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PubMed1997

Involvement of AP-2 in regulation of the R-FABP gene in the developing chick retina.

Little is known regarding the molecular pathways that underlie the retinal maturation process. We are studying the regulation of the retinal fatty-acid-binding protein (R-FABP) gene, highly expressed in retinal precursor cells, to identify DNA regulatory elements and transcriptional factors involved in retinal development. Although the upstream sequence of the R-FABP gene is extremely GC rich, CpG methylation in this region is not implicated in the regulation of this gene because the 5' flanking DNA remains unmethylated with tissue differentiation when there is a dramatic decrease in R-FABP transcript levels. Using a combination of DNase I hypersensitivity experiments, gel shift assays, and DNase I footprinting, we have found three sites of DNA-protein interaction within 205 bp of 5' flanking DNA in the undifferentiated retina and four sites in the differentiated retina. DNA transfection analysis indicates that the first two footprints located within 150 bp of 5' flanking DNA are required for high levels of transcription in primary undifferentiated retinal cultures. The first footprint includes a putative TATA box and Spl binding sites while the second footprint contains a consensus AP-2 DNA binding site. Supershift experiments using antibodies to AP-2 and methylation interference experiments indicate that an AP-2-like transcription factor present in both late-proliferative-stage retina and differentiated retina binds to the upstream region of the R-FABP gene. A combination of data including the expression profile of AP-2 during retinal development and DNA transfection analysis using constructs mutated at critical residues within the AP-2 binding site suggests that AP-2 is a repressor of R-FABP transcription.

Animals↗
PubMed2004

Comparison of gene expression changes induced in mouse and human cells treated with direct-acting mutagens.

Exposure to DNA-damaging agents can elicit a variety of stress-related responses that may alter the gene expression of numerous biological pathways. We used Affymetrix microarrays to detect gene expression changes in mouse lymphoma (L5178Y) and human lymphoblastoid (TK6) cells in response to methyl methanesulfonate (MMS; a prototypical alkylating agent) and bleomycin (a prototypical oxidative mutagen). Cells were treated for 4 hr, and RNA was isolated either at the end of the treatment or after a 20-hr recovery period. Two concentrations of each agent were used based on cytotoxicity levels and Tk mutant frequencies. Our microarray data analysis indicated that MMS and bleomycin gene expression responses were considerably different in mouse cells versus human cells. The results also suggested that more comprehensive cellular responses to MMS and bleomycin occurred in TK6 cells than in L5178Y cells. In contrast to L5178Y cells, the response of TK6 cells to MMS and bleomycin was characterized by the induction of p53-dependent genes that are involved in DNA repair, cell cycle regulation, and apoptosis. It appears that the induction of DNA damage by MMS in human TK6 cells mediated cytotoxicity and led to decreased cell survival. This may explain the greater sensitivity of TK6 cells to cytotoxic effects of MMS compared to L5178Y cells. Bleomycin exerted comparable cytotoxic effects in the two cell lines. Overall, these studies were unable to identify distinctive gene expression changes that differentiated bleomycin from MMS in either TK6 cells or mouse lymphoma cells.

Alkylating Agents↗
PubMed1999

Androgen receptor-mediated hypersensitivity to androgens in women with nonhyperandrogenic hirsutism: skewing of X-chromosome inactivation.

