HOX genes promote cell proliferation and are potential therapeutic targets in adrenocortical tumours.

BACKGROUND: Understanding the pathways that drive adrenocortical carcinoma (ACC) is essential to the development of more effective therapies. This study investigates the role of the transcription factor HOXB9 and other HOX factors in ACC and its treatment. METHODS: We used transgenic mouse models to determine the role of Hoxb9 in adrenal tumour development. Patient transcriptomic data was analysed for the expression of HOX genes and their association with disease. Drug response studies on various adrenocortical models were done to establish novel therapeutic options. RESULTS: Our human ACC dataset analyses showed high expression of HOXB9, and other HOX factors, are associated with poorer prognosis. Transgenic overexpression of Hoxb9 in the adrenal cortex of mice with activated Ctnnb1 led to larger adrenal tumours. This phenotype was preferentially observed in male mice and was characterised by more proliferating cells and an increase in the expression of cell cycle genes, including Ccne1. Adrenal tumour cells were found to be dependent on HOX function for survival and were sensitive to a specific peptide inhibitor. CONCLUSIONS: These studies show Hoxb9 can promote adrenal tumour progression in a sex-dependent manner and have identified HOX factors as potential drug targets, leading to novel therapeutic approaches in ACC.

Genes involved in the endoplasmic reticulum N-glycosylation pathway of the red microalga Porphyridium sp.: a bioinformatic study.

N-glycosylation is one of the most important post-translational modifications that influence protein polymorphism, including protein structures and their functions. Although this important biological process has been extensively studied in mammals, only limited knowledge exists regarding glycosylation in algae. The current research is focused on the red microalga Porphyridium sp., which is a potentially valuable source for various applications, such as skin therapy, food, and pharmaceuticals. The enzymes involved in the biosynthesis and processing of N-glycans remain undefined in this species, and the mechanism(s) of their genetic regulation is completely unknown. In this study, we describe our pioneering attempt to understand the endoplasmic reticulum N-Glycosylation pathway in Porphyridium sp., using a bioinformatic approach. Homology searches, based on sequence similarities with genes encoding proteins involved in the ER N-glycosylation pathway (including their conserved parts) were conducted using the TBLASTN function on the algae DNA scaffold contigs database. This approach led to the identification of 24 encoded-genes implicated with the ER N-glycosylation pathway in Porphyridium sp. Homologs were found for almost all known N-glycosylation protein sequences in the ER pathway of Porphyridium sp.; thus, suggesting that the ER-pathway is conserved; as it is in other organisms (animals, plants, yeasts, etc.).

Adrenal 20alpha-hydroxysteroid dehydrogenase in the mouse catabolizes progesterone and 11-deoxycorticosterone and is restricted to the X-zone.

The enzyme 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) is a progesterone-catabolizing enzyme that is highly expressed in mouse ovaries and adrenals. Although the functional significance of ovarian 20alpha-HSD for the induction of parturition has been defined, regulation and distribution of 20alpha-HSD in the adrenal gland has not been determined. We demonstrate that the expression of adrenal 20alpha-HSD is restricted to the X-zone, a transient zone between the adrenal cortex and the medulla of yet unknown function. Adrenal 20alpha-HSD activity in male mice peaks at 3 wk of age and disappears thereafter, whereas 20alpha-HSD enzyme activity is maintained in adrenals from nulliparous female animals. Testosterone treatment of female mice induces rapid involution of the X-zone that is associated with the disappearance of the 20alpha-HSD-positive cells. Conversely, reappearance of 20alpha-HSD expression and activity in male animals is evident after gonadectomy. Moreover, pregnancy, but not pseudopregnancy, is accompanied by X-zone regression and loss of 20alpha-HSD activity. Pregnancy-induced X-zone regression and -abolished 20alpha-HSD expression is partially restored in animals that were kept from nursing their pups. We found that in addition to its progesterone-reducing activity, 20alpha-HSD also functions as an 11-deoxycorticosterone-catabolizing enzyme. The unaltered growth kinetics of the X-zone in 20alpha-HSD knockout animals suggests that 20alpha-HSD is not required for the regulation of X-zone growth. However, 20alpha-HSD expression and enzymatic activity in all experimental paradigms is closely correlated with the presence of the X-zone. These findings provide the basis for 20alpha-HSD as a reliable marker of the murine X-zone.

Peripheral administration of the N-terminal pro-opiomelanocortin fragment 1-28 to Pomc-/- mice reduces food intake and weight but does not affect adrenal growth or corticosterone production.

