Exogenous ghrelin regulates proliferation and apoptosis in the hypotrophic gut mucosa of the rat.

Ghrelin is the natural endogenous ligand for growth hormone secretagogue receptors. This peptide regulates energy homeostasis and expenditure and is a potential link between gut absorptive function and growth. We hypothesized that ghrelin may induce a proliferative and antiapoptotic action promoting the recovery of the hypotrophic gut mucosa. Therefore, the aim of the study was to determine the action of exogenous ghrelin following gut mucosal hypotrophia in rats fed an elemental diet. An elemental diet provides readily absorbable simple nutrients and is usually given to patients with absorptive dysfunction. Male Wistar rats (n = 48) were fed the elemental diet for one week to induce mucosal hypotrophy and then treated for another week with systemic ghrelin and pair-fed with either a normoproteic or hyperproteic isocaloric liquid diet. Another group received a standard diet instead of the elemental diet and served as control (normotrophy). The elemental diet induced intestinal hypotrophia characterized by decreased proliferation in the ileum and increased apoptosis in jejunum and ileum. Ghrelin administration restored normal levels of proliferation in the ileum and apoptosis in the jejunum, with partial apoptosis restoration in the ileum. Ghrelin levels in plasma and fundus were increased in all groups, although the highest levels were found in rats treated with exogenous ghrelin. Ghrelin administration has a positive effect in the hypotrophic gut, regulating both proliferation and apoptosis towards a physiological balance counteracting the negative changes induced by an elemental diet in the intestines.

The genetics of malignant melanoma.

Melanoma probably is the most aggressive cancer in humans and remains one of the leading causes of cancer death in developed countries. This review summarizes the most important alterations in protooncogenes and tumor suppressor genes that contribute to the pathogenesis of malignant melanoma, with a special emphasis on the involved signaling pathways. Our knowledge of the molecular biology of melanoma has been benefited from recent advances on high-throughput technologies analyzing wide genomic and gene expression profiles that have uncovered unknown candidate genes. To test the interactions between distinct pathways and of those with the environment a wealth of genetically modified animal models has been generated over the past years. Other studies have focused on the isolation of melanoma stem cells and on the characterization of signaling pathways that contribute to their survival and maintenance. A consequence of all these studies is the emergence of potential new strategies that could improve the still inadequate arsenal of therapeutic tools to fight against this fatal disease.

Genetic and epigenetic alteration of the NF2 gene in sporadic meningiomas.

The role of the NF2 gene in the development of meningiomas has recently been documented; inactivating mutations plus allelic loss at 22q, the site of this gene (at 22q12), have been identified in both sporadic and neurofibromatosis type 2-associated tumors. Although epigenetic inactivation through aberrant CpG island methylation of the NF2 5' flanking region has been documented in schwannoma (another NF2-associated neoplasm), data on participation of this epigenetic modification in meningiomas are not yet widely available. Using methylation-specific PCR (MSP) plus sequencing, we assessed the presence of aberrant promoter NF2 methylation in a series of 88 meningiomas (61 grade I, 24 grade II, and 3 grade III), in which the allelic constitution at 22q and the NF2 mutational status also were determined by RFLP/microsatellite and PCR-SSCP analyses. Chromosome 22 allelic loss, NF2 gene mutation, and aberrant NF2 promoter methylation were detected in 49%, 24%, and 26% of cases, respectively. Aberrant NF2 methylation with loss of heterozygosity (LOH) at 22q was found in five cases, and aberrant methylation with NF2 mutation in another; LOH 22q and the mutation were found in 16 samples. The aberrant methylation of the NF2 gene also was the sole alteration in 15 samples, most of which were from grade I tumors. These results indicate that aberrant NF2 hypermethylation may participate in the development of a significant proportion of sporadic meningiomas, primarily those of grade I.

Molecular analysis of the erbB gene family calmodulin-binding and calmodulin-like domains in astrocytic gliomas.

