Wh. Rainey et al., TRANSFORMATION OF HUMAN GRANULOSA-CELLS WITH THE E6 AND E7 REGIONS OFHUMAN PAPILLOMAVIRUS, The Journal of clinical endocrinology and metabolism, 78(3), 1994, pp. 705-710
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.