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PubMed2005

Hypomethylation of the XIST gene promoter in prostate cancer.

In a process denoted "global hypomethylation" repetitive DNA sequences like LINE-1 retrotransposons become hypomethylated in human cancers, including a subset of prostate carcinomas. It is less well known to what extent single-copy sequences are affected by this phenomenon. Therefore, we have analyzed methylation and expression of the XIST gene by bisulfite sequencing and real-time RT-PCR. The promoter of this single-copy gene is strongly methylated in normal male cells, including leukocytes and normal prostate. In prostate cancer tissues and particularly in cell lines, partial hypomethylation was observed paralleling that of LINE-1 sequences. Weak XIST expression was found in normal prostate tissues, but none in leukocytes. Only slight increases in expression of this gene were found in cancer tissues and cell lines. Our data suggest that hypomethylation in prostate cancer is indeed "global," affecting repeat and unique sequences in parallel. Detection of partially hypomethylated XIST alleles in prostate cancer tissues might be useful for the identification of cases with pronounced hypomethylation, which tend to be more aggressive.

Aged↗
PubMed2003

Expression of tumor related genes NGX6, NAG-7, BRD7 in gastric and colorectal cancer.

AIM: NGX6, NAG-7 and BRD7 genes are tumor related genes, which have been newly cloned by positional candidate cloning strategy. This study was designed to investigate the expression levels of NGX6, NAG-7 and BRD7 genes in human gastric and colorectal cancer tissues, and their corresponding normal tissues, and to investigate whether these genes play a role in the pathogenesis of gastric and colorectal cancers. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), dot hybridization and Northern blot analysis were used to compare the expression levels of NGX6, NAG-7 and BRD7 genes in 34 gastric cancer tissues and 34 colorectal cancer tissues with their corresponding normal tissues of the same patients, respectively. RESULTS: Among the 34 colorectal cancer specimens and the 34 gastric cancer specimens, the expression of NGX6 in 25 colorectal cancer tissues was absent or very weak (73.5 %) by RT-PCR analysis. The down-regulation rate of NGX6 in colorectal cancer tissues was significantly higher than that in corresponding normal tissues (26.5 %,9/34) (P<0.005). Moreover, the down-regulation of NGX6 was significantly correlated with lymph node and/or distance metastases. Patients with lymph node and/or distance metastasis had much higher down-regulation rate of NGX6 than patients without metastases (93.8 % vs 55.6 %, P<0.05). However no correlation was found between the expression of NGX6 and pathologic type of colorectal cancer in this study, and also the expression of NGX6 did not display any difference between gastric cancer and corresponding normal tissues (58.8 % vs 70.6 %, P>0.25). Dot hybridization and Northern blot analysis confirmed the results of RT-PCR. Furthermore, NAG-7 and BRD7 mRNA was not up- or down-regulated in gastric and colorectal cancers compared with their corresponding normal tissues in our study. CONCLUSION: The down-regulation of NGX6 may be closely associated with tumorigenesis and metastasis of colorectal carcinoma. However, it may not contribute to the development and progression of gastric carcinoma. In addition, the expression levels of NAG-7, and BRD7 did not alter in gastric and colorectal cancers. This seems to suggest that NAG-7 and BRD7 genes may not play a role in gastric and colorectal carcinogenesis.

Blotting, Northern↗
PubMed2000

Telomere length maintenance in aging and carcinogenesis.

