Identification of SOCS2 and SOCS6 as biomarkers in human colorectal cancer.

BACKGROUND: Over the past years, some members of the family of suppressor of cytokine signalling (SOCS) proteins have emerged as potential tumour suppressors. This study aimed at investigating the clinical significance of SOCS proteins in colorectal carcinoma (CRC). METHODS: We integrated publicly available microarray expression data on CRC in humans, analysed the expression pattern of SOCSs and assessed the predictive power of SOCS2 and SOCS6 for diagnostic purposes by generating receiver operating characteristic curves. Using laser microdissected patient material we assessed SOCS expression on RNA and protein levels as well as their methylation status in an independent CRC patient cohort. Finally, we investigated the prognostic value of SOCS2 and SOCS6. RESULTS: The meta-analysis as well as the independent patient cohort analysis reveal a stage-independent downregulation of SOCS2 and SOCS6 and identify both molecules as diagnostic biomarkers for CRC. We demonstrate a different methylation pattern within the SOCS2 promoter between tumour tissue and normal control tissue in 25% of CRC patients. Furthermore, early CRC stage patients with low expression of SOCS2 display significantly shorter disease-free survival. CONCLUSIONS: Our data offers evidence that SOCS2 and SOCS6 levels are reduced in CRC and may serve as diagnostic biomarkers for CRC patients.

Time-resolved analysis of transcriptional events during SNAI1-triggered epithelial to mesenchymal transition.

The transcription regulator SNAI1 triggers a transcriptional program leading to epithelial to mesenchymal transition (EMT), providing epithelial cells with mesenchymal features and invasive properties during embryonic development and tumor progression. To identify early transcriptional changes occurring during SNAI1-induced EMT, we performed a time-resolved genome-scale study using human breast carcinoma cells conditionally expressing SNAI1. The approach we developed for microarray data analysis, allowed identifying three distinct EMT stages and the temporal classification of genes. Importantly, we identified unexpected, biphasic expression profiles of EMT-associated genes, supporting their pivotal role during this process. Finally, we established early EMT gene networks by identifying transcription factors and their potential targets which may orchestrate early events of EMT. Collectively, our work provides a framework for the identification and future systematic analysis of novel genes which contribute to SNAI1-triggered EMT.

Two G protein oncogenes in human endocrine tumors.

Somatic mutations in a subset of growth hormone (GH)-secreting pituitary tumors convert the gene for the alpha polypeptide chain (alpha s) of Gs into a putative oncogene, termed gsp. These mutations, which activate alpha s by inhibiting its guanosine triphosphatase (GTPase) activity, are found in codons for either of two amino acids, each of which is completely conserved in all known G protein alpha chains. The likelihood that similar mutations would activate other G proteins prompted a survey of human tumors for mutations that replace either of these two amino acids in other G protein alpha chain genes. The first gene so far tested, which encodes the alpha chain of Gi2, showed mutations that replaced arginine-179 with either cysteine or histidine in 3 of 11 tumors of the adrenal cortex and 3 of 10 endocrine tumors of the ovary. The mutant alpha i2 gene is a putative oncogene, referred to as gip2. In addition, gsp mutations were found in 18 of 42 GH-secreting pituitary tumors and in an autonomously functioning thyroid adenoma. These findings suggest that human tumors may harbor oncogenic mutations in various G protein alpha chain genes.

Rapid and reversible secretion changes during uncoupling of rat insulin-producing cells.

To determine whether insulin secretion is affected by a blockage of gap junctions between B cells, we have studied the secretion of rat pancreatic islets of Langerhans, primary dispersed islet cells, and cells of the RINm5F line, during short-term exposure to heptanol. Within minutes, this alkanol blocked gap junctions between the B cells of intact islets and abolished their normal secretory response to glucose. These two changes were rapidly and fully reversible after return of the islets to control medium. We further found that heptanol had no significant effect on the glucose-stimulated secretion of single B cells but inhibited that of B cell pairs. In the clone of RINm5F cells, whose junctional coupling and D-glyceraldehyde-induced stimulation of insulin release by aggregated cells were also inhibited by heptanol, this alkanol did not perturb intracellular pH and Ca2+ and the most distal steps of the secretion pathway. In summary, a gap junction blocker affected the secretion of insulin-producing cells by a mechanism which is dependent on cell contact and is not associated with detectable pleiotropic perturbations of the cell secretory machinery. The data provide evidence for the involvement of junctional coupling in the control of insulin secretion.

