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PubMed2000

Use of randomly amplified polymorphic DNA as a means of developing genus- and strain-specific Streptomyces DNA probes.

We have analyzed 20 randomly amplified polymorphic DNA (RAPD) primers against 36 Streptomyces strains, including 17 taxonomically undefined strains, 25 nonstreptomycete actinomycetes, and 12 outgroups consisting of gram-positive and -negative species. Most of the primers were useful in identifying unique DNA polymorphisms of all strains tested. We have used RAPD techniques to develop a genus-specific probe, one not necessarily targeting the ribosomal gene, for Streptomyces, and a strain-specific probe for the biological control agent Streptomyces lydicus WYEC108. In the course of these investigations, small-scale DNA isolations were also developed for efficiently isolating actinomycete DNA. Various modifications of isolation procedures for soil DNA were compared, and the reliability and specificity of the RAPD methodology were tested by specifically detecting the S. lydicus WYEC108 in DNA isolated from soil.

Blotting, Southern↗
PubMed1990

A constitutive damage-specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells.

Using a DNA band shift assay, we have identified a DNA-binding protein complex in primate cells which is present constitutively and has a high affinity for UV-irradiated, double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin have higher levels of this damage-specific DNA-binding protein complex, suggesting that the signal for induction can either be damage to the DNA or interference with cellular DNA replication. Physiochemical modifications of the DNA and competition analysis with defined substrates suggest that the most probable target site for the damage-specific DNA-binding protein complex is a 6-4'-(pyrimidine-2'-one)-pyrimidine dimer: specific binding could not be detected with probes which contain -TT- cyclobutane dimers, and damage-specific DNA binding did not decrease after photoreactivation of UV-irradiated DNA. This damage-specific DNA-binding protein complex is the first such inducible protein complex identified in primate cells. Cells from patients with the sun-sensitive cancer-prone disease, xeroderma pigmentosum (group E), are lacking both the constitutive and the induced damage-specific DNA-binding activities. These findings suggest a possible role for this DNA-binding protein complex in lesion recognition and DNA repair of UV-light-induced photoproducts.

Animals↗
PubMed1998

Multiple mechanisms regulate imprinting of the mouse distal chromosome 7 gene cluster.

Genomic imprinting is an epigenetic process that results in the preferential silencing of one of the two parental copies of a gene. Although the precise mechanisms by which genomic imprinting occurs are unknown, the tendency of imprinted genes to exist in chromosomal clusters suggests long-range regulation through shared regulatory elements. We characterize a 800-kb region on the distal end of mouse chromosome 7 that contains a cluster of four maternally expressed genes, H19, Mash2, Kvlqt1, and p57(Kip2), as well as two paternally expressed genes, Igf2 and Ins2, and assess the expression and imprinting of Mash2, Kvlqt1, and p57(Kip2) during development in embryonic and extraembryonic tissues. Unlike Igf2 and Ins2, which depend on H19 for their imprinting, Mash2, p57(Kip2), and Kvlqt1 are unaffected by a deletion of the H19 gene region, suggesting that these more telomeric genes are not regulated by the mechanism that controls H19, Igf2, and Ins2. Mutations in human p57(Kip2) have been implicated in Beckwith-Wiedemann syndrome, a disease that has also been associated with loss of imprinting of IGF2. We find, however, that a deletion of the gene has no effect on imprinting within the cluster. Surprisingly, the three maternally expressed genes are regulated very differently by DNA methylation; p57(Kip2) is activated, Kvlqt1 is silenced, and Mash2 is unaffected in mice lacking DNA methyltransferase. We conclude that H19 is not a global regulator of imprinting on distal chromosome 7 and that the telomeric genes are imprinted by a separate mechanism(s).

Animals↗
PubMed2006

PDLIM4 repression by hypermethylation as a potential biomarker for prostate cancer.