Normal somatic cells have a finite number of divisions, a limited capacity to proliferate. Human telomeres, the long DNA TTAGGG repeats at the ends of chromosomes, are considered a molecular clock marker. The gradual and progressive telomere shortening at each replicative cycle is associated, through the activation of pRB and p53 pathways and genomic instability, to the replicative senescence, a non-dividing state and widespread cell death. Activation of telomere maintenance [telomerase; or alternative lengthening of telomeres mechanisms (ALT), or other adaptive responses] can revert this program. Although not completely known, several mechanisms and modulating agents may be able to up and down-regulate telomere length and its maintenance. Chemopreventive therapies for the up-regulation of telomerase activity, able to prolong the life of cell cultures in a phenotypically youthful state, could have important applications in research and medicine. On the contrary the therapeutic down-regulation of telomerase activity may be used in cancer therapy. Telomerase expression per se is not oncogenic, but telomere shortening and maintenance seem to be crucial events in tumor formation. Thus a particular focus has been pointed out relatively to the immortalization of normal or potential pre-cancerous cells. With the extension of life span the probability to get in contact with carcinogens increases, genetic instability, oncogene activation and/or onco-suppressor gene inactivation (i.e. p53, pRB, ras): the cancer transformation can be then induced in predisposed cells, depending on their genetic context, by the activation of telomere maintenance. Pharmacological intervention may be able to modulate the rate of living, by increasing life span of few specific target cells, or decreasing it in proliferating . Because of the unknown state of the enormous cell number of the human organism, is it safe to extend the human life span by therapeutic agents?

Aging↗
PubMed2001

Combination therapy of 2-5A antisense against telomerase RNA and cisplatin for malignant gliomas.

2',5'-Oligoadenylate (2-5A) linked to an antisense oligonucleotide against human telomerase RNA (2-5A-anti-hTR) is a novel therapeutic modality we have recently developed. We designed the oligonucleotide to target telomerase which maintains chromosome integrity in cancer cells. We have already demonstrated the efficacy of this new therapy in malignant glioma cells with telomerase activity. In the present study, we investigated the effect of 2-5A-anti-hTR in combination with cisplatin, an anti-cancer drug commonly used for malignant glioma patients. Six human malignant glioma cell lines with telomerase activity were treated with 0.5 microM 2-5A-anti-hTR and/or cisplatin (0, 0.1, 1, 5, 10, or 20 microg/ml) for three days, and cell viability was measured using the MTT colorimetric assay. The combination therapy showed synergistic effect at 1 microg/ml cisplatin and additive effect at 5 microg/ml cisplatin. TUNEL staining of the treated cells showed significantly increased apoptotic cells after the combination therapy. Furthermore, tumor growth of subcutaneous xenografts of human malignant glioma cells in nude mice was effectively reduced after the combination therapy for seven days. Our study shows that the tumor-killing effects of the combined treatments are, at least in part, due to induction of apoptosis. 2-5A-anti-hTR is a promising therapy not only when used alone, but also in combination with cisplatin.

Animals↗
PubMed2001

Examination of the oncogenic potential of H19 gene in HeLa x normal human fibroblast hybrid cells.

Fusion of tumorigenic HeLa cells with human skin fibroblasts results in chromosomally stable non-tumorigenic hybrids. The studies of rare spontaneous tumorigenic segregants from the non-tumorigenic hybrid have implicated the loss of one copy of human fibroblast chromosome 11 with concomitant re-expression of tumorigenicity. In a previous study of differential display screening, we reported the tumorigenic-specific expression of H19 gene as a possible candidate for elicitation of tumorigenic phenotype. In this study, we examined the expression of H19 gene using gamma-ray-induced tumorigenic mutants (GIMs) and non-tumorigenic irradiated control cells (CONs) from the non-tumorigenic hybrids. H19 expression was recognized in all the GIMs with various expression levels, whereas no CONs expressed the H19 gene. To examine the tumorigenic potentials of H19 gene directly, we introduced an H19 gene expression vector into the non-tumorigenic hybrids and assayed the tumorigenicity of the transfectants by s.c. injection into athymic nude mice. However, no transfectants with stable H19 gene expression induced in vivo tumor growth. These results suggest that expression of the H19 gene may be necessary but is not sufficient to confer the tumorigenic phenotype in HeLa x fibroblast hybrids.

Animals↗
PubMed2005

Molecular genetics of spinocerebellar ataxia type 8 (SCA8).

