Pediatric adrenocortical tumors: molecular events leading to insulin-like growth factor II gene overexpression.

It has been previously shown that adrenocortical tumors (ACT) in adults exhibit structural abnormalities in tumor DNA in approximately 30% of cases. These abnormalities involve chromosome 11p15 and include loss of heterozygosity, paternal isodisomy, and overexpression of the gene for insulin-like growth factor II (IGF2), correlating with DNA demethylation at this locus. It has been hypothesized that these events occur late in the tumorigenic process in adults and seem to correlate with a worse prognosis. We present 4 pediatric cases of ACT diagnosed at 2.5 yr, 10 months, 12 yr, and 2.2 yr. All 4 patients presented with virilization, and 1 patient also showed signs and symptoms of glucocorticoid excess. The youngest patient's maternal aunt had surgical excision of a more than 15-cm ACT 18 yr previously, but the aunt is doing well at age 23 yr. They all had surgical removal of their tumors. The 2.5-yr-old child also received chemotherapy and radiotherapy because of capsular rupture and, after 3 local recurrences, died 3.3 yr after initial presentation. We investigated all 4 tumors for chromosome 11 structural abnormalities (11p15.5 to 11q23), IGF2 and H19 expression by competitive RT-PCR analysis, and IGF2 methylation patterns by Southern analysis. All 4 tumors (100%) showed a combination of structural abnormalities at the 11p15 locus with mosaic loss of heterozygosity involving 11p. All tumors also had significantly increased IGF2 messenger ribonucleic acid levels relative to normal adrenal (up to 36-fold) and significant IGF2 demethylation (mean, 87%). H19 messenger ribonucleic acid levels were undetectable in 3 of 4 tumors, explained in part by mosaic loss of the actively expressed maternal allele for this imprinted gene. By immunohistochemistry we were able to confirm increased IGF-II peptide levels within the tumor tissue in 10 pediatric patients, including the 4 patients described above. Concomitantly, we also observed nuclear accumulation of p53, suggesting somatic mutations. For the 10-month-old patient, sequencing revealed a p53 germline mutation. We therefore conclude that in pediatric ACT, structural abnormalities of tumor DNA and IGF2 overexpression as well as p53 mutations are very common and are therefore less useful for prognosis than in adults. Our findings support the theory that pediatric ACT, whose IGF2 expression and steroidogenesis evoke the phenotype of the fetal adrenal cortex, may arise because of defective apoptosis.

Novel promoter polymorphism in insulin-like growth factor-binding protein-3: correlation with serum levels and interaction with known regulators.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is a major determinant of circulating levels of the IGFs and is clinically useful for the evaluation of GH deficiency and for predicting the response to GH treatment. Recent studies provide evidence that the circulating level of IGFBP-3 is inversely related to the risk of several common cancers, and that antiproliferative agents such as antiestrogens and retinoids act in part by up-regulating IGFBP-3 gene (IGFBP3) expression. Although approximately 50% of the substantial interindividual variability in circulating IGFBP-3 levels is known to have a genetic basis, the specific loci involved are unknown. Direct sequencing of genomic DNA specimens from a multiethnic population identified several single nucleotide polymorphisms in the promoter region of IGFBP3. For the most common single nucleotide polymorphism (nucleotide -202) found to be in Hardy-Weinberg equilibrium, genotype was highly correlated to circulating level of IGFBP-3 in 478 men from the Physicians' Health Study. In vitro, we documented significantly higher promoter activity of the A allele at the -202 locus compared with the C allele, consistent with the relationship observed between genotype and circulating IGFBP-3 (AA > AC > CC). A positive correlation was observed between circulating retinol levels and circulating IGFBP-3 levels; subset analysis by genotype showed that this relationship was only present among individuals carrying an A allele at -202 (AA > AC > CC). Tall individuals or individuals with a body mass index of 27 or greater had levels of circulating IGFBP-3 that were significantly higher when they possessed at least one A allele (AA > AC > CC). The IGFBP3 promoter region deserves investigation as a locus where polymorphic variation occurs frequently and may influence GH responsiveness, somatic growth, and possibly cancer risk.

Genetic and nongenetic factors associated with variation of plasma levels of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in healthy premenopausal women.