Primarily involved in cell proliferation and differentiation processes, the plasma membrane-bound ErbB tyrosine kinase receptor family is formed by four members: erbB1/EGFR, erbB2/HER2/Neu, erbB3/HER3 and erbB4/HER4. Calmodulin (CaM) is a Ca2+-binding protein involved in the regulation of multiple intracellular processes that binds directly to EGFR in the presence of Ca2+, inhibiting its tyrosine kinase activity. Two main regions in the receptor have been implicated in this relationship: the calmodulin-binding domain (CaM-BD) and the calmodulin-like domain (CaM-LD); their sequences are highly conserved in other members of this family of receptors. The presence of mutations, amplification and/or overexpression and genomic rearrangement of these domains was investigated for all four erbB family genes in a series of 89 glial tumors, including 44 WHO grade IV glioblastomas, 21 WHO grade III anaplastic astrocytomas, and 24 WHO grade II astrocytomas. Gene alterations were only found in the regions of interest in EGFR. One glioblastoma showed an in frame tandem duplication of the intracellular region including CaM-LD (exons 18-25). CaM-BD gene overdose was evidenced in 18 tumors that showed EGFR amplification in other domains. Over-expression of CaM-BD and CaM-LD was detected in 6 and 17 cases, respectively, of the 19 tumors in which this study was performed. The other three genes coding for the ErbB receptors did not present point mutations, or rearrangements, and only a very low amplification rate was found for erbB2 (1 case) and erbB3 (4 cases). No overexpression of erbB2, erbB3 or erbB4 was detected. These findings suggest that EGFR is the main erbB gene family member non-randomly involved in malignant glioma development, and that the two domains under study, due to their high conservation and wide separation in the EGFR sequence, are good marker regions for evaluating EGFR/erbB1 gene amplification, as well as for analysing the presence of transcripts corresponding to truncated cytosolic forms of the receptor in these tumors.

DNA methylation of multiple promoter-associated CpG islands in meningiomas: relationship with the allelic status at 1p and 22q.

The purpose of this research was to examine the DNA methylation profile of meningiomas. Accordingly, we examined the DNA methylation status of ten tumor-related genes (RB1, p16(INK4a), p73, MGMT, ER, DAPK, TIMP-3, p14(ARF), THBS1, and Caspase-8) in 98 meningiomas (68 grade I; 27 grade II; and 3 grade III samples) using methylation-specific PCR and sequencing. The most frequently methylated genes were THBS1 (30%), TIMP-3 (24%), p16(INK4a) (17%), MGMT (16%), p73 (15%), ER (15%), and p14(ARF) (13%), whereas methylation was relatively rare in the other genes (<10%). Methylation occurred in at least one gene in 77.5% of the cases and in three or more genes in 25.5%. Methylation was tumor specific since it was absent in the controls: two non-neoplastic meningeal samples and two non-neoplastic brain samples. The frequency of aberrant gene methylation in grade I versus grade II-III tumors showed some differences for TIMP-3, THBS1, MGMT, p16(INK4a) and p73; these differences reached statistical significance for TIMP-3: 18% in grade I versus 40% in grade II-III (P < 0.02). Our previous loss of heterozygosity studies provided the allelic constitution at 1p and 22q for 60 of the 98 meningiomas included in this report. The level of aberrant promoter methylation increased in tumors (30 samples) displaying 1p loss (either isolated or as concurrent deletion at 1p/22q; P = 0.014). These meningiomas primarily accumulated the epigenetic changes of THBS1 (14/30; 47%; P < 0.005), TIMP-3 (12/30; 40%; P < 0.05), p73 (10/30; 26%; P < 0.02) and p14(ARF) /p16(INK4a)(7/30 each one; 23%; not significant). Our findings indicate that aberrant DNA methylation of promoter-associated CpG islands in meningiomas contributes to the development of these tumors.

Methylation status of TP73 in meningiomas.

Deletions at 1p are frequent in meningioma and represent a genetic marker associated with the genesis of atypical WHO grade II forms. Previous mutational analysis of TP73, a structurally and functionally TP53 homologous gene located at 1p36.33, failed to demonstrate a significant rate of sequence variations linked to gene inactivation in meningiomas with 1p loss. As an alternative, TP73 may be inactivated through aberrant 5' CpG island methylation, a primary mechanism participating in the inactivation of tumor suppressor genes during tumorigenesis. We determined the methylation status of the TP73 gene in a series of 60 meningiomas (33 grade I, 24 grade II, and 3 grade III samples), including tumors with deletion at 1p (n=30) and with intact 1p (n=30). Aberrant methylation was detected in 10 cases (33%) with 1p deletion and in 3 tumors (10%) with retention of alleles at this chromosome arm. The distribution of the 13 cases of methylation according to malignancy grade was 7 grade I, 5 grade II, and 1 grade III tumor. Accordingly, although TP73 aberrant methylation was more frequent in meningiomas with 1p deletion (P<0.05), no association with the grade of malignancy could be established. These findings, together with the previously reported increased TP73 expression in malignant meningiomas suggest that opposing functions of this gene may characterize distinct subsets of tumors: suppressed or reduced expression as a result of CpG methylation in some grade I-grade II tumors, and enhanced expression in some more malignant forms.