Spinocerebellar ataxias (SCAs) belong to a group of autosomal dominant, late-onset neurodegenerative disorders characterized by slowly progressive ataxia that eventually leads to severe gait, speech, coordination and sensory loss. The majority of these diseases result from expanded polyglutamine tracts in the encoded protein as seen in SCA1, SCA2, SCA3, SCA6, SCA7 and Dentatorubral-Pallidoluysian Atrophy (DRPLA). However, two novel forms of SCAs, SCA8 and SCA12, are associated with trinucleotide repeat expansions in non-translated regions of the genes. In the case of SCA8, the CUG expansion occurs at the 3' end of a processed non-coding RNA. While understanding of how expanded polyglutamine tracts compromise or alter protein function has advanced rapidly in the last five years, understanding of how trinucleotide repeat expansions alter the function of the non-coding SCA8 RNA and lead to human disease remains quite limited. Encouragingly, as discussed in this review, recent studies from murine and Drosophila models have provided new insights into both the cellular context in which SCA8 normally operates and the potential role of CTG expansion in the disease. Continued exploration of these genetically tractable model systems will further illuminate the biology underlying SCA8 disease, ultimately providing the necessary foundation on which to develop effective therapeutic interventions.

Animals↗
PubMed1999

Genomic imprinting of H19 and insulin-like growth factor-2 in pediatric germ cell tumors.

BACKGROUND: Insulin-like growth factor-2 (IGF2) and H19 are reciprocally imprinted genes on chromosome 11; IGF2 is expressed paternally and H19 is expressed maternally. Loss of imprinting (LOI) at both H19 and IGF2 has been reported in seven fully informative adult testicular germ cell tumors (GCTs) and may contribute to germ cell carcinogenesis. METHODS: Genomic DNA from 61 pediatric GCTs was amplified by polymerase chain reaction (PCR) and screened for heterozygosity at both IGF2 and H19 using either ApaI or RsaI, respectively. If heterozygous, polyadenylated RNA was isolated and reversed-transcribed into cDNA. cDNA then was amplified by PCR and the products were digested with restriction enzymes to evaluate GCT expression of IGF2 and H19. RESULTS: Eleven pediatric GCTs were fully informative for H19 and IGF2, including 5 ovarian GCTs, 2 testicular GCTs, and 4 extragonadal GCTs. Consistent with prior studies, both testicular GCTs showed LOI at both H19 and IGF2. In contrast, three of the five ovarian GCTs had LOI at both IGF2 and H19; one had LOI at IGF2 only, and one retained imprinting at both loci. Only one of the four extragonadal GCTs had LOI at IGF2 whereas three of the four had LOI at H19. CONCLUSIONS: These data suggest that LOI at H19 and IGF2 also may be common in pediatric testicular GCTs. However, ovarian and extragonadal pediatric GCTs showed variable patterns of LOI that may indicate differences in the timing of carcinogenesis in germ cells at these sites.

Adolescent↗
PubMed2000

Identification of a cell type-specific silencer in the first exon of the His-1 gene.

The His-1 gene is developmentally expressed in the murine choroid plexus but is silenced in the adult brain. To test the hypothesis that the gene contains cis-acting elements that contribute to this repression, we have analyzed segments of the proximal promoter for negative regulatory sequences by transient transfection analysis. The activity of the proximal promoter was moderately influenced by positively and negatively acting sequences located from -335 to -168 and -617 to -335, respectively. A strong His-1-positive regulatory element (HPRE, +18 to +29) was essential for maximal promoter activity and could also enhance the activity of the heterologous SV40 promoter in an orientation-dependent manner. The HPRE contains homology to the neuronal restrictive silencer element (NRSE) but interacted with nuclear proteins that were distinct from the NRSE-binding factor (NRSF). By contrast, a potent negative regulatory sequence (HNRE) was identified in the first exon that repressed either the His-1 or SV40 promoters by greater than 80%. This negative regulatory sequence interacted with nuclear proteins from cells that contain a silent His-1 gene but showed no interaction with nuclear proteins from cells that actively transcribe the endogenous gene. HNRE-mediated repression was orientation independent; most of this activity was mapped to a minimal 26-bp sequence. These findings suggest that the first exon of the His-1 gene contains a cell type-specific silencer that contributes to the regulation of His-1 transcription.