Activation of B-Raf and regulation of the mitogen-activated protein kinase pathway by the G(o) alpha chain.

Many receptors coupled to the pertussis toxin-sensitive G(i/o) proteins stimulate the mitogen-activated protein kinase (MAPK) pathway. The role of the alpha chains of these G proteins in MAPK activation is poorly understood. We investigated the ability of Galpha(o) to regulate MAPK activity by transient expression of the activated mutant Galpha(o)-Q205L in Chinese hamster ovary cells. Galpha(o)-Q205L was not sufficient to activate MAPK but greatly enhanced the response to the epidermal growth factor (EGF) receptor. This effect was not associated with changes in the state of tyrosine phosphorylation of the EGF receptor. Galpha(o)-Q205L also potentiated MAPK stimulation by activated Ras. In Chinese hamster ovary cells, EGF receptors activate B-Raf but not Raf-1 or A-Raf. We found that expression of activated Galpha(o) stimulated B-Raf activity independently of the activation of the EGF receptor or Ras. Inactivation of protein kinase C and inhibition of phosphatidylinositol-3 kinase abolished both B-Raf activation and EGF receptor-dependent MAPK stimulation by Galpha(o). Moreover, Galpha(o)-Q205L failed to affect MAPK activation by fibroblast growth factor receptors, which stimulate Raf-1 and A-Raf but not B-Raf activity. These results suggest that Galpha(o) can regulate the MAPK pathway by activating B-Raf through a mechanism that requires a concomitant signal from tyrosine kinase receptors or Ras to efficiently stimulate MAPK activity. Further experiments showed that receptor-mediated activation of Galpha(o) caused a B-Raf response similar to that observed after expression of the mutant subunit. The finding that Galpha(o) induces Ras-independent and protein kinase C- and phosphatidylinositol-3 kinase-dependent activation of B-Raf and conditionally stimulates MAPK activity provides direct evidence for intracellular signals connecting this G protein subunit to the MAPK pathway.

Retraction: miR-661 expression in SNAI1-induced epithelial to mesenchymal transition contributes to breast cancer cell invasion by targeting Nectin-1 and StarD10 messengers.

At the request of the University of Luxembourg and following an external investigation, the Editor and Publisher have agreed to retract this paper owing to unreliable data.

Preventive and chronic mineralocorticoid receptor antagonism is highly beneficial in obese SHHF rats.

BACKGROUND AND PURPOSE: Mineralocorticoid receptor (MR) activation contributes to heart failure (HF) progression. Its overactivity in obesity is thought to accelerate cardiac remodelling and HF development. Given that MR antagonists (MRA) are beneficial in chronic HF patients, we hypothesized that early MRA treatment may target obesity-related disorders and consequently delay the development of HF. EXPERIMENTAL APPROACH: Twenty spontaneously hypertensive HF dyslipidaemic obese SHHF(cp/cp) rats and 18 non-dyslipidaemic lean SHHF(+/+) controls underwent regular monitoring for their metabolic and cardiovascular phenotypes with or without MRA treatment [eplerenone (eple), 100 mg∙kg(-1) ∙day(-1) ] from 1.5 to 12.5 months of age. KEY RESULTS: Eleven months of eple treatment in obese rats (SHHF(cp/cp) eple) reduced the obesity-related metabolic disorders observed in untreated SHHF(cp/cp) rats by reducing weight gain, triglycerides and total cholesterol levels and by preserving adiponectinaemia. The MRA treatment predominantly preserved diastolic and systolic functions in obese rats by alleviating the eccentric cardiac hypertrophy observed in untreated SHHF(cp/cp) animals and preserving ejection fraction (70 ± 1 vs. 59 ± 1%). The MRA also improved survival independently of these pressure effects. CONCLUSION AND IMPLICATIONS: Early chronic eple treatment resulted in a delay in cardiac remodelling and HF onset in both SHHF(+/+) and SHHF(cp/cp) rats, whereas SHHF(cp/cp) rats further benefited from the MRA treatment through a reduction in their obesity and dyslipidaemia. These findings suggest that preventive MRA therapy may provide greater benefits in obese patients with additional risk factors of developing cardiovascular complications.