PURPOSE: We analyzed the expression of genes to identify reliable molecular markers in the diagnosis and progression of prostate cancer. EXPERIMENTAL DESIGN: Gene expression profiling was done using HG-U133 set microarrays in 32 prostate cancer and 8 benign tissues of patients with cancer. Expression levels of 11 genes were selected for quantitative real-time PCR evaluation in 52 prostate cancer and 20 benign tissues. Further, to assess transcriptional inactivation, we analyzed the promoter methylation of genes by quantitative methylation-specific PCR in 62 tumor and 36 benign tissues. RESULTS: Our results showed a significant down-regulation in the mRNA expression levels of PRIMA1, TU3A, PDLIM4, FLJ14084, SVIL, SORBS1, C21orf63, and KIAA1210 and up-regulation of FABP5, SOX4, and MLP in prostate cancer tissues by TaqMan real-time PCR. Quantitative methylation-specific PCR of PDLIM4, SVIL, PRIMA1, GSTP1, and PTGS2 detected prostate carcinoma with a sensitivity of 94.7%, 75.4%, 47.4%, 89.5%, and 87.7%, and a specificity of 90.5%, 75%, 54.2%, 95.8%, and 90.2%, respectively. Using this panel of methylation markers in combination, we were able to distinguish between prostate cancer and adjacent benign tissues with sensitivities and specificities of about 90% to 100%. Our data provide evidence of transcriptional repression of the putative tumor suppressor gene PDLIM4 by hypermethylation. CONCLUSIONS: Our analysis revealed differential expression of eight down-regulated and three up-regulated genes, implicating their role in prostate cancer development and progression. We further showed that the hypermethylation of PDLIM4 gene could be used as a sensitive molecular tool in detection of prostate tumorigenesis.

Aged↗
PubMed2005

The MLL partial tandem duplication: evidence for recessive gain-of-function in acute myeloid leukemia identifies a novel patient subgroup for molecular-targeted therapy.

MLL (ALL-1) chimeric fusions and MLL partial tandem duplications (PTD) may have mechanistically distinct contributions to leukemogenesis. Acute myeloid leukemia (AML) blasts with the t(9;11)(p22; q23) express MLL-AF9 and MLL wild-type (WT) transcripts, while normal karyotype AML blasts with the MLL(PTD/WT) genotype express MLL PTD but not the MLL WT. Silencing of MLL WT in MLL(PTD/WT) blasts was reversed by DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, and MLL WT induction was associated with selective sensitivity to cell death. Reduction of MLL PTD expression induced MLL WT and reduced blast colony-forming units, supporting opposing functions for MLL PTD and MLL WT whereby the MLL PTD contributes to the leukemic phenotype via a recessive gain-of-function. The coincident suppression of the MLL WT allele with the expression of the MLL PTD allele, along with the functional data presented here, supports the hypothesis that loss of WT MLL function via monoallelic repression contributes to the leukemic phenotype by the remaining mutant allele. These data from primary AML and the pharmacologic reversal of MLL WT silencing associated with a favorable alteration in the threshold for apoptosis suggest that these patients with poor prognosis may benefit from demethylating or histone deacetylase inhibitor therapy, or both.

Acute Disease↗
PubMed2003

CpG methylation and transcription factors Sp1 and Sp3 regulate the expression of the human secretin receptor gene.

The human secretin receptor (hSR) is an important glycoprotein receptor for regulating the secretion of pancreatic bicarbonate, water, and electrolytes. In this study we investigated the transcriptional regulation of the hSR gene. A minimal 106-bp promoter was identified, and it contains two GC boxes (GC box-A, -240 to -226; and GC box-B, -203 to -194, from the translation start site). EMSA and supershift analyses showed that both GC boxes interact with Sp1 and Sp3 transcription factors. Transient transfection in pancreas-derived human pancreatic ductule carcinoma (PANC)-1 and bovine pancreatic duct-1 cells showed that mutation of either GC box-A or -B reduced the promoter strength by 56-67%, whereas mutation of both GC boxes caused more than 90% reduction of promoter activity. Cotransfections of the hSR promoter with Sp1 and Sp3 expression vectors in Sp-deficient Drosophila SL-2 Schneider cells further demonstrated that the ratio of Sp1 to Sp3 is the key mechanism to modulate hSR gene expression. The methylation statuses of 27 CpG sites within the promoter region (-400 to -151 bp) were assessed in various human pancreas and liver cell lines. The hSR promoter is unmethylated (CAPAN-1, human pancreatic adenocarcinoma) or partially methylated (PANC-1 and HPAC, human pancreatic adenocarcinoma) in hSR-expressing cell lines but is completely methylated in hSR nonexpressing HepG2 cells. Methyltransferase inhibitor 5-aza-2'deoxycytidine increased hSR gene expression level in PANC-1 cells and induced hSR gene expression in HepG2 cells. Together, our study shows that, in addition to Sp1 and Sp3, promoter methylation also plays a role in the regulation of hSR gene expression.