Pro-opiomelanocortin (POMC) is a polypeptide precursor that undergoes extensive processing to yield a range of peptides with biologically diverse functions. POMC-derived ACTH is vital for normal adrenal function and the melanocortin alpha-MSH plays a key role in appetite control and energy homeostasis. However, the roles of peptide fragments derived from the highly conserved N-terminal region of POMC are less well characterized. We have used mice with a null mutation in the Pomc gene (Pomc(-/-)) to determine the in vivo effects of synthetic N-terminal 1-28 POMC, which has been shown previously to possess adrenal mitogenic activity. 1-28 POMC (20 mug) given s.c. for 10 days had no effect on the adrenal cortex of Pomc(-/-) mice, with resultant cortical morphology and plasma corticosterone levels being indistinguishable from sham treatment. Concurrent administration of 1-28 POMC and 1-24 ACTH (30 mug/day) resulted in changes identical to 1-24 ACTH treatment alone, which consisted of upregulation of steroidogenic enzymes, elevation of corticosterone levels, hypertrophy of the zona fasciculate, and regression of the X-zone. However, treatment of corticosterone-depleted Pomc(-/-) mice with 1-28 POMC reduced cumulative food intake and total body weight. These anorexigenic effects were ameliorated when the peptide was administered to Pomc(-/-) mice with circulating corticosterone restored either to a low physiological level by corticosterone-supplemented drinking water (CORT) or to a supraphysiological level by concurrent 1-24 ACTH administration. Further, i.c.v. administration of 1-28 POMC to CORT-treated Pomc(-/-) mice had no effect on food intake or body weight. In wild-type mice, the effects of 1-28 POMC upon food intake and body weight were identical to sham treatment, but 1-28 POMC was able to ameliorate the hyperphagia induced by concurrent 1-24 ACTH treatment. In a mouse model which lacks all endogenous POMC peptides, s.c. treatment with synthetic 1-28 POMC alone can reduce food intake and body weight, but has no impact upon adrenal growth or steroidogenesis.

Induction of Fc gammaRIIA expression in myeloid PLB cells during differentiation depends on cytosolic phospholipase A2 activity and is regulated via activation of CREB by PGE2.

Fc gammaRIIA expressed on neutrophils and monocytes has a fundamental role in combating bacterial infections. In the present study, the requirement of cytosolic phospholipase A2 (cPLA2) for induction of Fc gammaRIIA expression was studied in a model of cPLA2-deficient PLB-985 cells (PLB-D cells). Fc gammaRIIA was acquired only during differentiation of PLB but not of PLB-D cells induced by either 1,25-dihydroxyvitamin D3, retinoic acid, or interferon gamma. Addition of prostaglandin E2 (PGE2) to PLB-D cells undergoing differentiation restored the expression of Fc gammaRIIA protein, whereas addition of indomethacin to PLB cells during differentiation inhibited both the production of PGE2 and the expression of Fc gammaRIIA. Inhibition of PKA during PLB differentiation prevented Fc gammaRIIA expression, whereas dibutyryl cAMP (dbcAMP) induced its expression in both PLB and PLB-D cells. CREB phosphorylation and CREB-CRE interaction were detected only in differentiated PLB cells and not PLB-D cells and were inhibited by indomethacin. A reporter gene containing a Fc gammaRIIA gene promoter fragment with the CRE element was sufficient for CREB activation. Our results are the first to show that CREB activation is involved in up-regulation of Fc gammaRIIA expression in myeloid lineages. PGE2 formed via cPLA2 activates CREB through PKA and this process is dependent on development of PGE2 receptor 4.

Actin phylogeny and intron distribution in bangiophyte red algae(rhodoplantae).

The molecular phylogeny of red algal actin genes, with emphasis on the paraphyletic "Bangiophyceae," was examined and compared to the rhodophyte SSU rDNA phylogeny. Nineteen new genomic actin sequences and seven SSU rDNA sequences were obtained and subjected to diverse phylogenetic analyses (maximum likelihood, distance/neighbor-joining, maximum parsimony, Bayesian analyses, and, with respect to protein sequences, also quartet puzzling). The actin trees confirmed most of the major clades found in the SSU rDNA phylogenies, although with a lower resolution. An actin gene duplication in the florideophycean lineage is reported, presumably related to an increased complexity of sexual reproduction. In addition, the distribution and characteristics of spliceosomal introns found in some of the actin sequences were examined. Introns were found in almost all florideophycean actin genes, whereas only two bangiophyte sequences contained introns. One intron in the florideophycean actin genes was also found in metazoan, and, shifted by one or two nucleotides, in a glaucocystophyte, a cryptophyte, and two fungal actin genes, and thus may be an ancient intron.