Expression of mutationally activated G alpha s stimulates growth and differentiation of thyroid FRTL5 cells.

The alpha subunit of the GTP-binding protein Gs mediates hormonal stimulation of adenylyl cyclase. Human pituitary and thyroid tumours harbour mutations of G alpha s that constitutively activate the protein by inhibiting its intrinsic GTPase activity. We have investigated the mitogenic action of mutationally activated alpha s in thyroid FRTL5 cells, a cell line dependent upon thyroid-stimulating hormone (TSH) for both growth and differentiation. Introduction of alpha s carrying the substitution of glutamine-227 with leucine (Q227L alpha s) by retroviral infection of FRTL5 cells resulted in the expected stimulation of membrane adenylyl cyclase activity and in increased intracellular accumulation of cAMP. Measurements of cytosolic Ca2+ levels did not detect any concomitant effect on the polyphosphoinositide-Ca2+ signalling pathway. Expression of Q227L alpha s conferred to FRTL5 cells the ability to synthesize DNA in the absence of TSH, as revealed by [3H]thymidine incorporation experiments, and to proliferate independently of the mitogenic hormone, although with a rate of growth slower than that observed with TSH stimulation. The effect of Q227L alpha s on cell proliferation was associated with the constitutive activation of iodide uptake. The results indicate that expression of mutationally activated G alpha s is sufficient to bypass the requirement for TSH and promotes autonomous growth and activation of thyroid-specific differentiated functions in FRTL5 cells.

Cloning of an isoform of integrin-linked kinase (ILK) that is upregulated in HT-144 melanoma cells following TGF-beta1 stimulation.

We have shown previously that integrin-linked kinase (ILK) is upregulated in human HT-144 melanoma cells following TGF-beta1 stimulation. Using mRNA from TGF-beta1 stimulated HT-144 cells and reverse transcriptase polymerase chain reaction, we have isolated a cDNA encoding a protein highly homologous to ILK. Sequencing of the full-length 1359 base pair cDNA and polypeptide translation revealed that this protein, designated ILK-2, differs from the known ILK (hereafter called ILK-1) by only four amino acids, while the cDNA sequence diverges by 102 nucleotides, thus excluding that ILK-2 is an allelic variant of ILK-1. Expression of ILK-2 mRNA was observed in metastatic human HT-144 melanoma and HT-1080 fibrosarcoma cell lines, but not in normal human tissues. Moreover, stimulation of HT-144 cells with TGF-beta1, but not with EGF, PDGF-AB or insulin, induced a selective overexpression of ILK-2 mRNA as compared to ILK-1 mRNA. Bacterially-expressed GST/ILK-2 autophosphorylated and labeled myelin basic protein as well as a recombinant GST/beta3 integrin cytoplasmic tail peptide. Transfection of either ILK-2 or ILK-1 cDNA into the non-metastatic melanoma cell line SK-Mel-2, expressing exclusively ILK-1, induced anchorage independent cell growth and cell proliferation, as demonstrated by growth in soft agar. Our data provide evidence that ILK-2 is a new isoform of ILK-1 that is expressed in some highly invasive tumor cell lines but not in normal adult human tissues and whose expression is regulated by TGF-beta1.

Mechanisms of signal transduction at the dopamine D2 receptor.

D2 dopamine receptor activation induces inhibition of adenylate cyclase, with a rapid decrease of cAMP levels, and an ensuing blockade of IP3-dependent release of Ca2+ from intracellular stores. K+ channels are concomitantly activated and Ca2+ channels are possibly also inhibited. The increased K+ conductance causes hyperpolarization, which may be responsible for the abolition of Ca2+ action potentials and [Ca2+]i fluctuations occurring both at rest and after activation of receptors coupled to PIP2 hydrolysis. Lucia Vallar and Jacopo Meldolesi analyse this spectrum of intracellular signals which might be sufficient to sustain inhibition of secretion in pituitary lactotroph cells and possibly the other effects of D2 receptors in other cell systems.

