Alternative splicing of the imprinted candidate tumor suppressor gene ZAC regulates its antiproliferative and DNA binding activities.

ZAC encodes a zinc finger protein with antiproliferative activity, is maternally imprinted and is a candidate for the tumor suppressor gene on 6q24. ZAC expression is frequently lost in breast and ovary tumor-derived cell lines and down-regulated in breast primary tumors. In this report, we describe ZACDelta2, an alternatively spliced variant of ZAC lacking the sequence encoding the two N-terminal zinc fingers. Messenger RNAs encoding ZAC or ZACDelta2 were equally abundant and both proteins were nuclear. ZACDelta2 displayed an improved transactivation activity and an enhanced affinity for a ZAC binding site, suggesting that the two N-terminal zinc fingers negatively regulated ZAC binding to its target DNA sequences. Both proteins were equally efficient in preventing colony formation, indicating similar overall antiproliferative activities. However, these activities resulted from a differential regulation of apoptosis vs cell cycle progression since ZACDelta2 was more efficient at induction of cell cycle arrest than ZAC, whereas it was the reverse for apoptosis induction. Hence, these data further underline that ZAC gene is critically controlled, both at the transcriptional level through imprinting and at the functional level through alternative splicing.

Characterization of the methylation-sensitive promoter of the imprinted ZAC gene supports its role in transient neonatal diabetes mellitus.

ZAC is a recently isolated zinc finger protein that induces apoptosis and cell cycle arrest. The corresponding gene is imprinted maternally through an unknown mechanism and maps to 6q24-q25, within the minimal interval harboring the gene responsible for transient neonatal diabetes mellitus (TNDM) and a tumor suppressor gene involved in breast cancer. Because of its functional properties, imprinting status, and expression pattern in mammary cell lines and tumors, ZAC is the best candidate so far for both disease conditions. In the present work, we delineated ZAC genomic organization and mapped its transcriptional start site. It is noteworthy that the ZAC promoter localized to the CpG island harboring the methylation imprint associated with TNDM and methylation of this promoter silenced its activity. These data indicate that the methylation mark may have a direct effect on the silencing of the ZAC imprinted allele. Our findings further strengthen the hypothesis that ZAC is the gene responsible for TNDM and suggest a novel mechanism for ZAC inactivation in breast tumors.

Loss of expression of the candidate tumor suppressor gene ZAC in breast cancer cell lines and primary tumors.

Loss of chromosome 6q21-qter is the second most frequent loss of chromosomal material in sporadic breast neoplasms suggesting the presence of at least one tumor suppressor gene on 6q. We recently isolated a cDNA encoding a new zinc finger protein which we named ZAC according to its functional properties, namely induction of apoptosis and control of cell cycle progression. ZAC is expressed in normal mammary gland and maps to 6q24-q25, a recognized breast cancer hot spot on 6q. In the present report, we investigated the possible inactivation of ZAC in breast cancer cell lines and primary tumors. We detected no mutation in ZAC coding region in a panel of 45 breast tumors with allelic imbalance of 6q24-q25. However, a survey of eight breast cancer cell lines showed a deeply reduced (three cell lines) or complete loss of (five cell lines) ZAC expression. Treatment of three of these cell lines with the methylation-interfering agent 5-azacytidine induced ZAC re-expression. In addition, Northern blot and RNase protection assay analysis of ZAC expression in 23 unselected primary breast tumors showed a reduced expression in several samples. Together with its functional properties and chromosomal localization, these findings substantiate ZAC as a good candidate for the tumor suppressor gene on 6q24-q25.

hZAC encodes a zinc finger protein with antiproliferative properties and maps to a chromosomal region frequently lost in cancer.