Idiopathic hirsutism may result from an increase in the androgen receptor (AR)-mediated sensitivity of the hair follicle. The AR gene is located on the X-chromosome and contains a highly polymorphic trinucleotide repeat (CAGn) in its first exon, whose length and methylation pattern affect both AR expression and function. We analyzed these CAG repeats in the genomic DNA from 16 nonhyperandrogenic hirsute patients (Ferriman score: 16 +/- 4.7, mean +/- SD) and 10 normal controls (Ferriman score: 3 +/- 1.4), who were similar in their hormonal profiles. We found no difference in the number of CAG repeats between hirsute patients and controls, and no correlation between number of repeats and the Ferriman score or hormonal values. However, after DNA digestion with methylation-sensitive HpaII and measurement of the optical density, we found a marked decrease in the hirsute group (P < 0.0001), which was greater than in the control group (P = 0.0003). In addition, in the hirsute patients, the shorter of the two alleles was preferentially less methylated (P = 0.007), suggesting skewing of X-chromosome inactivation in the patients but not in the controls. When the mean optical density of both alleles was correlated with the Ferriman score, we observed a significant negative correlation (P = 0.02, r = -0.45), which became stronger when the shorter alleles were analyzed separately (P = 0.01; r = 0.48). We conclude that nonhyperandrogenic hirsutism is associated with skewing of X-chromosome inactivation in peripheral blood lymphocytes. This leads to the longer of the two AR alleles being preferentially methylated, allowing for the shorter (and presumably, more functional) allele to be expressed on the active X-chromosome. Further studies need to be performed to investigate whether this phenomenon is present in androgen-sensitive tissues in these patients.

Adult↗
PubMed2006

Proteomic profiling for cellular responses to different concentrations of N-methyl-N'-nitro-N-nitrosoguanidine.

Alkylating agent MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) can induce DNA damages which can lead to chromosomal aberrations, mutations, and cell death. Previous reports from our laboratory have found that low concentration of MNNG can induce nontargeted mutations (NTM) at undamaged bases in DNA, clustering of epidermal growth factor receptor (EGFR) and interference of EGFR mediated signaling, as well as activation of endoplasmic reticulum stress. Thus, the cellular responses to MNNG exposure are very complex, and can be triggered by signals originated from different compartments of the exposed cells. To further probe the molecular mechanisms involved in cellular responses to MNNG treatment, and to find potential biomarkers for MNNG induced stress condition, we performed proteomic analysis of whole cellular proteins from human amnion epithelial cells after exposing to MNNG at 3 different doses. More than 80 proteins were affected by MNNG treatment, and 71 proteins among them were identified using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. These proteins take part in a wide variety of cellular processes including regulation of transcription, metabolism, cytoskeleton organization, cell cycle, cell proliferation, signal transduction, transportation, etc. The significance of these proteins in the genesis of MNNG induced cellular defensive response and hazardous effect remains to be elucidated, the results may also give a clue for biomarker search for monitoring the exposure risk of MNNG.

Amnion↗
PubMed2026

Meningioma methylation profiling as a complement to WHO grading: a single-center experience.

OBJECTIVE: The methylation profile of meningiomas is a promising predictive tool that may improve risk stratification beyond WHO grading. This study aimed to evaluate the clinical relevance and real-world applicability of routine epigenetic testing in meningioma management. METHODS: The authors retrospectively analyzed patients who underwent meningioma resection between January 2021 and December 2023. Histopathological grading (WHO 2021) and methylation profiling (methylation class [MC]) with the MethylationEPIC v1.0 (850k) chip were performed by an independent neuropathologist. RESULTS: A total of 106 patients were included; 81 tumors (76%) were classified as WHO grade 1, 20 (19%) as grade 2, and 5 (5%) as grade 3. Epigenetically, 55 tumors (52%) were classified as benign, 18 (17%) as intermediate, and 2 (2%) as malignant; 31 (29%) could not be classified. Discordances between WHO grading and methylation profiling were observed in 18 of 74 cases. Tumor board decisions were made after a median of 8 days postoperatively, guided by WHO grading; however, the epigenetic report was only available after a median of 23 days. During follow-up, 20 patients experienced tumor progression. Progression was significantly associated with the MC (r = -0.4, p < 0.001) and tumor volume (r = 0.4, p = 0.0005), but not with WHO grading (r = 0.17, p = 0.084). However, the relatively high rate of unclassified tumors and delayed result availability limited the direct impact of MC profiling on immediate clinical decision-making. Interestingly, progression-free survival in MC-unclassified tumors mirrored that of the intermediate group. CONCLUSIONS: Methylation profiling demonstrates superior predictive accuracy for meningioma progression and complements WHO grading, especially in identifying malignant meningiomas. However, its current clinical utility is constrained by technical and logistical limitations. In real-world practice, epigenetic classification should therefore be considered a complementary tool rather than a replacement for established histopathological assessment.