3T3 Cells↗
PubMed2000

Loss of imprinting of the IGF-II and H19 genes in epithelial ovarian cancer.

To establish a possible role of genomic imprinting in the carcinogenesis of epithelial ovarian cancer, we determined the imprinting status of both IGF-II and H19 genes in 43 ovarian cancers, 7 low malignant potential ovarian tumors, and their matched normal tissues. In ovarian cancer, loss of heterozygosity (LOH) of IGF-II, H19 RsaI, and H19 AluI was found in 4 of 24 (16.7%), 3 of 20 (15%), and 1 of 16 (6.3%) samples, respectively. All patients with tumor specimens exhibiting LOH are of advanced clinical stages. Loss of imprinting (LOI) was found in 5 of 20 (25%) for IGF-II and in 4 of 17 (23.5%) and 1 of 15 (6.7%) for the RsaI and AluI sites of H19 gene with no LOH. However, no LOH was found in low malignant potential tumors, and only one of them showed LOI in H19 AluI site. Overexpression of IGF-II was demonstrated in all five LOI samples with normal expression of H19. Three of the five tumor specimens exhibiting LOI were transcribed from P1 promoter, whereas the remaining two were from the P3 promoter. These results suggested that LOH of both IGF-II and H19 genes was associated with advanced ovarian cancer. LOI of IGF-II and H19 genes may be involved in the development of ovarian cancer. Transcription of IGF-II from the P1 promoter may account for the biallelic expression of the IGF-II gene.

Female↗
PubMed2000

Characterization and clinical implications of marker chromosomes identified at prenatal diagnosis.

Eighteen fetuses with marker chromosomes were detected at diagnostic amniocentesis in our laboratory among 15 781 amniocentesis samples. Using combined approaches, conventional cytogenetics including special stain techniques and fluorescence in situ hybridization (FISH), we successfully characterized 15 of them, which assisted subsequent genetic counselling. Six marker chromosomes were of sex chromosome origin, each of which substituted a missing sex chromosome, and 12 were supernumerary marker chromosomes (SMCs). Nine of the SMCs were proven to be of autosomal origin. Of those autosomal SMCs, five originated from chromosome 15, two from chromosome 18, one from chromosome 12 and one from chromosome 1. Among 16 marker chromosomes with adequate follow-up information, 50% were benign including four sex chromosome markers and four autosomal markers. Two thirds of de novo marker chromosomes were associated with abnormal outcomes, while all inherited ones were benign regardless of their parental origin. Our study demonstrated that molecular characterization of prenatal marker chromosomes is of great significance in facilitating phenotype-genotype correlation.

Amniocentesis↗
PubMedDate not supplied

Telomerase activity is down regulated via decreases in hTERT mRNA but not TEP1 mRNA or hTERC during the differentiation of leukemic cells.

BACKGROUND: Recent studies have determined several telomerase-associated molecules, but the precise mechanisms regulating telomerase activity by those molecules has not been fully understood. MATERIALS AND METHODS: The telomerase activity was determined by TRAP assay. Using TaqMan RT-PCR, the quantitative and kinetic values of mRNA expression of the three telomerase-associated molecules were examined in HL60 cells differentiated with tumor necrosis factor mutant and all-transretinoic acid. RESULTS: The levels of telomerase activity in leukemic cell lines, leukemic cells from patients, and normal peripheral blood cells were distributed over a very wide range. Human telomerase catalytic subunit (hTERT) mRNA expression declined to nearly undetectable levels more rapidly than the inhibition of telomerase activity after treatment with these reagents in HL60 cells. Telomerase-associated protein (TEP1) mRNA increased approximately 6-fold over its level in untreated cells. The levels of human telomerase RNA component (hTERC) also increased approximately 2.7-fold at 5 days after treatment. CONCLUSIONS: These results suggest that telomerase activity is down-regulated mainly via decreases in hTERT, but not TEP1 and hTERC expression during the differentiation of leukemic cells.

