ACTH and PRL sensitivity of highly differentiated cell lines obtained by adrenocortical targeted oncogenesis.

We established cell lines from adrenal tumors of transgenic mice harboring the large T-antigen of simian virus 40 under the control of the adrenocortical specific promoter of the scavenger aldose reductase-like akr1b7 gene. Mass spectrometry analyses of serum-supplemented or serum-free culture media showed that ATC1 line secreted only corticosterone. These cells, propagated over 25 passages, were characterized with regard to ACTH and PRL responsiveness, as measured by increased corticosterone production, induction of genes involved in the different steps of steroidogenesis (cholesterol delivery, steroid biosynthesis and detoxification of by-products) and expression of transcriptional regulators (SF-1 and DAX1). Corticosterone secretion (RIA) in serum-free medium was stimulated over 12-fold after 6 h treatment with either 10(-9)M ACTH or PRL and both hormones seemed equivalent in promoting this secretion (149 +/- 14 ng and 145 +/- 18 ng/10(6) cells/6 h, respectively). As expected, Northern blots indicate that ATC1 cells expressed mRNAs for the enzymes of corticosterone metabolism CYP11B1 and CYP21A, as well as those for the proteins SIK, SRB1, StAR, CYP11A1, and AKR1B7. Interestingly, these cells have maintained not only the expression of SF-1 but also that of DAX1. No expression of the zona glomeruloza-specific cyp11b2 gene was detected. With the exception of cyp21a and mc2r genes which were constitutively expressed, most of the genes above mentioned were induced in a time- and dose-dependent fashion in response to ACTH or PRL while DAX1 was repressed. Importantly, hormone-mediated repression of DAX1 gene expression was also observed in vivo in mice adrenals. Altogether these data demonstrate that ATC1 line provided an unique model of well differentiated zona fasciculata immortalized cells suitable for the dissection of molecular events leading to ACTH and PRL regulation of adrenal functions.

Urinary steroids from a newborn human infant. Identification of 2 alpha-hydroxy-4-pregnene-3,20-dione, 3 beta,15 beta-dihydroxy-5-pregnen-20-one and 3 beta,15 alpha-dihydroxy-5-pregnen-20-one.

Urinary steroids from healthy newborn human infants were analyzed by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. The identification of 2 alpha-hydroxy-4-pregnene-3,20-dione and the characterization of its 2 beta-isomer is recorded here for the first time. Mass spectrometric evidence supporting the identification of 3 beta,15 beta-dihydroxy-5-pregnen-20-one and 3 beta,15 alpha-dihydroxy-5-pregnen-20-one is also presented. Furthermore, the following 15-hydroxylated steroids were also found and identified: 3 beta,15 epsilon,16 epsilon-trihydroxy-5-androsten-17-one, 5-androstene-3 beta,15 alpha,16 alpha,17 beta-tetrol, 3 beta,15 beta,17-trihydroxy-5-pregnen-20-one and 5-pregnene-3 beta,15 epsilon,17,20 epsilon-tetrol. The origin of these 2- and 15-hydroxylated urinary steroids is discussed in relation to current knowledge of 4-pregnene-3,20-dione and 3 beta-hydroxy-5-pregnen-20-one metabolism during the human perinatal period.

Identification of 2 alpha-hydroxy-4-pregnene-3,20-dione in human pregnancy urine.

2 alpha-Hydroxyprogesterone (2 alpha-hydroxy-4-pregnene-3,20-dione) was identified in human late pregnancy urine by liquid-gel chromatography, GLC and GC-MS. In addition, the following 2-hydroxylated C21 steroids were found and identified as 2 zeta-hydroxy-5 zeta-pregnane-3,20-dione, 2 zeta,20 zeta-dihydroxy-4-pregnen-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha- (and 5 beta)-pregnan-20-one, two isomers of pregnane-2,3,20-triol and 2 zeta,3 zeta,16 zeta-trihydroxy-5 zeta-pregnan-20-one.