Citral is a new inducer of caspase-3 in tumor cell lines.

Citral, 3,7-dimethyl-2,6-octadien-1-al, a key component of the lemon-scented essential oils extracted from several herbal plants such as lemon grass (Cymbopogon citratus), melissa (Melissa officinalis), verbena (Verbena officinalis) is used as a food additive and as a fragrance in cosmetics. In this study, we investigated the anti-cancer potential of citral and its mode of action. Concentrations of 44.5 muM, comparable to the concentration of citral in a cup of tea prepared from 1 g of lemon grass, induced apoptosis in several hematopoietic cancer cell lines. Apoptosis was accompanied by DNA fragmentation and caspase-3 catalytic activity induction. Citral activity (22.25 microM) was compared to a reference compound like staurosporine (0.7 microM), in respect to DNA fragmentation and caspase-3 enzymatic activity. The apoptotic effect of citral depended on the alpha,beta-unsaturated aldehyde group.

Regulation of progesterone levels during pregnancy and parturition by signal transducer and activator of transcription 5 and 20alpha-hydroxysteroid dehydrogenase.

The two highly related signal transducers and activators of transcription (Stats), Stat5a and Stat5b, are major mediators of prolactin signaling in both the mammary gland and in the ovary. Deficiencies in Stat5b, or in both Stat5a and Stat5b, result in loss of pregnancy during midgestation and are correlated with an increase in ovarian 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) and a decrease in serum progesterone, which normally declines only immediately before parturition. To determine the relative contribution of 20alpha-HSD to progesterone metabolism and Stat5 function during pregnancy and parturition, we created a 20alpha-HSD-deficient strain of mice by gene disruption. Mice deficient for 20alpha-HSD sustain high progesterone levels and display a delay in parturition of several days demonstrating that 20alpha-HSD regulates parturition downstream of the prostaglandin F2alpha receptor in an essential and nonredundant manner. Moreover, 20alpha-HSD deficiency partially corrected the abortion of pregnancies associated with Stat5b deficiency, supporting the concept that prolactin activation of Stat5b is important in suppressing 20alpha-HSD gene expression and thereby allowing the maintenance of progesterone levels that are required to sustain pregnancy.

HLA-G antigen and parturition: maternal serum, fetal serum and amniotic fluid levels during pregnancy.

OBJECTIVE: To determine whether soluble HLA-G1 (sHLA-G1) concentrations in maternal serum and in amniotic fluid are lower at term than in the second trimester. METHODS: In this prospective study amniotic fluid and maternal serum samples were aspirated from 21 pregnant women during genetic amniocentesis at 16-20 weeks' gestation, and from 19 women undergoing a cesarean section at term. In the latter group arterial umbilical cord blood was aspirated as well. sHLA-G1 levels were determined using ELISA assay. This assay included the anti-HLA-G monoclonal antibodies 87G and 16G1, both as capture antibodies and horseradish-peroxidase-labeled rabbit anti-human beta(2)-microglobulin antibodies, as the detection antibody. The relative concentrations of sHLA-G1 were measured from the absorbancy of the blue product at 650 nm. Student's t test was used for statistical analysis. RESULTS: sHLA-G1 levels in amniotic fluid were significantly lower at term than in the second trimester (0.160 +/- 0.05 vs. 0.272 +/- 0.150 OD units; p < 0.05). Levels of sHLA-G1 in maternal serum declined toward term, but the difference from the second trimester was not statistically significant (0.266 +/- 0.157 vs. 0.205 +/- 0.120 OD units; p = 0.193). There was a strong correlation of sHLA-G1 concentrations between cord serum and maternal serum (R(2) = 0.79; p < 0.001), but not between cord serum and amniotic fluid (R(2) = 0.00004) or amniotic fluid and maternal serum (R(2) = 0.02). CONCLUSIONS: sHLA-G1 antigen expression is higher in amniotic fluid than in maternal-fetal compartments and significantly decreases toward term. We speculate that the declining amniotic fluid sHLA-G1 levels may stimulate a maternal immunological response against the fetus and contribute to the initiation of parturition.