Adolescent↗
PubMed2000

Telomerase activity and expression of hTRT and hTR in gastrointestinal stromal tumors in comparison with extragastrointestinal sarcomas.

Stromal tumors of the gut (GISTs) have rarely been analyzed for genetic alterations. This study aimed at determining telomerase activity and the expression of the telomerase subunits human telomerase reverse transcriptase (hTRT) and human telomerase RNA (hTR) in GISTs and extragastrointestinal neurogenic or myogenic sarcomas. Telomerase activity was investigated using the telomeric repeat amplification protocol assay in 21 GISTs, recurrences and liver metastases from 16 patients, and in 22 leiomyosarcomas and 21 malignant peripheral nerve sheath tumors (MPNSTs), which served as reference tumors. Expression of hTRT and hTR mRNA was investigated using reverse transcription-PCR. Thirteen GISTs were localized in the stomach and three in the small intestine. Two tumors were benign. In one case, the biological behavior was uncertain. In 67% of GISTs, high telomerase activity was found, whereas high activity was noted in only 18% of leiomyosarcomas and in 48% of MPNSTs. There was no activity in two benign and two malignant GISTs. In one malignant tumor of the small intestine, the primary tumor showed no activity at first but a marked activity in its recurrence. In the tumor with uncertain behavior, telomerase activity and hTRT expression were only weak. In all GISTs showing telomerase activity, the catalytic subunit hTRT was expressed. All GISTs and extragastrointestinal sarcomas expressed hTR. In comparison with leiomyosarcomas and MPNSTs, malignant GISTs showed a higher telomerase activity, which, however, was not seen in benign GISTs. It is possible that telomerase activity occurs during the progression of malignant GISTs. There was a correlation between telomerase activity and the expression of hTRT.

Adult↗
PubMed2000

H19 and Igf2 monoallelic expression is regulated in two distinct ways by a shared cis acting regulatory region upstream of H19.

H19 and Igf2 are expressed in a monoallelic fashion from the maternal and paternal chromosomes, respectively. A region upstream of H19 has been shown to regulate such imprinted expression of both genes in cis. We have taken advantage of a loxP/cre recombinase-based strategy to delete this region in mice in a conditional manner to determine the temporal requirement of the upstream region in initiating and maintaining the imprinted expression of H19 and Igf2. Analysis of allele-specific expression of H19 and Igf2 and DNA methylation at the H19 promoter demonstrates that this region controls the monoallelic expression of the two genes in different ways, suggesting that it harbors two functionally distinct regulatory elements. Continued presence of the region is required to silence maternal Igf2 in accordance with its proposed role as an insulator. However, it does not have a direct role in keeping the paternal H19 promoter silenced. Instead, on the paternal chromosome, the upstream element mediates epigenetic modifications of the H19 promoter region during development, leading to transcriptional silencing of H19. Thereafter, its presence is redundant for preventing transcription. Presently, this temporal requirement of the silencing element appears to be a unique cis activity in the mammalian system. However, it is likely that other cis-acting elements, positive and negative, have the ability to effect stable changes in the chromatin structure and are not constantly required to give signals to the transcriptional machinery.

Alleles↗
PubMed2000

Severe phenotypes associated with inactive ring X chromosomes.

