Purification and partial sequencing of the major mitogen for human uterine smooth muscle-like cells in leiomyoma extracts.
We purified the major mitogen for human smooth muscle-like cells in leiomyoma extracts by sequential liquid chromatography on (a) carboxymethyl-Sepharose, (b) heparin-Sepharose columns, (c) cartridges of C18 silica, and (d) linear gradient reverse-phase high performance liquid chromatography. The mitogenic activity of the leiomyoma extract throughout purification was tested by tritiated thymidine incorporation and DNA content in NIH/3T3 fibroblasts and KW human smooth muscle-like cells. Purification of the leiomyoma-derived growth factor (LDGF) for KW smooth muscle-like cells confirmed that its partial NH2-terminal amino acid (aa) sequence (1-20 aa) was identical to 113-132 aa of the human cysteine-rich protein (hCRP). A synthetic peptide which was engineered based on the purified aa sequence, stimulated the proliferation and growth of KW cells. An oligonucleotide probe constructed by the cDNA of the hcrp gene that encodes this aa sequence depicted the expression of 1.9-kb LDGF mRNA in leiomyomas and myometrium. The expression of the LDGF mRNA was three to sixfold higher in leiomyomas compared with adjacent myometrium of women harboring leiomyomas by in situ hybridization analysis. These data suggest that LDGF may participate in the pathophysiology of uterine leiomyomas.
Sourla
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A
Molecular Endocrinology Laboratory, Centre Hospitalier de l'Universite Laval (CHUL), Quebec, Canada.
Koutsilieris
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M
D016475:3T3 Cells / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D005260:Female; D006801:Humans; D007889:Leiomyoma / Q000737:chemistry*; D051379:Mice; D008934:Mitogens / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D008969:Molecular Sequence Data; D009215:Myometrium / Q000737:chemistry / Q000187:drug effects*; D009363:Neoplasm Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D009687:Nuclear Proteins; D011506:Proteins; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014594:Uterine Neoplasms / Q000737:chemistry*; D016335:Zinc Fingers / Q000235:genetics
D008934:Mitogens; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011506:Proteins; D016271:Proto-Oncogene Proteins c-myc; C095024:leiomyoma-derived growth factor; C074370:cysteine-rich protein, mammalian