Humans↗
PubMed2004

DNA microarrays, a novel approach in studies of chromatin structure.

The DNA microarray technology delivers an experimental tool that allows surveying expression of genetic information on a genome-wide scale at the level of single genes--for the new field termed functional genomics. Gene expression profiling--the primary application of DNA microarrays technology--generates monumental amounts of information concerning the functioning of genes, cells and organisms. However, the expression of genetic information is regulated by a number of factors that cannot be directly targeted by standard gene expression profiling. The genetic material of eukaryotic cells is packed into chromatin which provides the compaction and organization of DNA for replication, repair and recombination processes, and is the major epigenetic factor determining the expression of genetic information. Genomic DNA can be methylated and this modification modulates interactions with proteins which change the functional status of genes. Both chromatin structure and transcriptional activity are affected by the processes of replication, recombination and repair. Modified DNA microarray technology could be applied to genome-wide study of epigenetic factors and processes that modulate the expression of genetic information. Attempts to use DNA microarrays in studies of chromatin packing state, chromatin/DNA-binding protein distribution and DNA methylation pattern on a genome-wide scale are briefly reviewed in this paper.

Chromatin↗
PubMed2005

Epigenetic status of the H19 locus in human oocytes following in vitro maturation.

Imprinting is an epigenetic modification that is reprogrammed in the germ line and leads to the monoallelic expression of some genes. Imprinting involves DNA methylation. Maternal imprint is reset during oocyte growth and maturation. In vitro maturation (IVM) of oocytes may, therefore, interfere with imprint acquisition and/or maintenance. To evaluate if maturing human oocytes in vitro would be hazardous at the epigenetic level, we first determined the methylation profile of the H19 differentially methylated region (DMR). The methylation status of the H19 DMR seems particularly vulnerable to in vitro culture conditions. We analyzed oocytes at different stages of maturation following IVM, germinal vesicle (GV), metaphase I (MI), and metaphase II (MII), using the bisulfite mutagenesis technique. Our results indicated that the unmethylated specific maternal profile for the H19 DMR was stably established at the GV stage. The majority of MI-arrested oocytes exhibited an altered pattern of methylation, the CTCF-binding site being methylated in half of the DNA strands analyzed. Of the 20 MII oocytes analyzed, 15 showed the normal unmethylated maternal pattern, while 5 originating from two different patients exhibited a methylated pattern. These findings highlight the need for extended analysis on MII-rescued oocytes to appreciate the epigenetic safety of the IVM procedure, before it becomes a routine and practical assisted reproductive procedure.

Adult↗
PubMed2003

Transcriptional regulation of sorghum defense determinants against a phloem-feeding aphid.

When attacked by a phloem-feeding greenbug aphid (Schizaphis graminum), sorghum (Sorghum bicolor) activates jasmonic acid (JA)- and salicylic acid (SA)-regulated genes, as well as genes outside known wounding and SA signaling pathways. A collection of 672 cDNAs was obtained by differential subtraction with cDNAs prepared from sorghum seedlings infested by greenbug aphids and those from uninfested seedlings. Subsequent expression profiling using DNA microarray and northern-blot analyses identified 82 transcript types from this collection responsive to greenbug feeding, methyl jasmonate (MeJA), or SA application. DNA sequencing analyses indicated that these encoded proteins functioning in direct defense, defense signaling, oxidative burst, secondary metabolism, abiotic stress, cell maintenance, and photosynthesis, as well as proteins of unknown function. In response to insect feeding, sorghum increased transcript abundance of numerous defense genes, with some SA-dependent pathogenesis-related genes responding to greenbug more strongly than to SA. In contrast, only weak induction of MeJA-regulated defense genes was observed after greenbug treatment. However, infestation tests confirmed that JA-regulated pathways were effective in plant defense against greenbugs. Activation of certain transcripts exclusively by greenbug infestation was observed, and may represent unique signal transduction events independent of JA- and SA-regulated pathways. Results indicate that plants coordinately regulate defense gene expression when attacked by phloem-feeding aphids, but also suggest that aphids are able to avoid triggering activation of some otherwise potentially effective plant defensive machinery, possibly through their particular mode of feeding.