G protein oncogenes in pituitary tumors.

G proteins are involved in the transduction of external signals from cell surface receptors to intracellular effectors. Somatic mutations activating the a-subunit of G(s) (the stimulatory regulatory protein of adenylyl cyclase) by inhibiting its intrinsic GTPase activity have been first identified in human GH-secreting adenomas and subsequently found in thyroid tumors and in McCune-Albright syndrome. It has been therefore proposed that the gene encoding the GS a-subunit may be converted into an oncogene (gsp for GS protein) in cell types that proliferate in response to cAMP. Since several G proteins mediate signaling pathways that are effective in coupling external stimuli to cell proliferation, it appears most likely that in the near future other G protein oncogenes will be identified in human tumors.

Activation of cyclic nucleotide phosphodiesterases in FRTL-5 thyroid cells expressing a constitutively active Gs alpha.

The expression of a constitutively activated Gs alpha protein in the rat thyroid cell line FRTL-5 causes an increase in the hormone-independent adenylyl cyclase activity and promotes TSH-independent growth of the cells. In spite of the constitutive activation of the adenylyl cyclase, the basal cAMP levels in these cells are only marginally increased. To define the role of phosphodiesterases (PDEs) in the genesis of this phenotype, cyclic nucleotide hydrolysis was determined in two cell lines expressing a mutated Gs alpha (Q227L). In these cells, the hydrolysis of both cAMP and cGMP was markedly increased in comparison with normal cells. This increase is the result of the activation of different forms of PDEs. Analysis of the cGMP hydrolysis and Ca++/calmodulin stimulation of the PDE activity indicated that the activity of a Ca++/calmodulin-stimulated PDE is increased in both cell lines. In addition, an increase in high-affinity, rolipram-sensitive cAMP-PDE activity was associated in both cell lines with the appearance of a 67-68 kilodalton (kDa) protein that cross-reacts with two antibodies against cAMP-PDEs. This form had the properties of ratPDE3.2/PDE4D2, a cAMP-PDE that is inducible by TSH in wild type cells. That an increase in cAMP-specific, rolipram-sensitive PDE plays a role in the phenotype induced by Q227L Gs alpha was confirmed by measurements of the mitogenic activity. Incubation with rolipram, which had no effect on wild type cells, caused an increase in cAMP levels and further stimulated TSH-independent proliferation in both cell lines carrying the mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of an adenylate cyclase dually regulated by somatostatin and human pancreatic growth hormone (GH)-releasing factor in GH-secreting cells.

We have investigated the effects of SRIF and human pancreatic GH-releasing factor-44 (hpGRF-44) on adenylate cyclase (AC) activity of male rat anterior pituitaries (in which somatotrophs are present in large proportion) and of human GH-secreting pituitary adenomas (which are almost homogeneously constituted by somatotrophs). The adenoma's responsiveness to both agents in terms of secretion was previously demonstrated in in vitro experiments. SRIF inhibited in a dose-dependent fashion the GH release from monolayer cultures of the tumors. The inhibition ranged from 32-66% at the maximal effective concentration (10(-6) M). hpGRF-44 stimulated GH release in a dose-dependent fashion. The stimulation was 78-172% at 10(-7) M. SRIF and hpGRF-44 markedly affected AC activity in both systems. SRIF elicited a pronounced inhibition of the enzyme activity in a dose-dependent manner. The inhibition was about 40% in the rat and ranged from 16-49% in adenomas at the maximal effective concentration (10(-5) M SRIF). The inhibitory effect was GTP-dependent. hpGRF-44 markedly stimulated AC activity. The stimulation was dose dependent and GTP dependent. The stimulation was about 650% in the rat and 26-350% in adenomas at the maximal effective concentration (10(-6) M). These results suggest the presence of a dually regulated (by SRIF and hpGRF-44) AC in GH-secreting cells; an involvement of cAMP in the intracellular mechanisms transducing the signals of SRIF and hpGRF-44 in somatotrophs.

