Fine analysis of the short arm of chromosome 1 in sporadic and familial pheochromocytoma.

OBJECTIVE: Despite the very recent discovery that about 25% of apparently sporadic forms of pheochromocytoma are actually due to germline mutations of RET, VHL, SDHB or SDHD genes, the genetic bases of the tumourigenesis of this type of cancer are still incompletely understood. Recent studies provided evidence that a new tumour suppressor gene, mapping on the short arm of chromosome 1, could be involved in early tumourigenesis of pheochromocytoma. DESIGN: We have performed a fine analysis of loss of heterozygosity (LOH) of this region. In particular, we have analysed 31 highly polymorphic microsatellites distributed at 3.8 Mege base (Mb) mean intervals along the short arm of the chromosome 1 in paired samples of DNA extracted from peripheral blood lymphocytes and tumour tissues. PATIENTS: The study was carried out on 38 patients with pheochromocytoma that had been grouped, by careful clinical and molecular investigation, in the following classes: 21 sporadic, five multiple endocrine neoplasia type 2 (MEN2), two type 1 neurofibromatosis (NF1), five von Hippel-Lindau (VHL), one somatic VHL mutated and four nonsyndromic familial cases. RESULTS: In 12/21 sporadic cases (57.1%), in 4/5 MEN2 (80%), 2/4 non-syndromic familial cases (50%), and in 2/2 NF1 (100%), the entire short arm was deleted, while in 6/21 sporadic (28.6%) and 1/5 MEN2 (20%) cases a partial deletion was detected. On the other hand, none of the five cases due to VHL mutation (either germline or somatic) had LOH at chromosome 1. In total, complete or partial deletion of 1p was detected in 27/38 (71%) of the cases. The most frequently deleted marker was D1S2890, which maps at 1p32.1. This region, which spans from 50 to 62 Mb from telomere, was therefore further investigated with markers located at a mean interval of 1.3 Mb in the subset of cases that showed a partial deletion of 1p. This analysis showed that a small region between 55.1 and 59.0 Mb was most frequently missing, which could therefore contain a novel pheochromocytoma locus. CONCLUSIONS: The results presented here confirm that the short arm of chromosome 1 harbours one or more genes responsible for the development of pheochromocytoma and suggest that one of them could map in a 3.9-Mb fragment between 1p32.3 and 1p32.1.

Functional interactions of L-162,313 with angiotensin II receptor subtypes and mutants.

A nonpeptide ligand, L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-is obutyl-3-thienyl]phenyl]methyl]imidazo[4,5,6]pyridine) was characterized on the angiotensin II receptors. This compound displaced [125I][Sar1]angiotensin II from rat angiotensin AT1A, AT1B or AT2 receptor individually expressed in COS-7 cells (Ki = 207 nM, 226 nM and 276 nM, respectively). In monkey kidney cells expressing angiotensin AT1A or AT1B receptors, it stimulated inositol phosphate accumulation, but the maximal response was 34.9 and 23.3%, respectively, of those of angiotensin II. Furthermore, an antagonist effect of L-162.313 was observed in response to angiotensin II. Single-point substitutions in the second and third transmembrane domains of the rat angiotensin AT1A receptor, which impaired the binding of losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1[(1H-tetrazol-5-yl)biphenyl-4 -yl)methyl]imidazole), also affected the binding of L-162,313. Losartan and L-162,313 require some common structural determinants for non-peptide recognition on the angiotensin AT1 receptor. Furthermore, some of these substitutions, which impaired the inositol phosphate accumulation in response to angiotensin II, also impaired the response to L-162,313. Angiotensin II and L-162,313 require common critical residues for angiotensin AT1 receptor activation.

Angiotensin II receptors in cortical and medullary adrenal tumors.