We previously reported the identification of mZac, a novel mouse zinc finger protein that shared with p53 the ability to regulate concomitantly apoptosis and cell cycle progression. We describe here the isolation, chromosomal localization, and functional in vitro characterization of its human homolog. hZAC is a widely expressed zinc finger protein that reveals transactivation and DNA-binding activity. hZAC inhibits tumor cell growth through induction of apoptotic cell death and G1 arrest. Thus hZAC, like its mouse counterpart, displays antiproliferative properties through pathways known to be central to the activity of p53. We mapped hZAC on chromosome 6q24-q25, a region frequently deleted in many solid tumors. Indeed, allelic loss at 6q24-q25 has been shown in breast and ovary cancers, melanomas, astrocytomas, and renal cell carcinomas. Furthermore, Abdollahi et al. [Abdollahi, A., Godwin, A. K., Miller, P. D., Getts, L. A., Schultz, D. C., Tagushi, T., Testa, J. R. & Hamilton, T. C. (1997) Cancer Res. 57, 2029-2034] recently isolated ZAC through its loss of expression in a surface epithelial ovary tumor model and accordingly named it Lot for "lost on transformation." In view of these observations, the functional properties we report here provide further arguments to consider hZAC as a tumor suppressor gene candidate.

Expression of G protein alpha-subunits in bovine parathyroid.

A G protein-coupled Ca(2+)-sensing receptor was recently cloned from bovine parathyroid and shown to mediate divalent cation regulation of PTH secretion. To define which G proteins might be coupled to the Ca(2+)-sensing receptor in parathyroid cells, we determined which G protein alpha-subunit messenger RNAs (mRNAs) are expressed in the parathyroid. We also considered the possibility that a novel parathyroid-specific G alpha might be present. We, therefore, used the reverse transcription-polymerase chain reaction to study the expression of G alpha subunits in a bovine parathyroid mRNA preparation. Degenerate primers, corresponding to two regions conserved in every G alpha subunit, the G3 and G4 sequences, were used to amplify G alpha complementary DNA fragments that were subcloned and sequenced. We found that mRNAs corresponding to G alpha s, G alpha i2, G alpha 11, G alpha 12, and G alpha z are the predominant G alpha mRNAs expressed in the bovine parathyroid. No novel G alpha mRNA was identified. Northern blots confirmed the expression of the cloned G alpha subunits and showed lower expression of G alpha o and G alpha i1 mRNAs. Immunoblots confirmed abundant expression of G alpha s, G alpha i2, and G alpha 11 and provided evidence for expression of G alpha i1 and G alpha i3, but not G alpha o. G alpha q mRNA was not identified by the degenerate primer reverse transcription-polymerase chain reaction strategy, but the immunoblot detected G alpha q protein, albeit at considerably lower levels than G alpha 11. The abundance of G alpha 11 relative to G alpha q in bovine parathyroid is consistent with but does not prove a role for G alpha 11 in coupling the Ca(2+)-sensing receptor to phospholipase C.

5-Hydroxytryptamine1A receptor synthetic peptides. Mechanisms of adenylyl cyclase inhibition.

The 5-hydroxytryptamine1A receptor (5-HT1AR) is a G-protein-coupled receptor negatively coupled to adenylyl cyclase (AC). We have studied the functional domains of 5-HT1AR using synthetic peptides to block or mimic receptor function. The entire second intracellular loop (5-HT1AR-i2) and the carboxyl end of the third intracellular loop (5-HT1AR-i3-C) strongly inhibited forskolin-stimulated AC activity. These effects were not additive with those of 5-HT. Like 5-HT, the peptides 5-HT1AR-i3-C and -i2 weakly inhibited AIF4- and Mn2+ stimulated AC activity. 5-HT1AR binding assays indicated that peptides could interact with the same G-protein pool as the 5-HT1AR. 5-HT1AR-i3-C- and -i2-stimulated [35S]guanosine 5'-O-(thiotriphosphate) binding on Go/Gi proteins. Only 5-HT1AR-i3-C partially adopted an alpha-helical conformation in solution. These data show that different domains in the 5-HT1AR second and third intracellular loops can couple to and activate Gi proteins in order to mediate AC inhibition. Peptide-induced AC inhibition was not sensitive to pertussis toxin as opposed to the 5-HT1AR-mediated effect. Our data show that the 5-HT1AR and the 5-HT1AR peptides activate Gi proteins in a slightly different manner.