Effect of calcium on adenylate cyclase of rat anterior pituitary gland.

The effect of free calcium (Ca2+) on adenylate cyclase (AC) activity of rat anterior pituitary gland have been investigated in order to shed some light on the interrelationships between the two second messengers (cAMP and calcium) which operate in pituitary cells. Anterior pituitary homogenates or crude membranes preparations (obtained using buffers free of divalent cation chelators) were assayed and the concentrations of Ca2+ in the assay mixture containing EGTA were calculated by a computer program for each addition of CaCl2. A wide range of Ca2+ concentrations (from 2 X 10(-9) to 6 X 10(-4)M) was spanned. Ca2+ was found to markedly inhibit pituitary AC and the mathematical analysis of data indicated the presence of two inhibition The two KiS were: 1.78 +/- 0.48 X 10(-7) M and 2.47 +/- 0.52 X 10(-4) M for the homogenates and 1.71 +/- 0.45 X 10(-7) M and 3.15 +/- 0.85 X 10(-4) M for the membrane preparations. No stimulation of the enzyme could be detected at any Ca2+ concentration tested. Furthermore, because of our experimental conditions it is unlikely that there was substantial loss of endogenous calmodulin, or other calcium binding protein(s) required to mediate AC stimulation by calcium. The lack of a calcium-calmodulin stimulation of pituitary AC was confirmed by experiments with anticalmodulin drugs (trifluoperazine and calmidazolium, R24571) and experiments with EGTA-washed membranes in the presence of exogenous calmodulin. At any Ca2+ concentration, the same AC activity was observed in the presence and in the absence of anticalmodulin drugs or added calmodulin. The mechanism of pituitary AC inhibition by Ca2+ was investigated focusing on a range of Ca2+ concentrations near the Ki for the high affinity calcium site and thus similar to the intracellular Ca2+ concentrations. Ca2+ was found to act as a competitive inhibitor of the Mg2+ activation of AC and as a noncompetitive inhibitor with respect to the MgATP2-, the substrate of the enzyme. The effects of Ca2+ on AC were also studied in cell populations and tissues extremely rich in PRL-secreting cells (cell fractions purified from rat anterior pituitaries and human prolactinomas). The pattern of Ca2+ action was found to be nearly superimposable on that observed in total pituitary.(ABSTRACT TRUNCATED AT 400 WORDS)

Alpha 1-adrenergic stimulation of in vitro growth hormone release and cytosolic free Ca2+ in rat somatotrophs.

The effects of alpha 1-adrenergic agents on GH release and intracellular free Ca2+ concentration ([Ca2+]i) were investigated in purified rat somatotroph preparations. Phenylephrine (PHE) stimulated in vitro GH release; the maximal effect (2.5-fold stimulation) occurred at 1 microM PHE. The effect was completely blocked by the alpha-adrenergic antagonist phentolamine and partially counteracted by the beta-antagonist propranolol. Experiments with the fluorescent Ca2+ probe fura 2 show that PHE causes [Ca2+]i to rise from 178 +/- 31 nM (mean +/- SE; n = 25) to 370 +/- 55 nM (n = 9). This effect was complete within 20 sec and was maintained for at least 5-10 min. The rise was rapidly interrupted by administration of 1 microM phentolamine. The beta-receptor agonist isoproterenol caused a small [Ca2+]i rise due to action on alpha 1-adrenoreceptors. The PHE-induced [Ca2+]i rise showed two components: an initial peak due to Ca2+ mobilization from intracellular stores and a subsequent rise due to Ca2+ influx from the extracellular space. Somatostatin (SRIF) lowered both resting [Ca2+]i and Ca2+ influx stimulated by PHE. Pertussis toxin pretreatment did not modify PHE-induced [Ca2+]i changes, while it completely prevented the effect of SRIF on both resting and triggered [Ca2+]i, thus suggesting that a GTP-binding protein sensitive to the toxin is involved in the transduction of SRIF action. The increase in cAMP induced by cholera toxin pretreatment modified neither PHE nor SRIF action on [Ca2+]i. In conclusion, in rat somatotrophs Ca2+ mobilization and influx are stimulated by alpha 1-adrenergic agents, and this triggered [Ca2+]i rise results in a stimulation of GH release. In these cells SRIF is able to reduce both resting [Ca2+]i levels and [Ca2+]i increases induced by alpha 1-adrenergic activation.