CpG island methylation in sporadic and neurofibromatis type 2-associated schwannomas.

PURPOSE: The purpose of this research was to examine the DNA methylation profile of schwannomas. EXPERIMENTAL DESIGN: We examined the DNA methylation status of 12 tumor-related genes (NF2, RB1, p14(ARF), p16(INK4a), p73, TIMP-3, MGMT, DAPK, THBS1, caspase-8, TP53, and GSTP1) in 44 sporadic and/or NF2-associated schwannomas using methylation-specific PCR. RESULTS: The most frequently methylated genes were THBS1 (36%), p73 (27%), MGMT (20%), NF2 (18%), and TIMP-3 (18%). The RB1/p16INK4a gene pair displayed aberrant methylayed alleles in 15% of cases, whereas methylation was relatively rare in the other genes (<5%). Methylation was tumor specific because it was absent in two nonneoplastic nerve sheath samples and two nonneoplastic brain samples studied as controls. CONCLUSIONS: Our findings indicate that aberrant methylation seems to be a mechanism for NF2 gene inactivation, considered an early step in schwannoma tumorigenesis, and as well, aberrant hypermethylation of other tumor-related genes might represent secondary events that also contribute to the development of these tumors.

Aberrant CpG island methylation in neurofibromas and neurofibrosarcomas.

Aberrant methylation of the promoter CpG island of human genes is an alternative gene inactivation mechanism that contributes to the carcinogenesis of human tumours. We have determined the methylation status of the CpG island of 11 tumour-related genes (RB1, p14ARF, p16INK4a, p73, TIMP-3, MGMT, DAPK, THBS1, caspase 8, TP53 and GSTP1) in 18 neurofibromas (including one plexiform neurofibroma) and three neurofibrosarcomas, as well as two non-neoplastic peripheral nerve sheath samples, using methylation-specific polymerase chain reaction. The series included sporadic and neurofibromatosis type 1-associated tumours. The incidence of aberrant methylation in the tumour samples was 52% for THBS1, 43% for MGMT, 33% for TIMP-3, 19% each for p16INK4a and p73, 14% for RB1, 5% for p14ARF, and 0% for DAPK, caspase 8, TP53 and GSTP1. No methylation of these genes was detected in the two samples of non-neoplastic peripheral nerve sheath. All but three samples in the study displayed aberrant methylation in at least one of the studied genes, and there was no correlation between methylation status and the patients' clinical parameters. These findings suggest that methylation of some tumour-related genes may play a significant role in the tumourigenesis of neurofibromas/neurofibrosarcomas.

Aberrant promoter methylation of multiple genes in oligodendrogliomas and ependymomas.

Promoter hypermethylation represents a primary mechanism in the inactivation of tumor suppressor genes during tumorigenesis. To determine the frequency and timing of hypermethylation during carcinogenesis of nonastrocytic tumors, we analyzed promoter methylation status of 10 tumor-associated genes in a series of 41 oligodendrogliomas (22 World Health Organization [WHO] grade II; 13 WHO grade III; 6 WHO grade II-III oligoastrocytomas) and 7 WHO grade II-III ependymomas, as well as 2 nonneoplastic brain samples, by a methylation-specific polymerase chain reaction. Aberrant CpG island methylation was detected in 9 of 10 genes analyzed, and all but one sample displayed anomalies in at least one gene. The frequencies of hypermethylation for the 10 genes were as follows, in oligodendrogliomas and ependymomas, respectively: 80% and 28% for MGMT; 70% and 28% for GSTP1; 66% and 57% for DAPK; 44% and 28% for TP14(ARF); 39% and 0% for THBS1; 24% and 28% for TIMP3; 24% and 14% for TP73; 22% and 0% for TP16(INK4A); 3% and 14% for RB1; and 0% in both neoplasms for TP53. No methylation of these genes was detected in normal brain tissue samples. We conclude that a high frequency of aberrant methylation of the 5' CpG island of the MGMT, GSTP1, TP14(ARF), THBS1, TIMP3, and TP73 genes is observed in nonastrocytic neoplasms. This aberration seems to occur early in the carcinogenesis process (it is already present in the low-grade forms), although in some instances (DAPK, THBS1, and TP73) it appears also associated with the genesis of anaplastic forms.