Differential coupling of 5-HT1A receptors occupied by 5-HT or 8-OH-DPAT to adenylyl cyclase.

Human serotonin (5-hydroxytryptamine, 5-HT)-1A receptors have been transfected in NIH-3T3 cells, and their coupling to adenylyl cyclase was analysed depending on 1) the number of receptor expressed, 2) the experimental conditions used, 3) the nature of the agonists. Two monoclonal cell lines were used, expressing low (45 fmol/mg) and high (500 fmol/mg) levels of 5-HT1A receptor. Two methods were tested to study the negative coupling of the transfected 5-HT1A receptors to adenylyl cyclase: 1) measurement of cAMP production in intact cells, 2) measurement of adenylyl cyclase activity in vitro on membrane preparations. Studies on intact cells revealed that an increase in the receptor concentration was followed by 1) an increase in the efficacies of 5-HT, 5-CT (5-carboxamidotryptamine) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), 2) a 2 to 3-fold increase in the potency of 5-CT and 8-OH-DPAT, but no change in the potency of 5-HT. In membrane preparations, 8-OH-DPAT dose-response curve was shifted leftwards when the receptor concentration became higher whereas the corresponding shift was smaller for 5-HT and absent for 5-CT. Surprisingly, on membrane preparations, 8-OH-DPAT was a partial agonist relative to 5-HT. The relative efficacy of 8-OH-DPAT was lower in the clone expressing the lowest level of receptor. This partial agonist behavior of 8-OH-DPAT could be modulated by the ionic conditions under which the adenylyl cyclase activity was measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of 5-HT1A receptors expressed in NIH-3T3 cells induces focus formation and potentiates EGF effect on DNA synthesis.

NIH-3T3 fibroblasts have been transfected with human serotonin 5-HT1A receptors. Clonal cell lines expressed between 40 and 500 fmol receptor/mg. 5-HT1A agonists strongly inhibited nonstimulated- as well as forskolin- or isoproterenol-stimulated adenylyl cyclase. The effects of 5-HT1A receptor activation on cell growth were investigated. 5-HT1A agonists accelerated cell division, generated foci, and increased DNA synthesis. The stimulation of [3H]thymidine incorporation was much stronger when tyrosine kinase receptors were activated concomitantly. Cyclic AMP (cAMP) elevating agents inhibited DNA synthesis induced by all mitogens tested. The mitogenic activity of 5-HT1A agonists did not seem to be linked to adenylyl cyclase inhibition because 1) we were not able to measure any decrease in intracellular cAMP levels under the conditions of DNA synthesis assay and 2) 2',5'-dideoxyadenosine, which strongly inhibited adenylyl cyclase, was not mitogenic and did not modify the mitogenic effects of 5-HT1A agonists. Pertussis toxin completely blocked potentiation of epidermal growth factor effect induced by 8-hydroxy-di-(n-propyl)aminotetralin, a 5-HT1A agonist, but only partially blocked the one induced by insulin. In conclusion, in transfected NIH-3T3 cells, transforming and mitogenic effects of 5-HT1A agonists involve a pertussis toxin-sensitive G protein but do not seem to be linked to adenylyl cyclase inhibition.

Transfection of human 5-hydroxytryptamine1A receptors in NIH-3T3 fibroblasts: effects of increasing receptor density on the coupling of 5-hydroxytryptamine1A receptors to adenylyl cyclase.