Morphological studies on mixed growth hormone (GH)- and prolactin (PRL)-secreting human pituitary adenomas. Coexistence of GH and PRL in the same secretory granule.

A morphological study was carried out on five mixed GH- and PRL-secreting pituitary adenomas, surgically removed from acromegalic patients with hyperprolactinemia, in order to verify whether the two hormones were contained in the same cell or in different cells. Double labeling with the protein A-gold immunotechnique was used to visualize the ultrastructural localization of the two hormones on ultrathin sections of the tumors. By means of this high resolution technique we found in all adenomas the presence of numerous (from 50-80% of the whole cell population) mammosomatotrophs, i.e. cells containing simultaneously PRL and GH. The occurrence of cells producing only GH (in four tumors) or only PRL (in one tumor) was also observed. In mixed cells GH and PRL were segregated in the same mixed granule. In one tumor granules positive only for GH together with mixed granules were found in the same cell. Immunofluorescence studies, at the light microscopic level, allowed us to clearly identify mammosomatotrophs only in two tumors. Double labeling using the gold immunotechnique appears therefore to be the most suitable experimental approach to detect the existence of mixed cells in plurihormonal adenomas. Our results support the idea that the frequency of mixed adenomas with mixed cells may be higher than that believed previously. The simultaneous presence of two hormones in the same secretory granule could explain why, in patients having mixed tumors, factors able to stimulate or inhibit the release of one hormone can also stimulate or inhibit the secretion of the other.

Lack of desensitization of adenomatous somatotrophs to growth-hormone releasing hormone in acromegaly.

The study was undertaken to investigate whether GHRH causes desensitization in GH-secreting adenomas in analogy to the normal situation. For this purpose, GH secretion after repeated GHRH administration to acromegalic patients in vivo and cultured adenomatous somatotrophs in vitro was studied. Six acromegalic patients and 6 normal subjects received 3 consecutive 50 micrograms GHRH rapid iv injections at 2-h intervals. Blood samples were drawn at 0, 15, 30, 60, and 120 min after each dose. The acromegalic patients had variable responses to the first injection and in each patient the second and third injections elicited serum GH responses that were quite similar to those after the first one. The GH increment, evaluated as net incremental area under the curve (mean +/- SE; nanograms per ml/120 min) was 2660 +/- 1501 after the first injection, 2176 +/- 1378 after the second injection, and 1978 +/- 1191 after the third injection; P = NS. On the contrary, in normal subjects a marked elevation of GH was observed only after the first injection (net incremental area under the curve, mean +/- SE after the first injection: 710 +/- 154; after the second injection: 6 +/- 39 and after the third injection: 108 +/- 28, P less than 0.01 vs. the first dose). The effect of in vitro GHRH pretreatment on the subsequent response to GHRH was evaluated in monlayer cultures from 12 GH-secreting adenomas. At 10(-8) M, GHRH significantly stimulated GH release from 8 adenomas. The GHRH pretreatment that was effective in inducing desensitization in cultured rat anterior pituitary cells, i.e. preexposure of the cultured cells to 10(-8) M GHRH for 4 h at 37 C, did not abolish the GH response to the subsequent challenge with GHRH (mean percent stimulation: 150 +/- 14% in untreated cells vs. 153 +/- 30% in pretreated cells; P = NS). Only in one adenoma, did GHRH-promoted desensitization occur. No modification was found in GHRH unresponsive adenomas. In the adenomas, not only the efficacy but also the potency of GHRH on GH release after GHRH pretreatment was of the same order of magnitude as in untreated cells. No desensitization to GHRH action occurred when the pretreatment time was prolonged up to 8 h and the GHRH concentration in the preincubation medium was increased to 10(-7) M.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential transduction of dopamine signal in different subtypes of human growth hormone-secreting adenomas.