Animals↗
PubMed2005

Upstream CpG island methylation of the PAX3 gene in human rhabdomyosarcomas.

BACKGROUND: Adult tumors can be characterized by hypermethylation of CpG islands associated with 5'-upstream and coding regions of specific genes. This hypermethylation can also be part of the aging process. In contrast, much less is known about gene hypermethylation in childhood cancers, where methylation changes are not part of the aging process but likely represent developmental dysregulation. PAX3 is an important gene in muscle development and muscle-producing neoplasms such as rhabdomyosarcomas. PROCEDURES: We examined the methylation status of a PAX3 5'-CpG island in rhabdomyosarcoma subtypes and in normal fetal skeletal muscle. PAX3 methylation was analyzed in 15 embryonal rhabdomyosarcomas, 12 alveolar rhabdomyosarcomas, and in six normal skeletal muscle samples, using semi-quantitative PCR analysis of DNA digested with methyl-sensitive restriction enzymes. RESULTS: The CpG island in the upstream region of the human PAX3 gene was hypermethylated in the majority of ERMS examined (13 of 15 tumors, mean of 52% methylation), whereas most ARMS (9 of 12 tumors) and all normal muscle samples showed relative hypomethylation (both 18% mean methylation). Various CpG sites differ in contribution to overall PAX3 CpG island methylation, with methylation at a HaeII site being inversely correlated with PAX3 expression. CONCLUSIONS: PAX3 CpG island methylation appears to distinguish embryonal subtype of rhabdomyosarcoma from alveolar, and methylation at certain sites within this CpG island is inversely correlated with PAX3 expression. In addition to exemplifying developmental dysregulation, methylation of PAX3 has potential in the development of an epigenetic profile for the diagnosis of rhabdomyosarcoma.

Child↗
PubMed1990

DNA damage induced in mouse peritoneal exudate cells after in vivo administration of chemical and physical agents as determined by alkaline elution.

The alkaline elution technique for detecting DNA strand breaks has been applied to the study of DNA damage in mouse peritoneal exudate cells resulting from the in vivo administration of chemical and physical agents. The direct methylating agents methyl methanesulphonate and N-methyl-N-nitrosourea induced extensive breakage in samples taken 2 h after administration. The direct ethylating agents ethyl methanesulphonate and N-ethyl-N-nitrosourea also induced DNA strand breaks, but to a lesser extent than the methylating agents. The indirect methylating agent dimethylnitrosamine showed hardly any effect in this system. A weak but positive response was observed upon treatment with the anti-neoplastic alkylating agent procarbazine hydrochloride. The whole-body irradiation of mice with 60Co gamma-rays also induced DNA strand breaks. The elution profiles for gamma-ray irradiation were different from those of alkylating agents, and indicate that alkylating agents produce many more secondary lesions leading to DNA strand breaks than gamma-rays. N-methyl-N-nitrosourea produced slightly more DNA strand breaks in mutagen-sensitive mice, which are derived from the CD-1 strain, than in ICR mice.

Alkylating Agents↗
PubMed2002

Use of CpG island microarrays to identify colorectal tumors with a high degree of concurrent methylation.

We provide a comprehensive description of our microarray-based technique for the simultaneous detection of multiple CpG islands in cancer. Amplicons from tumor and control samples were pools of differentially methylated CpG island fragments hybridized to a panel of approximately 8000 CpG island tags. Data analysis identified 694 CpG island loci hypermethylated in a group of 14 colorectal tumors. The Stanford hierarchical cluster algorithm segregated the tumors into two subgroups, one of which exhibited a high level of concurrent hypermethylation while the other had little or no methylation. This is in agreement with previous observations of a CpG island methylation phenotype present in colorectal tumors. The present study demonstrates that this microarray-based technique is useful in classifying tumors according to their methylation profiles.