Several pieces of evidences suggest that angiotensin II (Ang II) has mitogenic effects, and a link between Ang II receptors and adrenal tumors can be suggested. In various adrenal tumors, aldosterone-producing adenoma (APA), Cushing's adrenal adenomas (Cush), pheochromocytomas (Pheo), and adrenal carcinomas, we studied the density, affinity, and subtype of Ang II receptors. Ang II binding was tested in cell membrane homogenates. [125I]Ang II was used as ligand, and Losartan and CGP 42112 were used as selective Ang II type 1 and type 2 antagonists, respectively. In APA, Ang II receptor density was 178.5 +/- 82.7 fmol/mg: however, due to the high degree of variability, the receptor density was not significantly higher than that in nontumorous adrenal cortex (59.3 +/- 8.4 fmol/mg). In Cush, the receptor density (27.6 +/- 8.2 fmol/mg; P < 0.05) was significantly lower than that in controls, whereas in Pheo and cortical carcinoma, Ang II binding was very low and in several cases almost undetectable. There was no remarkable difference in the Ang II receptor affinity among all tissues tested. The ratio between type 1 and type 2 Ang II receptors showed a large prevalence of type 1 in controls, APA, and three cases of Cush; in two cases of Cush, this ratio was reversed. In conclusion, our data indicate that Ang II receptors are normally expressed in APA and can also be detected in Cush, whereas they have a very low density in Pheo and adrenal carcinoma. Therefore, Ang II receptors are not involved in the lack of response to Ang II that is characteristic of APA; additionally, a reduction of Ang II receptors can be associated with dedifferentiation or malignancy of adrenal tumors. Further investigation of the expression and functional characterization of Ang II receptors is required to better clarify their possible role in adrenal tumorigenesis.

Angiotensin II receptors: protein and gene structures, expression and potential pathological involvements.

Two distinct types of cell-surface angiotensin II receptors (AT1 and AT2) have been defined pharmacologically and cDNAs encoding each type have been identified by expression cloning. These pharmacological studies showed the AT1 receptors to mediate all the known functions of angiotensin II in regulating salt and fluid homeostasis. Further complexity in the angiotensin II receptor system was revealed when homology cloning showed the existence of two AT1 subtypes in rodents and in situ hybridization and reverse transcription-polymerase chain reaction analyses showed their level of expression to be regulated differently in different tissues: AT1A is the principal receptor in the vessels, brain, kidney, lung, liver, adrenal gland and fetal pituitary, while AT1B predominates in the adult pituitary and is only expressed in specific regions of the adrenal gland (zona glomerulosa) and kidney (glomeruli). Expression of AT1A appears to be induced by angiotensin II in vascular smooth-muscle cells but is inhibited in the adrenal gland. Preliminary analysis of the AT1 promoters is also suggestive of a high degree of complexity in their regulation. Investigation of a potential role for altered AT1 receptor function has commenced at a genetic level in several diseases of the cardiovascular system. No mutations affecting the coding sequence have been identified in Conn adenoma and no linkage has been demonstrated with human hypertension by sib-pair analysis. None the less, certain polymorphisms that do not alter the protein structure have been found to be associated with hypertension and to occur at an increased frequency in conjunction with specific polymorphisms in the ACE gene in individuals at increased risk for myocardial infarction. Further characterization of the regions of the AT1 gene that regulate its expression are therefore needed. The physiological importance of the AT2 gene product still remains a matter of debate.

Expression of type 1 angiotensin II receptors in human aldosteronomas.

Type 1 angiotensin II (AII) receptors (AT1 receptors), besides stimulation of aldosterone secretion, seem to transduce the growth factor-like activity of AII on glomerulosa cells. Although a local renin-angiotensin system and AII synthesis have been found in human adrenals and aldosteronomas, it is unclear whether aldosteronomas express AT1 receptors. Utilizing polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) with primers complementary to both genomic and cDNA sequences of human AT1 receptor, we have amplified and cloned a 734 bp fragment of the AT1 coding region. This DNA, after cloning and sequencing, was used for Northern analysis. Total RNA was extracted from 5 non-tumorous adrenals and 5 aldosteronomas. AT1 mRNA (approximately 2.4 kb) was expressed in all the aldosteronomas tested. Densitometric analysis of AT1 signals, corrected by beta actin expression, when compared to non-tumorous adrenals, did not show significant differences. AT1 receptor density and affinity in cell membrane obtained from 9 non-tumorous adrenal cortex and 8 aldosteronomas were also studied. 125I-AII was used as ligand and Dup 753 as AT1 antagonist: AT1 receptor density and affinity were not significantly different in aldosteronomas vs non-tumorous adrenal cortex. In conclusion, the expression of AT1 gene and the formation of an apparently normal receptor suggest that AT1 receptor should have a role in aldosteronoma cell biology.