This article reports the effect of dopamine (DA) on adenylyl cyclase (AC) activity and intracellular free calcium concentration ([Ca2+]i) in 20 GH-secreting pituitary adenomas exclusively composed of somatotrophs (GH-omas) and 3 tumors largely constituted by mammosomatotrophs (MS-omas). DA (between 10 nmol/L and 100 mumol/L) did not reduce AC activity in any GH-omas, whereas the amine caused a significant inhibition in membranes from all MS-omas. The effect was detectable at DA concentrations higher than 0.1 mumol/L, and maximal inhibition (ranging from 24-30%) was reached at 10 mumol/L. The ergot derivative CH 29717 and l-sulpiride demonstrated potent agonist and antagonist activities, respectively. Somatostatin reduced AC activity in all tumors; the percent inhibition values (between 17-34%) were similar in GH-omas and MS-omas. In both GH-omas and MS-omas, DA (1 mumol/L) caused a significant [Ca2+]i reduction (between 17-44%) that was essentially due to the block of Ca2+ influx from the extracellular spaces. The receptors involved in this effect showed the pharmacological properties of D2 receptors. In conclusion, the DA effect in tumoral somatotrophs is defective; DA fails to exert an inhibitory action on AC activity. In mammosomatotrophs, the typical D2 receptor-effector coupling is retained, resulting in decreased AC activity in these cells.

Activating mutations of the Gs alpha gene are associated with low levels of Gs alpha protein in growth hormone-secreting tumors.

Evidence suggests the existence of a direct relationship between cellular Gs alpha content and activation of the adenylyl cyclase system. Data on Gs alpha levels in endocrine tumors that depend on cAMP for growth, particularly pituitary adenomas, are still limited. The levels of Gs alpha protein were evaluated in 11 GH-secreting adenomas with Gs alpha mutations (gsp+) and 15 without (gsp). Complementary DNAs from gsp+ tumors contained very low amounts of wild-type Gs alpha sequences, indicating a preponderance of the mutant Gs alpha transcripts in these tumors. Immunoblotting of Gs alpha protein showed that the two isoforms were present at high levels in all gsp-, but were undetectable or barely detectable in gsp+. The low Gs alpha content in gsp+ tumors was not due to a reduction in ribonucleic acid synthesis or stability, as Gs alpha messenger ribonucleic acid levels were similar in wild-type and mutant tissues. Treatment of gsp- cells with cholera toxin caused a marked reduction of Gs alpha levels. As in other cell systems cholera toxin increases Gs alpha degradation, our data are consistent with an accelerated removal of mutant Gs alpha. This may represent an additional mechanism of feedback response to the constitutive activation of cAMP signaling in pituitary tumors with mutations in the Gs alpha gene.

Clinical, biochemical, and morphological correlates in patients bearing growth hormone-secreting pituitary tumors with or without constitutively active adenylyl cyclase.

Somatic mutations in the alpha-chain (alpha s) of the stimulatory regulatory protein of adenylyl cyclase (Gs) causing constitutive activation of the enzyme have been identified in a subset of human GH-secreting pituitary adenomas. This study reports on the differences between acromegalic patients bearing tumors without (group 1; n = 51) or with (group 2; n = 29) this alteration. No difference in age, sex, clinical features, duration of the disease, or cure rate was observed between the two groups. By contrast, group 2 patients had higher basal GH levels than group 1. Moreover, a significant difference in sellar morphology was found; group 2 patients more frequently showed sellas of normal size (grade I) than group 1. Hypersecretory activity of group 2 tumors was also apparent at electron microscopy; contrary to those of group 1, cells of group 2 tumors were densely granulated and showed prominent rough endoplasmic reticulum and Golgi complex. With respect to group 1, group 2 patients were less responsive to GH-releasing hormone, while they were more sensitive to somatostatin- and dopamine-induced GH inhibition. These results suggest that patients with constitutively active adenylyl cyclase have hyperactive tumors; the sensitivity of these tumors to inhibitory agents (somatostatin and dopamine), possibly counteracting the expression of activating mutations, might explain the low rate of tumor growth.