Human serotonin [5-hydroxytryptamine (5-HT)1A] receptors have been transfected in NIH-3T3 cells, and their pharmacology and coupling to adenylyl cyclase have been analyzed. Three cellular preparations were used, 1) monoclonal cell lines (clones 6, 2B, and 4B), expressing 45, 280, and 500 fmol of 5-HT1A receptors/mg of protein, respectively; 2) clones 6, 2B, and 4B in which the concentration of 5-HT1A receptors was increased after stimulation of the glucocorticoid-inducible promoter with dexamethasone; and 3) polyclonal cell lines that expressed an increasing amount of 5-HT1A receptor as a function of cell passage. The transfected 5-HT1A receptors inhibited basal, forskolin-stimulated, and isoproterenol-stimulated adenylyl cyclase. The inhibition was dependent on the receptor density expressed, increasing from 60% at low density (45 fmol/mg) to 90% at a density higher than 280 fmol/mg. The pharmacology of the 5-HT1A receptor was studied, with particular attention being paid to the behavior of some agonists. These pharmacological characteristics are similar to those of 5-HT1A receptors in hippocampus but different from those of 5-HT1A in cerebral cortex. Analysis of the potencies and efficacies of the full agonist 5-HT and the partial agonist ipsapirone, as a function of receptor density in the three cellular populations used, revealed that 1) the efficacies of the full and partial agonists increased with the receptor density; 2) the EC50 values of the full and partial agonists were not shifted to the left when the receptor density was increased (based on the increase in efficacy and considering the classical pharmacological models of receptor-drug action, a 9-10-fold shift was expected); and 3) the ratio between the efficacies of the full agonist 5-HT and the partial agonist ipsapirone was not modified when the receptor concentration was increased or when the GTP-binding protein availability was decreased. The results indicate that neither the classical nor the operational model of drug-receptor action can be used to describe the coupling of 5-HT1A receptors to adenylyl cyclase in transfected NIH-3T3 cells. One of the explanations could be that 5-HT1A receptors and GTP-binding proteins are coupled in functional domains (almost precoupled), rather than distributed in homogeneous compartments in which they are free to diffuse.

5-HT1A-sensitive adenylyl cyclase of rodent hippocampal neurons: effects of antidepressant treatments and chronic stimulation with agonists.

The effects of chronic treatment with desimipramine (a tricyclic antidepressant), fluoxetine [a specific 5-hydroxytryptamine (5-HT) uptake inhibitor], clorgyline (a specific monoamine oxydase inhibitor of A type), ipsapirone (a specific 5-HT1A receptor agonist) as well as electroconvulsive shock treatment were investigated on rat hippocampal 5-HT1A receptors negatively coupled to adenylyl cyclase. Drugs were injected intraperitoneally in rats for 2 or 3 weeks, and biochemical determinations were made 4 to 72 hr after the final dose. Chronic treatments with desimipramine, ipsapirone and fluoxetine did not induce any change in the 5-HT1A-induced inhibition of the adenylyl cyclase activity. In contrast, chronic treatment with clorgyline and electroconvulsive shock treatment induced a slight but significant reduction of 5-HT's ability to inhibit hippocampal adenylyl cyclase. This indicates that, at least in hippocampal neurons, the 5-HT1A receptor coupled to adenylyl cyclase is not easily desensitized. This was verified in vitro on murine hippocampal neurons in culture, by measuring the effects of intense stimulation (1 and 2 hours), with 5-HT, ipsapirone and 8-hydroxy-2-(di-n-propylamino)tetralin. Indeed, such stimulations did not significantly affect the 5-HT1A receptor-induced inhibition of cAMP production in these hippocampal neurons in culture. Our results indicate that it is not the post-synaptic 5-HT1A receptor of hippocampus that is modified during antidepressant treatments, at least at the level of its coupling to adenylyl cyclase.

Does age influence the blood pressure and sodium excretion responses to sodium depletion in rats?

Experiments were performed in 3- and 24-month-old male Wistar rats in order to assess the influence of age on the response by systolic blood pressure and sodium excretion to abrupt sodium restriction. In response to the sodium deprivation, urinary sodium excretion was markedly reduced in all animals; however, the cumulative sodium excretion (from days 1 to 6 of depletion) was significantly higher in aged rats (1299 +/- 243 mumol) than in young rats (757 +/- 70 mumol). After 6 days of a low-sodium diet, the systolic blood pressure was similar in both groups. Plasma renin activity was higher in young compared with aged rats (27 +/- 5 versus 14 +/- 3 ng/ml per h). These results indicate that ageing is associated with a significant disturbance in the renal but not the systemic response to an abrupt restriction in sodium intake.