Leiomyoma, also known as uterine fibroids, are benign smooth muscle cell tumors that originate from the myometrium. Approximately 70% of pre-menopausal women develop leiomyomas in the United States. Data indicate that progesterone can promote the growth of leiomyomas, however, the pathways involved are relatively unclear. The PI3K/AKT pathway is commonly hyperactivated in human tumors and we have recently shown that leiomyoma cells have increased basal AKT phosphorylation compared to matched myometrial cells. In addition, progestins can rapidly phosphorylate AKT in primary leiomyoma cells. In order to decipher the mechanisms involved in the rapid activation of AKT by progesterone, we utilized both primary and immortalized leiomyoma cells as well as primary myometrial cells and first determined the protein expression of the players in the PI3K/AKT pathway: p85, p110, AKT1, AKT2, and AKT3. The regulatory subunit of PI3K, p85, and catalytic subunit, p110 were expressed in primary and immortalized leiomyoma cells as well as primary myometrial cells. Progestin treatment over 48 hours did not alter p85 or p110 protein levels in these cells. Primary and immortalized leiomyoma cells and primary myometrial cells expressed AKT1 in the presence and absence of serum. AKT2 and AKT3 expression, however, varied depending on the patient in primary leiomyoma cells. AKT2 was expressed in the absence of serum in immortalized leiomyoma cells and primary myometrial cells. Primary myometrial cells also expressed AKT3 in the absence of serum; however, immortalized leiomyoma cells expressed AKT3 only in the presence of serum. Levels of total AKT protein decreased in immortalized leiomyoma cells in response to R5020 or RU486 alone as well as in combination. In primary myometrial cells, total AKT proteins decreased in response to the combination of R5020 and RU486. Finally, we demonstrated that progesterone rapidly phosphorylates AKT, within 15minutes, in the immortalized leiomyoma cell line as it does in the primary leiomyoma cells. Furthermore, the highest level of p-AKT was observed with 10nM and 100nM progesterone. In summary, all three cell types expressed p85, p110 and AKT 1. AKT 2 and AKT 3 expressions were variable, depending on the patient. While long term progestin treatment did not affect p85 or p110 protein levels, total AKT protein was differentially regulated by progestins and serum in the leiomyoma compared to the myometrial cells. In conclusion, not only can progesterone rapidly activate the AKT pathway in leiomyoma cells but it may regulate AKT expression after long term treatment. In addition, AKT isoform expression in primary and immortalized leiomyoma cells may depend on the availability of growth factors and hormones to regulate cell survival. Research supported by a grant from the Friends of Prentice. (poster)