Pim-1 kinase and p100 cooperate to enhance c-Myb activity.

The pim-1 oncogene is regulated by hematopoietic cytokine receptors, encodes a serine/threonine protein kinase, and cooperates with c-myc in lymphoid cell transformation. Using a yeast two-hybrid screen, we found that Pim-1 protein binds to p100, a transcriptional coactivator that interacts with the c-Myb transcription factor. Pim-1 phosphorylated p100 in vitro, formed a stable complex with p100 in animal cells, and functioned downstream of Ras to stimulate c-Myb transcriptional activity in a p100-dependent manner. Thus, Pim-1 and p100 appear to be components of a novel signal transduction pathway affecting c-Myb activity, linking all three to the cytokine-regulated control of hematopoietic cell growth, differentiation, and apoptosis.

Suppression of gonadotropins inhibits gonadal tumorigenesis in mice transgenic for the mouse inhibin alpha-subunit promoter/simian virus 40 T-antigen fusion gene.

We have previously developed a transgenic (TG) mouse model expressing the Simian virus 40 T-antigen (Tag), driven by a 6-kb fragment of the mouse inhibin alpha-subunit promoter (inh-alpha). The mice develop metastasizing gonadal tumors, of granulosa/theca or Leydig cell origin, with 100% penetrance by the age of 5-8 months. In the present study, we examined whether the appearance and growth of the gonadal tumors are dependent on gonadotropins. Gonadotropin suppression was achieved either by treatment of 3-month-old mice for 2-3 months with a GnRH antagonist (Cetrorelix, SB-75), or by cross-breeding the TG mice to the genetic background of the gonadotropin-deficient hypogonadal mutant mouse (hpg). Gonadal tumor growth was clearly inhibited by SB-75 treatment in one of the TG mouse lines (IT6-M), as indicated by the absence of macroscopically visible tumors and by reduced gonadal weights. Despite the suppressed gonadotropin secretion and Tag expression, hyperplasia of testicular Leydig, and ovarian stromal cells persisted in some of the treated mice. In another TG mouse line (IT6-F), with more aggressive tumorigenesis, the SB-75 treatment only partially inhibited gonadal tumor growth. None of the hypogonadotropic TG mice, homozygous for the hpg mutation, developed gonadal tumors. Their gonadal histology was indistinguishable from that of the non-TG hpg mice, suggesting total inhibition of gonadal tumorigenesis in the absence of gonadotropin stimulation. Tag expression and Leydig cell hyperplasia were apparent already in the postnatal TG mice but absent in those TG mice homozygous for the hpg mutation. In conclusion, the present results indicate that the gonadal tumorigenesis in our TG mouse model starts in early age as hyperplasia in specific somatic cells. Both this, and the subsequent malignant tumor growth, are gonadotropin dependent. A sufficient level of Tag expression, a prerequisite for gonadal tumorigenesis, only occurs upon gonadotropin stimulation.

Gonadectomy permits adrenocortical tumorigenesis in mice transgenic for the mouse inhibin alpha-subunit promoter/simian virus 40 T-antigen fusion gene: evidence for negative autoregulation of the inhibin alpha-subunit gene.

We have developed a transgenic (TG) mouse model for gonadal tumorigenesis expressing the Simian virus 40 T-antigen (Tag) under the mouse inhibin alpha-subunit promoter. Gonadal tumors appear with 100% penetrance by the age of 5-8 months in the TG mice. When 1-month-old TG mice were gonadectomized, adrenal gland tumors were observed in each animal (12 females, 11 males) at the age of 6-8 months. No adrenal tumors were detected in gonadectomized non-TG mice (nine females, nine males) or in the intact TG mice (n > 100). The tumors appeared to originate from the X zone of the adrenal cortex. The TG mice with adrenocortical tumors had elevated serum levels of progesterone, estradiol, and immunoreactive inhibin (including dimeric forms), but corticosterone secretion was reduced. The lack of adrenal tumors in intact TG mice suggested that the tumorous gonads secrete factor(s) inhibiting adrenal tumorigenesis. As a candidate molecule, we studied the effects of inhibin, which was high in the serum of control females and TG females with ovarian tumors, as well as in TG males with testicular tumors. The DNA synthesis, as well as the levels of inhibin-alpha and Tag mRNA expression, were significantly reduced by recombinant human inhibin A in cell cultures derived from the adrenal tumors. In accordance, the expression level of inhibin-alpha mRNA in the normal adrenal gland was elevated 2 weeks after gonadectomy. These findings suggest that gonadal inhibin can down-regulate the expression of the inhibin alpha-subunit gene in the adrenal gland. When circulating inhibin is eliminated by gonadectomy, Tag expression and tumorigenesis are stimulated in the adrenal glands of the TG mice. The results demonstrate a novel mechanism of autoregulation in inhibin alpha-subunit gene expression.

Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin alpha-subunit promoter/simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin-responsive granulosa cell lines.

To establish in vivo gonadal tumor models and permanent lines of gonadal somatic cells we produced transgenic (TG) mice expressing the Simian virus (SV) 40 T-antigens (T-ag), driven by 6 or 2.1 kilobase fragments of the mouse inhibin alpha-subunit promoter. Hitherto, altogether 44 TG mice, one of which carried the shorter transgene, have produced gonadal tumors. Two founder females expressing the longer transgene, KK1 and KK3, and three established TG mouse lines were studied in detail. Penetrance of the phenotype in IT6-M and IT6-F mouse lines was 100% (tumors/TG: IT6-M 22/22, IT6-F 14/14). The T-ag mRNA was strongly expressed in the gonads, adrenal glands, pituitary, and brain. The KK-1 and KK-3 ovarian tumor cells immunostained with anti-SV40 large-T antibody. The KK-1 cells possessed high-affinity LH receptors [equilibrium association constant (Ka = 7.8 x 10(10) liters/mol] and responded to human CG by elevated cAMP and progesterone production. Also FSH slightly stimulated their cAMP and estradiol production (P < 0.01). These cells expressed cytochrome P450arom and inhibin alpha mRNA, but not cytochrome P450c17 alpha. In conclusion, the KK-1 cells are immortalized luteinizing granulosa cells expressing endogenous gonadotropin receptors, steroidogenic enzymes, and inhibin alpha. These cells will be useful in studies on the molecular aspects of granulosa cell function. The present study indicates that the 6-kilobase fragment of the inhibin alpha promoter described in this article contains the elements directing tissue-specific expression in vivo and is useful for targeted expression of other genes in the gonads.