Circulating levels of insulin-like growth factor-I (IGF-I) and insulin-like growth factor-binding protein 3 (IGFBP-3) vary considerably between normal individuals. Recent epidemiological studies have provided evidence that these levels are predictive of risk of several common cancers. To evaluate possible sources of variation of the levels of circulating IGF-I and IGFBP-3 in females, we studied specific candidate genetic and nongenetic factors in 311 nulliparous, premenopausal Caucasian women, 17-35 years of age. Women who used oral contraceptives (OC) had reduced levels of IGF-I (269 versus 301 ng/ml; P = 0.001 adjusted for age) and increased levels of IGFBP-3 (4213 versus 4009 ng/ml; P = 0.002, adjusted for age) compared with nonusers. The ratio of IGF-I:IGFBP-3 was associated with the dose of estrogen contained in the OC (P(trend) = 0.006, adjusted for age). We identified a novel single bp polymorphism in the promoter region of the gene encoding IGFBP-3. This polymorphism was related to the level of IGFBP-3 in the circulation. Mean IGFBP-3 levels were 4390, 4130, and 3840 ng/ml for the AA, AC, and CC genotypes, respectively (P(trend) = 0.006, adjusted for age and OC use). We observed no effect of a recently described polymorphism in the promoter region of the gene encoding IGF-I on the plasma IGF-I level, but there was evidence for a modifying effect of this locus on the influence of OC on the IGF-I level. Our results support the view that circulating IGF-I levels and IGFBP-3 levels are complex traits and are influenced by a number of interacting genetic and nongenetic factors.

Identification and characterization of novel genes modulated in the thyroid of dogs treated with methimazole and propylthiouracil.

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific stimulation or repression of a large number of genes. To better understand differentiated epithelial cell growth regulation, we have initiated a study to identify genes which are regulated by the thyrotropin-dependent mitogenic pathway in dog thyroid cells. A thyroid cDNA library was prepared from a methimazole and propylthiouracil-treated dog and differentially screened with probes derived from control or stimulated thyroids. Among 19 clones isolated, 6 encode known proteins (inwardly rectifying potassium channel, nucleosome assembly protein, ribosomal protein L7, elongation factor 1alpha, non-muscle myosin light chain, and heat shock protein 90beta). The 13 others correspond to proteins whose function is unknown. Among them, 5 correspond to mRNAs whose expression was modulated by mitogenic stimulation of thyrocytes in primary culture. A preliminary characterization of two of these cDNAs is reported: clone 5, which might represent a novel, atypical protein kinase, and clone 3, which contains ankyrin-like repeats, suggesting that it might interact with other proteins.

Phosphorylation of mitogen-activated protein kinases is involved in the epidermal growth factor and phorbol ester, but not in the thyrotropin/cAMP, thyroid mitogenic pathway.

In dog thyroid epithelial cells (thyrocytes) in primary culture, thyrotropin (TSH) acting through cAMP induces proliferation and differentiation expression, whereas epidermal growth factor (EGF) and tumor-promoting phorbol esters induce proliferation and dedifferentiation. In these cells we have demonstrated mitogen-activated protein (MAP) kinase phosphorylation by 32P labeling and two-dimensional gel electrophoresis and by immunodetection with anti-MAP kinase and anti-phosphotyrosine antibodies after one- or two-dimensional gel electrophoresis. MAP kinase localization was demonstrated by immunochemical staining. We show the following results. (i) As in other systems, EGF and phorbol esters induced p42 and p44 MAP kinases phosphorylation on tyrosine, serine, and threonine. This effect was rapid, peaking after 5 and 15 min, respectively, followed by a slow decline thereafter. It preceded a translocation of MAP kinase immunoreactivity from cytoplasm to nucleus. (ii) Carbamylcholine, a potent stimulator of the Ca(2+)-phosphatidylinositol cascade which is unable to induce DNA synthesis, stimulated MAP kinases phosphorylation and nuclear staining with kinetics similar to those observed after EGF action, indicating that MAP kinase phosphorylation was not sufficient for mitogenesis. (iii) The cAMP-dependent mitogenic cascade elicited by TSH and forskolin did not involve the phosphorylation and nuclear translocation of p42 and p44 MAP kinases at any time during the entire prereplicative phase. Activation of MAP kinases by phosphorylation is therefore not a necessary step in the G0-G1 transition in this mitogenic cascade.

The P2Y11 receptor mediates the ATP-induced maturation of human monocyte-derived dendritic cells.

Recently, it has been shown that ATP and TNF-alpha synergize in the activation and maturation of human dendritic cells (DC); the effect of ATP was reproduced by hydrolysis-resistant derivatives of ATP and was blocked by suramin, suggesting the involvement of a P2 receptor, but the particular subtype involved was not identified. In this report we confirm that ATP and various derivatives synergize with TNF-alpha and LPS to induce the maturation of human monocyte-derived DC, as revealed by up-regulation of the CD83 marker and the secretion of IL-12. The rank order of potency of various analogs (AR-C67085 > adenosine 5'-O-(3-thiotriphosphate) = 2'- and 3'-O-(4-benzoyl-benzoyl) ATP > ATP > 2-methylthio-ATP) was close to that of the recombinant human P2Y11 receptor. Furthermore, these compounds activated cAMP production in DC, in a xanthine-insensitive way, consistent with the involvement of the P2Y11 receptor, which among P2Y subtypes has the unique feature of being dually coupled to phospholipase C and adenylyl cyclase activation. The involvement of the P2Y11/cAMP/protein kinase A signaling pathway in the nucleotide-induced maturation of DC is supported by the inhibitory effect of H89, a protein kinase A inhibitor. Taken together, our results demonstrate that ATP activates DC through stimulation of the P2Y11 receptor and subsequent increase in intracellular cAMP.

