GLI2 induces genomic instability in human keratinocytes by inhibiting apoptosis.

Abnormal Sonic Hedgehog signalling leads to increased transcriptional activation of its downstream effector, glioma 2 (GLI2), which is implicated in the pathogenesis of a variety of human cancers. However, the mechanisms underlying the tumorigenic role of GLI2 remain elusive. We demonstrate that overexpression of GLI2-ß isoform, which lacks the N-terminal repressor domain (GLI2ΔN) in human keratinocytes is sufficient to induce numerical and structural chromosomal aberrations, including tetraploidy/aneuploidy and chromosomal translocations. This is coupled with suppression of cell cycle regulators p21(WAF1/CIP1) and 14-3-3σ, and strong induction of anti-apoptotic signalling, resulting in a reduction in the ability to eliminate genomically abnormal cells. Overexpression of GLI2ΔN also rendered human keratinocytes resistant to UVB-mediated apoptosis, whereas inhibition of B-cell lymphoma 2 (BCL-2) restored endogenous (genomic instability (GIN)) and exogenous (UVB) DNA damage-induced apoptosis. Thus, we propose that ectopic expression of GLI2 profoundly affects the genomic integrity of human epithelial cells and contributes to the survival of progenies with genomic alterations by deregulating cell cycle proteins and disabling the apoptotic mechanisms responsible for their elimination. This study reveals a novel role for GLI2 in promoting GIN, a hallmark of human tumors, and identifies potential mechanisms that may provide new opportunities for the design of novel forms of cancer therapeutic strategies.

Fingerprinting genomic instability in oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a high-risk pre-cancerous condition where 7-13% of these patients develop head and neck squamous cell carcinoma (HNSCC). To date there is no cancer predictive markers for OSF patients. Genomic instability hallmarks early genetic events during malignant transformation causing loss of heterozygosity (LOH) and chromosomal copy number abnormality. However, to date there is no study on genomic instability in OSF. Although this condition is known as a high-risk pre-cancerous condition, there is no data regarding the genomic status of this disease in terms of genetic susceptibility to malignant transformation. METHODS: In this study, we investigated the existence of genetic signatures for carcinogenesis in OSF. We employed the high-resolution genome-wide Affymetrix Mapping single nucleotide polymorphism microarray technique to 'fingerprint' global genomic instability in the form of LOH in 15 patient-matched OSF-blood genomic DNA samples. RESULTS: This rapid high-resolution mapping technique has revealed for the first time that a small number of discrete hot-spot LOH loci appeared in 47-53% of the OSF tissues studied. Many of these LOH loci were previously identified regions of genomic instability associated with carcinogenesis of the HNSCC. CONCLUSION: To our knowledge, this is the first evidence that genomic instability in the form of LOH is present in OSF. We hypothesize that the genomic instability detected in OSF may play an important role in malignant transformation. Further functional association studies on these putative genes may reveal potential predictive oral cancer markers for OSF patients.

Upregulation of HIF-1alpha in malignant transformation of oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous condition showing extensive fibrosis of the submucosa and affects most parts of the oral cavity, including pharynx and upper third of the oesophagus. The molecules involved in the biological pathways of the fibrotic process appeared to be either down- or upregulated at different stages of the disease. Despite the precancerous nature, malignant transformation of the epithelium in the background of fibrosis has not been studied in detail. HIF-1alpha is a known transcription factor that is induced by hypoxia. AIMS: To test the hypothesis that hypoxia plays a role in malignant transformation and progression of OSF. MATERIALS AND METHODS: We used both formalin-fixed and frozen samples of OSF and normal mucosa to investigate the relationship between HIF-1alpha and epithelial dysplasia using immunohistochemistry and RT-PCR. CONCLUSIONS: Our data indicate that HIF-1alpha is upregulated at both protein and mRNA levels in OSF and the correlation with epithelial dysplasia is statistically significant (P < 0.001). We propose that HIF-1alpha may play a role in malignant transformation of OSF. Further, over-expression of HIF-1alpha may contribute to the progression of fibrosis. It may be possible to use HIF-1alpha as a marker for malignant transformation of OSF.

An endogenous 5-HT(7) receptor mediates pigment granule dispersion in Xenopus laevis melanophores.

