMCs were established from hysterectomy specimens. DNA synthesis assays showed that PDGF (0.1-10 ng/ml) and EGF (5-100 ng/ml) both caused a dose-dependent increase in cell proliferation of these SMCs after 24 hr of treatment. Cells were then treated with either 10 ng/ml PDGF or 100 ng/ml EGF for 0, 5, 10, 15 min and loaded with 10 M dihydroethidium dye. This dye fluoresces upon oxidation by ROS within cells and becomes intercalated into the cellular DNA. Results showed that both growth factors caused marked increases in intracellular fluorescence of myometrial and leiomyoma SMCs and these increases in fluorescence were dependent on the length of exposure to the growth factors. Treatment of cells with a ROS inhibitor, diphenyleneiodonium chloride (a NADPH oxidase inhibitor), at concentrations ranging from 10-100 M caused a dose dependent inhibition of growth factor stimulated DNA synthesis. Addition of exogenous H₂O₂ (10-100 M) stimulated a 30-50% increase in DNA synthesis in uterine SMCs. The results of our studies show that ROS are a necessary part of the intracellular signaling pathways for PDGF and EGF in uterine SMCs. Future work will focus on identifying subsequent steps in the mitogenic signaling pathway that are altered by ROS particularly the activation of mitogen activated protein kinases.

KEY WORDS: leiomyoma, ros, egf, pdgf