Mental retardation and congenital malformations in individuals with small ring X chromosomes are often due to the functional disomy that results from failure of these chromosomes to undergo X inactivation. Such chromosomes either lack the XIST locus or do not express it. We have carried out genetic analysis of the ring X chromosomes from two girls with a 45,X/46,X,r(X) karyotype, mental retardation, and a constellation of abnormalities characteristic of the severe phenotype due to X disomy. In each case the ring X chromosome included an intact XIST locus which was expressed; the breakpoints were distal to DXS128, and therefore outside the XIC region; transcription analysis of alleles at the androgen receptor locus confirmed that these were inactive chromosomes. The characteristics of the XIST RNA were similar to the wild-type. Additional studies in cultured fibroblasts showed a second ring in a small percentage of the cells. The association of severe phenotype with an inactive X chromosome most likely reflects the presence of a second ring X chromosome which was active at least in some tissues during embryogenesis, but is no longer prominent in the tissues we analyzed.

Dosage Compensation, Genetic↗
PubMed1999

Telomerase hTR and hTRT gene expression in oral precancerous lesions and squamous cell carcinomas.

OBJECTIVE: To investigate the expression of telomerase genes in oral precancerous lesions and squamous cell carcinomas and to research the relationship between telomerase gene expression and carcinogenesis of oral mucosa epithelium. MATERIALS AND METHODS: Eighty two cases were detected for hTR and hTRT gene by in situ hybridization techniques--7 cases of normal oral mucosa, 7 cases of hyperplasia lesions, 30 cases of oral precancerous lesions (dysplasia lesions), 8 cases of oral mucosa carcinomas in situ, and 30 cases of oral squamous cell carcinomas. RESULTS: Weaker signals of hTR and hTRT gene expression were observed in normal oral epithelia and hyperplasia lesions; hTR and hTRT were positive 28.6% (4 of 14) and 21.4% (3 of 14), respectively. The expression of telomerase genes in the precancerous lesions became stronger due to phenotypic progression and the increasing degree of dysplasia; hTR and hTRT were positive 60.0% (18 of 30) and 46.7% (14 of 30), respectively. Stronger hTR and hTRT expressions were observed in squamous cell carcinoma, with an equal positivity of 81.6% (31 of 38). CONCLUSION: Telomerase gene expression is closely related to the malignant degree of oral mucosa. Telomerase is reactivated frequently during late stages of oral precancerous lesions and may play a crucial role in progression of oral cancer.

Carcinoma, Squamous Cell↗
PubMed2000

[Wilms' tumors and malformation complexes].

Wilms' tumor is an embryonal tumor which is derived from metanephric metanephric blastema. The occurrence of both sporadic and hereditary forms, along with various congenital abnormalities of Wilms' tumor suggest that the tumors develop when a predisposing germ line mutation is accompanied by a second mutation. The existence of both gross chromosomal abnormalities has led to the genetic characterization of a number of loci involved in the development of Wilms' tumor. A tumor suppressor gene for Wilms' tumor, WT1, has been isolated from the 11p13 region. The product of this gene is a transcription factor with four zinc fingers. Because of expression of WT1 is limited to the developing glomeruli of the kidneys and the genital ridge, it is thought to have a functional role in renal and gonadal organogesis. Thus dysfunction of WT1 causes loss of normal regulation of proliferation and leads to tumor formation and occurrence of Wilms' tumor anomaly complexes. The role of the imprinting genes, H19 and IGF2 in oncogenesis of Wilms' tumors are also discussed.

Chromosome Aberrations↗
PubMed2000

A transcriptional insulator at the imprinted H19/Igf2 locus.

Igf2 and H19 exhibit parent-of-origin-specific monoallelic expression. H19 is expressed from the maternal chromosome and Igf2 from the paternal. The two genes share enhancer elements and monoallelic expression of both genes is dependent on cis-acting sequences upstream of the H19 promoter. In this work we examine the mechanisms by which this region silences the maternal Igf2 allele and we demonstrate that deletion of this region can result in high levels of activation of both H19 and Igf2 from a single chromosome. Moreover, by inserting this cis element between a promoter and its enhancer at a heterologous position, we demonstrate that the sequences carry both insulator activity and the ability to be stably imprinted. We also characterize the insulator in vitro and show that it is neither enhancer nor promoter specific.

Animals↗