CHARACTERIZATION OF GONADAL SEX CORD-STROMAL TUMOR-CELL LINES FROM INHIBIN-ALPHA AND P53-DEFICIENT MICE - THE ROLE OF ACTIVIN AS AN AUTOCRINE GROWTH-FACTOR

Inhibin-alpha-deficient mutant mice have been generated by a targeted deletion of the inhibin-alpha gene through homologous recombination in murine embryonic stem cells. Essentially all of the homozygous mutant s develop gonadal sex cord-stromal tumors. To investigate their endocr ine and proliferative characteristics, gonadal tumor cells were mainta ined in vitro. Cells from inhibin-alpha-deficient mice multiplied poor ly; however, cells from mice deficient in both inhibin-alpha and p53 p roliferated rapidly and showed higher saturation density and plating e fficiency, thus allowing the establishment of clonal tumor cell lines. Although negligible estrogen and testosterone was produced by the clo nal cells, high levels of progesterone were secreted. A clonal testis tumor cell line (inhibin-alpha/p53 deficient) showed no response to ex ogenous FSH, human CG (hCG), or inhibin A but exhibited a 6- to 8-fold increase in progesterone production in response to forskolin treatmen t. The stimulatory effect of forskolin was, however, partially blocked by activin treatment. Northern blot analysis revealed inhibin beta(A) and beta(B) mRNA expression in these cells. Furthermore, Western blot analyses indicated the secretion of the beta(A)-subunit protein. We f urther tested the role of activin on tumor cell growth. Treatment with follistatin, an activin-binding protein, inhibited tumor cell replica tion in a dose-dependent manner. In contrast, treatment with activin A stimulated tumor cell growth by itself and partially blocked follista tin action. Incorporation of thymidine into DNA of these cells was als o stimulated by activin. In addition, treatment with antiactivin A ser um inhibited tumor cell replication and blocked the stimulatory action of activin on cell growth. The activin action is likely mediated by s pecific receptors because cross-linking of [I-125]activin to the 50-55 kilodalton type I and 75-80 kilodalton type II receptors was found us ing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis . Northern blot analysis also revealed follistatin mRNA expression in the tumor cells, suggesting these cells are related to granulosa cells . Out findings indicate that activin can act as an autocrine growth fa ctor in stimulating the proliferation of gonadal tumor cell lines deri ved from inhibin-alpha and p53-deficient mice and inhibits progesteron e production. These tumor cell lines are useful for studies on the reg ulation of gonadal cell proliferation and steroidogenesis as well as t he signaling pathway mediating activin action.