Melatonin (5-methoxy N-acetyltryptamine) and serotonin (5-HT) exert rapid, but opposite effects on pigment granule distribution in Xenopus laevis melanophores. Low concentrations of melatonin (10(-11) - 10(-9) M) cause a dramatic perinuclear aggregation of the melanin-containing granules, while 5-HT (10(-8) - 10(-5) M) disperses pigment granules throughout the cell. The present study found that pharmacological doses of melatonin (> or =10(-6) M) induced a time- and concentration-dependent pigment granule dispersion, which was mediated by an endogenous melanophore 5-HT receptor. 5-HT produced a concentration-dependent elevation of melanophore cyclic AMP, and 5-HT-induced dispersion was blocked by H89 (10(-4) M), an inhibitor of protein kinase A (PKA), but not by a PKC inhibitor (Ro 31-8220, 10(-5) M), indicating a vital role for cyclic AMP in 5-HT-induced dispersion. 5-HT-mediated dispersion was not blocked by antagonists selective for G(s)-coupled 5-HT(4) (GR113808) or 5-HT(6) (Ro 04-6790, Ro 63-0563, olanzepine) receptors, nor by 5-HT(1 - 3) (pindolol, ketanserine, metoclopramide, MDL72222, tropisetron) receptor antagonists, but was inhibited by a selective 5-HT(7) receptor antagonist, DR4004, and other antagonists with a high affinity for 5-HT(7) receptors. The rank order of antagonist potency was: risperidone (mean pK(B) 7.82)>methiothepin (7.43)>DR4004 (6.92)>mesulergine (6.83)>methysergide (6.60)>[+/-]-sulpiride (5.81)>spiperone (5.52). The agonist potency order [mean pEC(50), 5-CT (8.68)>5-HT (7.13)>5-MT (6.94)>8-OH-DPAT (4.79)>sumatriptan (<4)] was also consistent with an action on 5-HT(7) receptors. RT - PCR confirmed that melanophores express 5-HT(7) receptor mRNA. The pigment dispersing effect of high melatonin concentrations in melanophores is most likely mediated by activation of 5-HT(7) receptors. Conceivably some of the effects attributed to pharmacological doses of melatonin in mammals may be mediated by activation of 5-HT(7) receptors.

Mapping the melatonin receptor. 6. Melatonin agonists and antagonists derived from 6H-isoindolo[2,1-a]indoles, 5,6-dihydroindolo[2,1-a]isoquinolines, and 6,7-dihydro-5H-benzo[c]azepino[2,1-a]indoles.

6H-Isoindolo[2,1-a]indoles (5, 7, 10, 13), 5,6-dihydroindolo[2, 1-a]isoquinolines (20, 21), and 6,7-dihydro-5H-benzo[c]azepino[2, 1-a]indoles (23, 25, 27, 30) have been prepared as melatonin analogues to investigate the nature of the binding site of the melatonin receptor. The affinity of analogues was determined in a radioligand binding assay using cloned human mt(1) and MT(2) receptor subtypes expressed in NIH 3T3 cells. Agonist and antagonist potency was measured using the pigment aggregation response of a clonal line of Xenopus laevis melanophores. The 2-methoxyisoindolo[2, 1-a]indoles (7a-d) showed much higher binding affinities than the parent isoindoles (5a-e), and whereas 7a-c were agonists in the functional assay, 7d and 5a-e were antagonists. The 2-ethoxyisoindolo[2,1-a]indoles (10a-d) showed reduced binding affinities compared to their methoxy analogues, while the 5-chloro derivative 13 showed a considerable reduction in binding affinity and potency compared to 7a. The 10-methoxy-5,6-dihydroindolo[2, 1-a]isoquinolines (21a-c) had higher binding affinities than the corresponding parent indoloisoquinolines (20a-c) in the human receptor subtypes, and the parent compounds were antagonists whereas the 10-methoxy derivatives were agonists in the functional assay. The N-cyclobutanecarbonyl derivatives of both the parent (20d) and 10-methoxyl (21d) series had similar binding affinities and were both antagonists with similar potencies. The 11-methoxy-6, 7-5H-benzo[c]azepino[2,1-a]indoles (25a-d) had higher binding affinities than the corresponding parent compounds (23a-d) at the MT(2) receptor but similar affinities at the mt(1) site; all of the compounds were antagonists in the functional assay. Changing 11-methoxy for 11-ethoxy decreased the binding affinity slightly, and this was more evident at the MT(2) receptor. All of the derivatives investigated had either the same or a greater affinity for the human MT(2) receptor compared to the mt(1) receptor (range 1:1-1:132). This suggests that the mt(1) and MT(2) receptor pockets differ in their ability to accommodate alkyl groups in the indole nitrogen region of the melatonin molecule. Two compounds (7c and 25c) were tested in functional assays on recombinant mt(1) and MT(2) melatonin receptors. Compound 7c is a potent agonist with some selectivity (44-fold) for the MT(2) receptor, while 25c is an MT(2)-preferring antagonist. Increasing the carbon chain length between N-1 of indole and the 2-phenyl group from n = 1 through n = 3 leads to a fairly regular decrease in the binding affinity, but, remarkably, when n = 3, it converts the methoxy compounds from melatonin agonists to antagonists. The Xenopus melatonin receptor thus cannot accommodate an N-n-alkyl chain attached to a 2-phenyl substituent with n > 2 in the required orientation to induce or stabilize the active receptor conformation.