Frequent death-associated protein-kinase promoter hypermethylation in brain metastases of solid tumors.

Death-associated protein (DAP) kinase is a gene that participates in apoptosis induced by interferon gamma. It appears to play a role in lung cancer metastasis in animal models, suggesting that DAP-kinase may function as a metastasis suppressor by inducing apoptosis. Expression silencing through CpG island methylation of DAP-kinase has been frequently found in connection with adverse survival, as cells lacking DAP-kinase appear to be more invasive and more metastatic in lung cancer. The purpose of this study was to analyze the promoter methylation status of DAP-kinase gene in brain metastases of solid tumors. Methylation-specific PCR was performed on ten brain metastasis samples derived from malignant melanoma (three cases), lung cancer (two), breast carcinoma (two), ovarian carcinoma (two) and colon carcinoma (one case), and in corresponding peripheral blood DNA samples. Two normal brain tissue samples were also analyzed, no promoter hypermethylation was observed in either case. DAP-kinase hypermethylated alleles were identified in nine metastases (90%), and in peripheral blood lymphocytes DNA from four cases. Our data suggest that silencing of DAP-kinase through promoter hypermethylation is a common event in the multistep process of tumor metastasis, including brain involvement.

Epigenetic changes in pilocytic astrocytomas and medulloblastomas.

Aberrant methylation of CpG islands located in promoter regions represents one of the major mechanisms for silencing of cancer-related genes in tumour cells. We determined the frequency of aberrant CpG island methylation of several tumour-associated genes: MGMT, GSTP1, DAPK, p14ARF, THBS1, TIMP-3, p73, p16INK4A, RB1 and TP53 in 24 neurogenic tumours consisting of pilocytic astrocytomas (n=13) and medulloblastomas (n=11). The methylation index (number methylated genes/total genes analysed) displayed slight differences (0.18 and 0.25, respectively), and the profile of methylated genes in the two neoplasms was distinct, as predicted. The main differences involved the methylation rate of GSTP1 (0% in pilocytic astrocytomas vs. 18% medulloblastomas) and p14ARF (0% in pilocytic astrocytomas vs. 45% in medulloblastomas) genes. Pilocytic astrocytomas also demonstrated some differences when compared to methylation data from other astrocytic tumours, primarily regarding the MGMT methylation rate. Despite the fact that these differences do not show specific tumour-associated gene methylation patterns, our findings should help us understand the pathogenic mechanisms of both neurogenic neoplasm types.

CpG island methylation of tumor-related genes in three primary central nervous system lymphomas in immunocompetent patients.

We have determined the promoter CpG island methylation status of O(6)-methylguanine-DNA methyltransferase (MGMT), glutathione-S-transferase P1 (GSTP1), death-associated protein kinase (DAPK), p14(ARF), thrombospondin-1 (THBS1), tissue inhibitor of metalloproteinase-3 gene (TIMP-3), p73, p16(INK4A), RB1, and TP53 genes in three primary central nervous system lymphomas (PCNSL). Five genes (GSTP1, DAPK, TIMP-3, p16(INK4A), and RB1) were hypermethylated in two samples, whereas MGMT, THBS1, and p73 were aberrantly methylated in only one sample. No case presented CpG island methylation for the p14(ARF) and TP53 genes. These findings concur with previous data suggesting a frequent inactivation of p16(INK4A) and very limited involvement of TP53 in PCNSL and also provide insights into the epigenetic molecular involvement of other tumor-related genes in this neoplasm.

Promoter hypermethylation of multiple genes in astrocytic gliomas.

