TRANSFORMATION OF HUMAN GRANULOSA-CELLS WITH THE E6 AND E7 REGIONS OFHUMAN PAPILLOMAVIRUS

Ovarian granulosa cells are the primary site of estrogen and progester one synthesis and play an essential role in the maturation of the deve loping ovum. Freshly isolated granulosa cells are often used to study the regulation of steroid and protein biosynthesis, but the small numb er of cells available for these cultures has proven inadequate for man y detailed gene regulatory studies. The goal of this study was to deve lop human granulosa (HG) cell lines that maintain differentiated funct ion. The E6 and E7 open reading frames of high risk strains of human p apillomavirus have been used to produce immortalized cell lines. Prima ry cultures of human luteinized granulosa cells were infected with def ective retroviruses containing the E6 and E7 regions of human papillom avirus 16 and with the neomycin phosphotransferase gene to confer G418 resistance. Three of eight clones;that were isolated after selection in medium containing G418 were found to produce progesterone following treatment with forskolin or dibutyryl cAMP for 48 h. Forskolin caused these cells to retract in the characteristic rounding response, as de scribed in primary HG cultures. One clone, HGL5, was used for a detail ed characterization of differentiated function. HGL5 cells retained th e ability to increase progesterone production and convert exogenously added androstenedione to estradiol in response to agonists of the prot ein kinase-A pathway (forskolin and dibutryl cAMP), but were not respo nsive to FSH or LH treatment. A key enzyme in the production of estrad iol, cytochrome P450 aromatase, has proven difficult to maintain in lo ng term cultures of granulosa cells. For that reason, we examined the expression of aromatase in the transformed HGL5 clone by monitoring mR NA levels. Aromatase mRNA increased by 4- to 5-fold after forskolin tr eatment, as determined by Northern analysis. This human granulosa cell culture line maintains many of the functions of normal cells and shou ld provide an important model to study the molecular events controllin g granulosa cell differentiation and function.