H19 sense and antisense transgenes modify insulin-like growth factor-II mRNA levels.

The oppositely-imprinted genes insulin-like growth factor-II (IGF2) and H19, a putative tumor suppressor, often show coordinate, reciprocal regulation and are believed to play a role in carcinogenesis. To explore the possible interactions between these genes, we stably transfected diHepG2 cells with a plasmid containing either the sense or the antisense H19 cDNA sequences and verified their expression by Northern analysis and by RNase protection analysis. Levels of H19, IGF2 and gamma-actin mRNA were quantified by competitive RT-PCR analysis. Although H19 sense transgene overexpression (n = 24 clones) did not decrease the low, basal levels of IGF2 mRNA compared to control cells, levels of IGF2 mRNA were positively correlated with the levels of H19 antisense mRNA (P < 0.0001, n = 40 clones). Furthermore, the increase in IGF2 mRNA level was accompanied by an elevation of IGF-II peptide in conditioned media. To see if H19 mRNA had a specific effect on transcription, we also performed transient transfections with reporter gene constructs containing IGF2 promoter 3 in the presence of sense or antisense H19 cDNA sequences under control of a cytomegalovirus promoter. We show a lower reporter gene activity from reporter gene constructs in the presence of sense H19 cDNA than from those with antisense or neomycin. Our results suggest that H19 participates in the repression of IGF2, at least in part through effects on IGF2 transcription, an effect which may contribute to its action as a tumor suppressor.

Cloning of cDNA specifically involved in the thyroid cAMP mitogenic pathway.

The activation of the cyclic AMP cascade in dog and human thyroid cells in primary culture induces the expression of differentiated gene expression, hyperfunction and proliferation. These programs are developed simultaneously in quiescent dedifferentiated cells. In this paper the strategy followed by our group to define the genes involved in the cAMP mitogenic cascade is outlined.

Characterization of a phosphoprotein whose mRNA is regulated by the mitogenic pathways in dog thyroid cells.

We have isolated cDNA clones encoding the dog and human forms of a novel protein whose function is still unknown. Sequence analysis indicates that dog clone c5fw protein contains 343 amino acid residues. several potential phosphorylation sites. and two of the 12 conserved subdomains (VIII and IX) that fold into a common catalytic core structure of the large family of protein kinases. Human clone c5fw shares 95% amino acid identity with its dog counterpart. We have also isolated another human-related clone c5fw sharing 70% amino acid identity with the dog sequence. We transiently expressed c-myc epitope-tagged clone c5fw protein in COS-7 cells and infected thyrocytes in primary culture with a recombinant adenovirus containing clone c5fw cDNA (adenovirus c5fw). In both experiments, a 46-kDa protein was detected and subsequently more extensively characterized. By two-dimensional gel electrophoresis and V8 protease digestion, we showed that this overexpressed protein is phosphorylated on different sites. Moreover, cells stimulated with thyrotropin or epidermal growth factor, thyrotropin and fetal calf serum increased the level of clone c5fw protein produced after infection by adenovirus containing clone c5fw. The disappearance of this 46-kDa protein after 1 h of puromycin treatment indicates that it is a labile protein. Immunofluorescence and subcellular fractionation analysis have revealed that c-myc-tagged clone c5fw was insoluble and localized mainly in the cytoplasm, in the form of granules.

Molecular characterization of a pediatric pheochromocytoma with suspected bilateral disease.

An 11-year-old boy with hypertension was suspected of having bilateral adrenal pheochromocytomas and hyperplasia. Molecular analysis of specific tumor suppressor genes and oncogenes excluded the familial syndromes, von Hippel-Lindau (VHL) disease and multiple endocrine neoplasia (MEN) type 2A. Further evaluation identified a unilateral adrenal pheochromocytoma with a VHL heterozygous somatic mutation (G695A) and loss of the maternal allele at 11p15.5-11p14 exclusively in the tumor tissue. Both reverse-transcriptase polymerase chain reaction and immunohistochemistry confirmed increased expression of IGF2 within the tumoral tissue, relative to a normal control adrenal gland. These results ruled out familial syndromes and suggested that the VHL mutation and the loss of maternal allele on chromosome 11 could have contributed to tumor development.