Algorithms↗
PubMed2005

Pattern of nonspecific (or global) DNA methylation in oral carcinogenesis.

BACKGROUND: Although alterations in nonspecific (or global) DNA methylation (GDM) in specific cells are known to be involved in the process of lung carcinogenesis, similar associations have not been evaluated in other smoking-related cancers of the head and neck. METHODS: We evaluated the status of GDM by using monoclonal antibodies specific for 5-methylcytosine (5-mc) in oral squamous cell carcinoma (SCC) specimens of 48 cigarette smokers who had SCC develop and in 93 age-, race-, and sex-matched smokers who did not. RESULTS: Percentages of cells positive for 5-mc immunostaining of DNA of SCC and dysplastic lesions were significantly higher than those of normal oral epithelial cells from cancer subjects and from noncancer subjects. The degree of DNA methylation was unrelated to DNA content. CONCLUSIONS: The pattern of GDM in oral SCCs is different from that of lung SCCs. The differences in nutrient risk factor profiles that are related to GDM and differential activity of DNA methyltranferases between oral and lung SCCs may explain these observations.

5-Methylcytosine↗
PubMed2006

E-cadherin transcriptional downregulation by promoter methylation but not mutation is related to epithelial-to-mesenchymal transition in breast cancer cell lines.

Using genome-wide expression profiling of a panel of 27 human mammary cell lines with different mechanisms of E-cadherin inactivation, we evaluated the relationship between E-cadherin status and gene expression levels. Expression profiles of cell lines with E-cadherin (CDH1) promoter methylation were significantly different from those with CDH1 expression or, surprisingly, those with CDH1 truncating mutations. Furthermore, we found no significant differentially expressed genes between cell lines with wild-type and mutated CDH1. The expression profile complied with the fibroblastic morphology of the cell lines with promoter methylation, suggestive of epithelial-mesenchymal transition (EMT). All other lines, also the cases with CDH1 mutations, had epithelial features. Three non-tumorigenic mammary cell lines derived from normal breast epithelium also showed CDH1 promoter methylation, a fibroblastic phenotype and expression profile. We suggest that CDH1 promoter methylation, but not mutational inactivation, is part of an entire programme, resulting in EMT and increased invasiveness in breast cancer. The molecular events that are part of this programme can be inferred from the differentially expressed genes and include genes from the TGFbeta pathway, transcription factors involved in CDH1 regulation (i.e. ZFHX1B, SNAI2, but not SNAI1, TWIST), annexins, AP1/2 transcription factors and members of the actin and intermediate filament cytoskeleton organisation.

Breast Neoplasms↗
PubMed1990

Identification and purification of a Bombyx mori homologue of FTZ-F1.

Extracts from embryos and from posterior and middle silk glands of the silkworm, Bombyx mori contain a sequence specific DNA binding factor termed BmFTZ-F1. The factor binds to the recognition site of FTZ-F1, a positive regulator of the fushi tarazu gene in Drosophila melanogaster. BmFTZ-F1 and FTZ-F1 share the same methylation interference patterns, the same chromatographic behaviors and similar protease digestion profiles. Anti-FTZ-F1 cross reacts with BmFTZ-F1. These results indicate that BmFTZ-F1 is a B. mori homologue of FTZ-F1. The mobility of the factor-DNA complex formed in the silk gland extract changes depending on the developmental stages. Purification of BmFTZF1 to an almost homogeneous state reveals that the factor is a 73 kd protein.

Animals↗
PubMed1990

Thalidomide induced alteration in secondary structure of rat embryonic DNA in vivo.