Design of subtype selective melatonin receptor agonists and antagonists.

Studies of the physiological actions of melatonin have been hindered by the lack of specific, potent and subtype selective agonists and antagonists. In the present study, we describe the utility of a melanophore cell line from Xenopus laevis for exploring structure-activity relationships among novel melatonin analogues and report a novel MT2-selective agonist (IIK7) and MT2-selective receptor antagonist (K185). IIK7 is a potent melatonin receptor agonist in the melanophore model, and in NIH3T3 cells expressing human mt1 and MT2 receptor subtypes. In radioligand binding experiments IIK7 is 90-fold selective for the MT2 subtype. K185 is devoid of agonist activity, but acts as a competitive melatonin antagonist in melanophores. A low concentration (10(-9) M) antagonizes melatonin inhibition of forskolin stimulation of cyclic AMP in NIH3T3 cells expressing human MT2 receptors, but has no effect in cells expressing mt1 receptors. In binding assays, K185 is 140-fold selective for the MT2 subtype.

The putative melatonin receptor antagonist GR128107 is a partial agonist on Xenopus laevis melanophores.

1. GR128107 (3-(1-acetyl-3-methyl-piperidine)-5-methoxyindole) has previously been reported to be a competitive melatonin receptor antagonist in blocking melatonin inhibition of [3H]-dopamine release from rabbit retina, a response mediated by the MT2 receptor subtype. 2. GR128107, like melatonin, induced a rapid (maximum response in 60-90 min) pigment aggregation in a clonal line of Xenopus laevis melanophores. GR128107 behaved as a partial agonist (pEC50 8.58+/-0.03, n=3) with an Emax of 0.83 (relative to melatonin, pEC50 10.09+/-0.03, n=3). 3. The concentration-response curve for pigment granule aggregation to both melatonin and GR128107 was displaced in a parallel, rightward manner by melatonin receptor antagonists with very similar potencies; estimated pKB RJ252 (against melatonin 4.60/against GR128107 4.54) < GR135533 (6.40/6.14) < Luzindole (6.45/6.49) < S20929 (6.58/6.65) < 4-P-PDOT (6.73/6.85). 4. Both melatonin- and GR128107-induced pigment granule aggregation was prevented by pretreatment of melanophores with pertussis toxin (10-1000 ng ml(-1)). 5. Prolonged pre-treatment of melanophores with melatonin desensitized the pigment aggregation response to GR128107. In desensitized cells, the maximal aggregation produced by GR128107 was only 0.27+/-0.01 (n=4) and the pEC50 was reduced (vehicle 8.57+/-0.12; melatonin pre-treated 7.84+/-0.09, n=4). The maximal response to melatonin in desensitized melanophores was unchanged but the pEC50 was reduced (vehicle 10.49+/-0.03; melatonin pre-treated 9.83+/-0.04, n=4). 6. These results demonstrate that GR128107 induces pigment granule aggregation in Xenopus melanophores by activating a cell membrane melatonin receptor coupled via a pertussis toxin-sensitive G-protein. 7. The partial agonist activity of GR128107 in melanophores may be apparent because of the very high density of melatonin receptors in these cells (Bmax 1223 fmol mg protein(-1)) compared to the low density of sites in rabbit retina (Bmax 3.1 fmol mg protein(-1)). This suggestion is supported by the finding that GR128107, like melatonin, acted as a full agonist and inhibited forskolin-stimulation of cyclic AMP accumulation in NIH-3T3 cells expressing a high density of human mt1 or MT2 receptors.

Comparison of the structure-activity relationships of melatonin receptor agonists and antagonists: lengthening the N-acyl side-chain has differing effects on potency on Xenopus melanophores.

The potency and affinity of two series of melatonin receptor ligands were examined using the pigment aggregation response in a clonal line of Xenopus laevis melanophores and radioligand binding assays on native receptors in chicken brain, recombinant human mt1 and MT2 and Xenopus laevis mel1c receptor subtypes. One series was based on melatonin and had a methoxy group at the 5-position of the indole ring, while the other was based on luzindole and lacked this substituent but did have a 2-benzyl moiety; the N-acyl group of each series of analogues was varied from one to five carbon atoms. All analogues in the melatonin series were full agonists in melanophores (pEC50 7.76-10.24), while all compounds in the luzindole series were competitive melatonin antagonists (pA2 5.47-6.60). With the agonist series, increasing the N-acyl side-chain from one to three carbon atoms was well tolerated in both the functional and binding assays, but further lengthening of the side-chain progressively and dramatically reduced potency and affinity. In contrast, for the antagonist series neither potency nor binding affinity changed substantially with the length of the N-acyl chain, except at the recombinant MT2 subtype where two of the analogues had a lower affinity. In binding assays, three of the five antagonists were MT2-selective; the most selective analogue (N-pentanoyl 2-benzyltryptamine, MT2 pKi 8.03) having 89- and 229-fold higher affinity than at mt1 or mel1c receptor subtypes. The different structure-activity relationships of these receptor agonists and antagonists is discussed with regard to the possible binding sites of agonists and antagonists within the receptor protein.

