Endocrine Abstracts

To date, the in depth analysis of the key molecular mechanisms involved in functional autonomy and tumor formation in aldosterone producing adenomas has been hampered by the rarity of the disease and the lack of adequate tumor cell lines. Herein, we cultivated a primary cell culture of an aldosterone producing adenoma taken from a 40 year old male patient with a left sided adrenal tumor mass. The cells have been passaged 24 times and still continue to grow after nearly 11 months in a stable, cell line-like fashion. Adherent monolayer growth was observed, when the cell were cultured in serum containing media, whereas they adopted spheroid-like structures in EGF and FGF supplemented serum free media. Aldosterone output was measurable in spheroids (S) as well as in monolayer (M) cells (3107±297.0 vs 706±7.0 pg/ml, P=0.01), and could be further increased by ACTH stimulation (3382±245.4 pg/ml vs 946±29.2, P=0.01). Real-time PCR analyses revealed that in comparison to a normal (N) human adrenal gland, mRNA levels of 3ß-HSD and StAR - normalized to HPRT – were significantly lower in the cultured cells (3ß-HSD: 3.9±0.13; P<0.01 (S) and 0.2±0.003; P<0.01 (M) versus 1380±219.6 (N), StAR: 475±67.6; P<0.01(S) and 85±22.4; P<0.01 (M) versus 7970±740.6 (N)). However, these expression levels were similar to those measured in the established adrenocortical cancer cell line NCIh295R (3ß-HSD: 5.4±0.38; P<0.01; StAR: 170±28.7; P<0.01). This holds also true for P450scc, since only slight differences in mRNA expression between monolayer cells, spheroids, and NCIh295R cells could be detected (234±5.1; P<0.01 (S) and 321±9.6; P=0.01 (M) versus 294±3.7 NCI). These results were supported by a clearly positive immunohistochemical staining for P450scc on embedded spheroids and monolayer cells, also verifying the adrenocortical origin of the cultured cells. Currently we are aiming at further defining the in vitro characteristics of the cultured aldosteronoma cells, especially regulatory pathways involved, the reaction to different stimuli, and eventually the ability for in vivo engraftment.