Teratogenicity of thalidomide was demonstrated in Wistar rats following a single maternal intravenous injection during the embryonic organogenetic period. When compared to day 13 embryonic DNA isolated from untreated control mothers, differences were observed in the mean wet weights of day 13 embryos from rats treated with thalidomide during days 10 or 11 of gestation, and significantly less amounts of embryonic DNA were recovered from mothers similarly treated on days 10 or 12 of their respective gestation periods. Rat embryonic DNA may be separated into two fractions by stepwise elution from benzoylated DEAE-cellulose (BD-cellulose) columns with 1.0 M NaCl (SE-DNA) and 1.8% (w/v) caffeine (CE-DNA) solutions, respectively. Other studies using bacterial, yeast, and rat liver DNA suggested that the first fraction contains native DNA, whereas the second may exhibit some degree of single-stranded character. Similar reproducible chromatographic profiles were obtained using a novel "batch method" developed for general application. Rat embryonic DNA was monitored by labelling in vivo with an i.p. injection of [methyl-3H]-thymidine (3H-TdR) during days 5, 6, and 7 of the gestation period. All samples were analysed on day 13 of gestation. A simple increase in percentage of caffeine-eluted DNA was not detected in thalidomide treated samples; however, diversity of percent (%) CE-DNA within litter was noted. Briefly, the percent CE-DNA values for embryos in one litter were ranked and arbitrarily grouped in classes with limits of mean +/- 1 SD, mean +/- 2 SD, and so on to generate a characteristic profile of percent CE-DNA distribution. The number of embryos within the range of each SD unit was expressed as a percentage of each litter. A plot of the ranges of percent CE-DNA versus percentage of each litter was used to illustrate the distribution profile of the particular litter and to be used for comparison between samples from untreated control and thalidomide and/or dimethylformamide (DMF) treated DNA. Treatment of day 12 mothers with thalidomide produced a majority of embryos having percent CE-DNA values similar to those of untreated controls with the exception of the inclusion of a second population of embryos with much higher percent CE-DNA values than those of the untreated controls. Similar treatment of day 11 animals produced a majority of embryos still having percent CE-DNA values similar to those of untreated controls and also having a second group of embryos with a lower percent CE-DNA values than those of untreated controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2005

Frequent downregulation of the runt domain transcription factors RUNX1, RUNX3 and their cofactor CBFB in gastric cancer.

Our previous studies suggest that lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer, but potential roles of other members of the RUNX family genes have not yet been reported. We examined the expression of 3 Runt-related (RUNX) genes, RUNX1, RUNX2 and CBFB, in gastric cancer cell lines and primary gastric cancer specimens and compared them to those of RUNX3 reported earlier in conjunction with clinicopathologic factors. Expression of RUNX family genes in 9 gastric cancer cell lines, 56 primary gastric cancer specimens and surrounding normal gastric mucosa were estimated by Northern blot analysis, quantitative RT-PCR and in situ hybridization. Northern blot analysis in gastric cancer cell lines showed downregulation of RUNX1 and RUNX3 in 67% and 78% of the cell lines tested, respectively. The ratio of the average RUNX mRNA/beta-actin mRNA ratio (x10(3)) for RUNX1 was 48.0 +/- 21.1 vs. 21.4 +/- 8.1; RUNX2, 1.1 +/- 0.3 vs. 1.0 +/- 0.2; RUNX3, 9.2 +/- 6.3 vs. 3.1 +/- 1.3 and CBFB, 42.0 +/- 19.4 vs. 21.0 +/- 8.4 (normal vs. tumor, respectively, average +/-SD). The basal RUNX2 expression was very weak, and there was no significant change in gastric cancers. Both RUNX1 and RUNX3 showed remarkable downregulation in 62% and 69%, respectively, of surgically resected specimens compared to surrounding mucosa analyzed by quantitative RT-PCR (p < 0.01). Furthermore, CBFB, the gene encoding the cofactor of RUNX1, -2, -3, was also downregulated in significant fraction (32%, p < 0.05). The percentage of downregulation of RUNX1, RUNX3 and CBFB increased as the cancer stage progressed. Tricostatin A and 5'-azacitidin reactivate RUNX3 expression, but they could not reactivate expression of RUNX1 and CBFBeta in gastric cancer cells, suggesting that the downregulation was due to mechanisms other than methylation of the promoter region. These findings suggest that RUNX1 and CBFBeta in addition to RUNX3 play some roles in gastric cancers and that roles of RUNX gene family in gastric cancer are more widespread and complex than previously realized.