Mapping the melatonin receptor. 5. Melatonin agonists and antagonists derived from tetrahydrocyclopent[b]indoles, tetrahydrocarbazoles and hexahydrocyclohept[b]indoles.

Tetrahydrocyclopent[b]indoles, tetrahydrocarbazoles, and hexahydrocyclohept[b]indoles have been prepared as melatonin analogues to investigate the nature of the binding site of the melatonin receptor. The affinity of analogues was compared in a radioligand binding assay using chicken brain membranes and agonist and antagonist potency measured in clonal Xenopus laevis melanophore cells. Comparison of the N-acyl-3-amino-6-methoxytetrahydrocarbazoles (2) with N-acyl-4-(aminomethyl)-6-methoxy-9-methyltetrahydrocarbazoles (9) showed that the latter have much higher binding affinities for the chicken brain receptor. Comparison of N-acyl-1-(aminomethyl)-7-methoxy-4-methyltetrahydrocyclopent[b]ind oles (10), 6-methoxytetrahydrocarbazoles (9), and N-acyl-10-(aminomethyl)-2-methoxy-5-methylhexahydrocyclohept[b]ind oles (11) showed that the tetrahydrocarbazoles had the highest binding affinity with the cyclohept[b]indoles and the cyclopent[b]indoles having rather lower affinities. All of these observations are in agreement with our postulated model of melatonin orientation at the binding pocket in which the 3-amidoethane side chain is in a conformation close to the 5-methoxyl group, as is shown in the X-ray crystallographic structure of 9m and in the energy-minimized computed structures. Separation of the enantiomers of members from each of these three systems was accomplished by chiral HPLC. It was found that in all cases the (-)-enantiomer had a higher binding affinity than the (+)-enantiomer. An X-ray crystallographic analysis of the two enantiomers of 9a showed that the (+)-enantiomer had the (R) absolute stereochemistry. Since the sign of the Cotton curves, determined from circular dichroism studies, was the same for all (+)-enantiomers, it is assumed that the absolute stereochemistry at these centers is identical. In the Xenopus melanophore assay, the tetrahydrocarbazoles 2 (R = H) were mainly weak antagonists, while those with R = OMe were agonists. The biological behavior of the tetrahydrocarbazoles 9 (R = H) depended on R1, some being agonists and some antagonists, whereas those with R = OMe were generally agonists. Variation of the R and R1 groups in compounds of type 9 produced both agonists and antagonists. The tetrahydrocylopentaindoles 10 had similar biological properties to the corresponding analogues of 9, but the hexahydrocycloheptaindoles 11 showed a much greater propensity to be antagonists. In all cases the (S)-enantiomers were found to be more potent agonists than the (R)-enantiomers.

Melatonin receptor pharmacology: toward subtype specificity.

The recent cloning of three distinct melatonin receptor subtypes (Mel1a, Mel1b and Mel1c) which are part of a new family of G-protein coupled receptors, and probably mediate the physiological actions of the hormone, has spurred interest in the design of analogues with subtype selectivity. The 5-methoxyl and N-acetyl groups of melatonin are important for binding to and activation of the receptor. The indole nucleus serves to hold these two groups at the correct distance from one another and allows them to adopt the required orientation for interaction with the receptor binding pocket. We have investigated the subtype selectivity of a number of analogues of melatonin in which the structure has systematically been modified in order to probe the similarities and differences in the interaction of ligand and receptor subtype. At all three subtypes 5-methoxyl and N-acetyl groups of melatonin are important for high affinity binding. However, replacing the 5-methoxyl group (eg with 5-H, 5-OH, 5-Me or 5-BzO) reduces affinity much less at the Mel1b receptor subtype than at either Mel1a or Mel1c cloned subtypes. This suggests differences between the Mel1b and Mel1a/1c subtypes in the size and shape of the binding pocket or in the manner in which melatonin interacts with the receptor at this position. Further studies have revealed that analogues with longer N-acyl carbon chains behave similarly at each subtype. These observations suggest that the 'pocket' into which the N-acetyl group fits is very similar for each subtype. Substitutions at the 2-position on the indole ring improved affinity at each receptor subtype but did not give selective analogues. The systematic 'mapping' of the requirements for binding at each receptor subtype should allow the design of more selective agonists and antagonists, which will be valuable tools for the characterization and classification of functional melatonin receptors.