Promoter hypermethylation represents a primary mechanism in the inactivation of tumor suppressor genes during tumorigenesis. To determine the frequency and timing of hypermethylation during carcinogenesis of astrocytic tumors, we analysed promoter methylation status of ten tumor-associated genes (MGMT, GSTP1, DAPK, p14ARF, THBS1, TIMP-3, p73, p16INK4A, RB1 and TP53) in a series of 88 astrocytic gliomas, including 24 diffuse astrocytomas; 21 anaplastic astrocytomas, and 43 glioblastomas (33 primary and 10 secondary), as well as two non-neoplastic brain samples, by methylation-specific PCR. Aberrant CpG island methylation was detected in all ten genes analysed, and all but one sample displayed anomalies in at least one gene. The methylation index (number methylated genes/total genes analysed) was 0.3, 0.38, 0.33 and 0.29 for diffuse astrocytomas, anaplastic astrocytomas and secondary and primary glioblastomas, respectively. Some differences may be established regarding the methylation profiles of specific genes and tumor types: MGMT, THBS1, TIMP-3, and p16INK4A appear hypermethylated in low-grade tumors (at least in 45% of cases), whereas GSTP1, DAPK, and p14ARF are mostly changed in 15-50% of the higher grade forms versus <10% in low-grade tumors. Some variation also exists regarding the methylation values for p73 and RB1 (10-40% of cases) among all groups. TP53 presented hypermethylation rates <10% in all tumor subtypes. Our findings thus suggest that methylation represents a common mechanism that contributes to inactivating cancer-related genes in astrocytic neoplasms. This epigenetic change is, in general, an early event in the development of astrocytic neoplasms but this gene silencing mechanism may also appear as a late event involving some loci.

Retinoblastoma-related gene RB2/p130 exons 19-22 are rarely mutated in glioblastomas.

The retinoblastoma gene family RB1, p107 and RB2/p130 cooperate to regulate cell cycle progression through the G1 phase of the cell cycle. Previous data demonstrated that RB2/p130 inhibits proliferation of the glioblastoma cell line T98G, which is resistant to the growth suppressive effects of both RB1 and p107, and that RB2/p130 gene overexpresion induces astrocyte differentiation. We screened by single-strand conformation polymorphism and sequence analysis the structure of exons 19 through 22 of the RB2/p130 gene, which encodes the B domain and C terminus, in a series of 42 glioblastomas (32 primary and 10 secondary). Sequence variations were identified in one tumor, suggesting that mutation inactivation of RB2/p130 is a rare event in glioblastoma.

Analysis of the NF2 gene in oligodendrogliomas and ependymomas.

Allelic losses of chromosome 22 are commonly found in ependymomas and oligodendrogliomas, suggesting that at least one tumor suppressor gene on chromosome 22 must be inactivated during the multistep process of tumorigenesis in these glial tumors. The neurofibromatosis 2 gene (NF2) located at 22q12, is a candidate tumor suppressor gene potentially involved in the pathogenesis of gliomas. Because there have been only a few studies of the NF2 gene in glial tumors other than astrocytoma, we screened the entire 17 NF2 exons for mutations in a series of 47 nonastrocytic tumors, including 40 oligodendrogliomas and 7 ependymomas. Only one mutation was detected, a 59-base pair insertion in exon 3 from a spinal anaplastic ependymoma. These results concur with previous findings proposing preferential inactivation of the NF2 gene in a subgroup of ependymomas, and suggest that the NF2 gene is not the target of chromosome 22 aberrations in oligodendrogliomas.

Loss of chromosome 22 and absence of NF2 gene mutation in a case of multiple meningiomas.

Multiple meningiomas are rare, and only 13 cases have been subjected to molecular genetic analysis to detect mutations of the tumor-suppressor gene neurofibromatosis type 2 (NF2) located on chromosome 22. Most of these cases display NF2 gene mutations parallel to loss of the chromosome 22 homolog, indicating that inactivation of this gene may represent an early event in the development of multiple meningiomas. We report a case of a 61-year-old woman who developed multiple (dorsal and intracranial) meningiomas. Cytogenetic and molecular genetic studies demonstrated the loss of a copy of chromosome 22 in the 5 meningiomas studied and the absence of NF2 gene mutations in 4 of those available for this molecular analysis. These findings, together with similar data from 2 previously reported cases, suggest the participation of a tumor-suppressor gene other than NF2 on chromosome 22 in the pathogenesis of a subgroup of multiple meningiomas.