Aged↗
PubMed2005

Aberrant methylation of secreted frizzled-related protein genes in esophageal adenocarcinoma and Barrett's esophagus.

Hypermethylation of secreted frizzled-related proteins (SFRP) genes frequently occurs with several cancers but has not been studied in esophageal adenocarcinoma or its precursor-Barrett's esophagus. To explore the role of SFRP methylation in the neoplastic progression of Barrett's esophagus and to evaluate methylated SFRP genes as biomarkers for Barrett's esophagus and cancer, methylation of SFRP genes was determined in esophageal adenocarcinomas, Barrett's esophagus and normal epithelia using methylation-specific PCR. Protein expression of SFRP genes was then assessed in these tissues by immunohistochemistry. The mRNA expression of SFRP genes was quantified by real-time reverse-transcription PCR in esophageal adenocarcinoma cell lines with and without demethylation by 5-aza-2'deoxycytidine and inhibition of deacetylation by trichostatin A treatment. Hypermethylation of SFRP1, 2, 4 and 5 was detected in 93%, 83%, 73% and 85% of 40 cancers; 81%, 89%, 78% and 73% of 37 Barrett's epithelia; 25%, 64%, 32% and 21% of 28 adjacent normal epithelia from Barrett's patients; and 10%, 67%, 0% and 13% of 30 normal esophagogastric epithelia from healthy individuals, respectively (p < 0.001 for SFRP1, 4 and 5; p < 0.05 for SFRP2). Protein expression of SFRP1, 2 and 4 was downregulated in 87%, 67% and 90% of cancers, and expression correlated inversely with grade and stage of cancers and with grade of dysplasia. Expression of SFRP2 and SFRP4 proteins was lower in cancers with corresponding gene methylation (p < 0.05). Demethylation treatment effectively re-expressed SFRP mRNA in cancer cell lines. Thus, hypermethylation of SFRP genes is a common early event in the evolution of esophageal adenocarcinoma, and methylation of SFRP1, 4 and 5 might serve as biomarkers for Barrett's neoplasia. Aberrant promoter methylation appears to functionally silence SFRP gene expression in esophageal adenocarcinoma.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Promoter hypermethylation of the potential tumor suppressor DAL-1/4.1B gene in renal clear cell carcinoma.

Renal clear cell carcinoma (RCCC) is a malignant tumor with poor prognosis caused by the high incidence of metastasis to distal organs. Although metastatic RCCC cells frequently show aberrant cytoskeletal organization, the underlying mechanism has not been elucidated. DAL-1/4.1B is an actin-binding protein implicated in the cytoskeleton-associated processes, while its inactivation is frequently observed in lung and breast cancers and meningiomas, suggesting that 4.1B is a potential tumor suppressor. We studied a possible involvement of 4.1B in RCCCs and evaluated it as a clinical indicator. 4.1B protein was detected in the proximal convoluted tubules of human kidney, the presumed cell of origin of RCCC. On the other hand, loss or marked reduction of its expression was observed in 10 of 19 (53%) renal cell carcinoma (RCC) cells and 12 of 19 (63%) surgically resected RCCC by reverse transcription-PCR. Bisulfite sequencing or bisulfite SSCP analyses revealed that the 4.1B promoter was methylated in 9 of 19 (47%) RCC cells and 25 of 55 (45%) surgically resected RCCC, and inversely correlated with 4.1B expression (p < 0.0001). Aberrant methylation appeared to be a relatively early event because more than 40% of the tumors with pT1a showed hypermethylation. Furthermore, 4.1B methylation correlated with a nuclear grade (p = 0.017) and a recurrence-free survival (p = 0.0036) and provided an independent prognostic factor (p = 0.038, relative risk 10.5). These results indicate that the promoter methylation of the 4.1B is one of the most frequent epigenetic alterations in RCCC and could predict the metastatic recurrence of the surgically resected RCCC.

Aged↗