ISOLATION AND CHARACTERIZATION OF HEPARIN-BINDING GROWTH-FACTORS IN HUMAN LEIOMYOMAS AND NORMAL MYOMETRIUM

Uterine leiomyomas (fibroids) are benign, smooth muscle cell (SMC) tum ors of the myometrium containing abundant extracellular matrix (ECM). Heparin-binding growth factors present in leiomyoma and normal myometr ial fresh tissue were isolated using heparin-affinity fast protein liq uid chromatography, Purification of these growth factors was monitored by the stimulation of [H-3]thymidine incorporation into BALBc-3T3 cel ls and myometrial SMC, Western blot analysis confirmed that two consis tent peaks of growth factor activity (eluting at 0.5 M NaCl and 1.7 M NaCl) were platelet-derived growth factor (PDGF), 31 kDa, and basic fi broblast growth factor (bFGF), 18 kDa, respectively. Northern blot ana lysis of leiomyoma and myometrial tissue revealed three RNA transcript s (2.8, 2.3, and 1.9 kb) for PDGF-A chain, one RNA transcript (4.0 kb) for PDGF-B chain, and two RNA transcripts (3.7 and 3.5 kb) for bFGF, RNase protection assay showed elevated expression of the bFGF mRNA tra nscript in leiomyomas in 3 out of 5 patients. Immunoperoxidase stainin g of paraffin-embedded tissue showed that PDGF was predominantly intra cellular in both vascular and myometrial SMC, Basic FGF, by contrast, was found primarily bound to the ECM of myometrium and fibroids. Leiom yomas showed much stronger staining for bFGF due to the large areas of ECM in these tumors. A third mitogenic peak eluting at 1.1 M NaCl was also seen in both myometrial and leiomyoma tissue, This peak was not definitively identified by Western blotting, However, Northern analysi s for heparin binding-epidermal growth factor (HBEGF), which also elut es at 1.1 M NaCl, detected one RNA transcript for HBEGF (2.5 kb) in no rmal myometrium but little or no expression in the corresponding leiom yoma tissue. Immunoperoxidase staining showed that HBEGF was a cell-me mbrane-associated protein in both normal myometrial and leiomyoma SMC with more intense staining in normal myometrium. These results show th at both leiomyomas and myometrium synthesize a number of heparin-bindi ng growth factors, The enhanced growth of leiomyomas may be due, in pa rt, to the presence of large quantities of bFGF that are stored in the ECM of these tumors, In addition, the level of HBEGF mRNA declines du ring the transformation of myometrial SMC into leiomyomas.