Adenocarcinoma↗
PubMed2006

[Research advances on the relationship of PI3-kinase/Akt/mTOR pathway and epigenetic modification].

The mammalian target of rapamycin (mTOR) has been shown to link growth factor signaling and posttranscriptional control of protein translation through activation of the PI3K/Akt pathway, which is frequently involved in cell cycle progression. The inhibition of mTOR has promising potential in anticancer and immunosuppressive therapies, and additional phase II clinical trials are ongoing. Epigenetic modification, which involves DNA methylation, histone modification and chromatin remodeling, as well as the recently described RNAi mechanism, can initiate the formation of silenced chromatin. Persistent activation or inhibition of the mTOR pathway may affect epigenetic modification. In this paper, we reviewed the research advances in the relationship between PI3K/Akt/mTOR and epigenetic modification.

Animals↗
PubMed2006

A genome-wide screen for promoter methylation in lung cancer identifies novel methylation markers for multiple malignancies.

BACKGROUND: Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The "rules" governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets. METHODS AND FINDINGS: In an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5' CpG islands, are induced from undetectable levels by 5-aza-2'-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors. CONCLUSIONS: By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention.

Adenocarcinoma↗
PubMedDate not supplied

The role of combined metal interactions in metal carcinogenesis: a review.

Exposures to complex mixtures of metals in the workplace or environment are more likely to occur than exposures to a single metal alone. The evidence shows that exposures to complex metal mixtures can enhance the risk of cancer in certain human populations. The findings of several studies have suggested, however, that certain metal-metal interactions can inhibit carcinogenic activity. The mechanisms of metal-metal interactions in human carcinogenesis are relatively unknown. Metals represent a highly diverse group of agents: each metal can act through different mechanisms and in one or more steps of the carcinogenic process. Some potential mechanisms may involve direct reactions of the metal with DNA or indirect mechanisms that include modification of DNA repair, DNA methylation status, and metabolic processes involved in DNA replication and expression. Lipid peroxidation and the generation of free radicals induced by certain metals can affect DNA integrity. This review will address the role of metals in carcinogenesis and how concomitant exposure to metal mixtures can influence cancer induction. The most current mechanistic data regarding metal interactions and its implications in human carcinogenesis will be discussed. Furthermore, research gaps will be identified to provide data that will improve risk assessments for complex metal mixtures encountered in the workplace and environment.

Cell Transformation, Neoplastic↗
PubMed2026

Somatic genetic alterations in pituitary neuroendocrine tumors.

The molecular characterization of pituitary neuroendocrine tumors (PitNETs) has progressed pronouncedly in recent years, unraveling the molecular pathways driving initiation and progression of different PitNET types and allowing a better understanding of their biology. The most frequent recurring somatic driver alterations were recognized in corticotroph PitNETs (USP8, USP48, BRAF) and somatotroph PitNETs (GNAS) and, much less frequently, in lactotroph PitNETs (SF3B1). Additional well-characterized somatic driver alterations, including TP53, ATRX, and DAXX, are enriched in aggressive corticotroph tumors. Identification of new molecular markers and delineation of their clinical phenotypes are enabling further subclassification of PitNETs based on tumor molecular profiles, with earlier recognition of more aggressive variants. These molecular markers also provide an opportunity for new targeted therapies. Beyond single-gene alterations, epigenetic modifications, such as DNA methylation, histone modifications, and noncoding RNA dysregulation, are emerging as important contributors to PitNET pathogenesis and potential therapeutic targets. Multi-omics approaches encompassing genomics, transcriptomics, epigenomics, and proteomics are transforming PitNET classification. In this review, we provide a comprehensive, data-driven update on somatic driver alterations, epigenetic alterations, converging signaling pathways, and the related emerging therapeutic targets in PitNETs, integrating pooled analyses from published cohorts.

Humans↗
PubMed1995

Possible role of oxidative damage in metal-induced carcinogenesis.

This review presents and evaluates evidence relevant to the mechanisms of metal carcinogenicity with special emphasis on the emerging hypothesis of the oxidative nature of metals' effect on DNA. The carcinogenic transition metals are capable of in vivo binding with the cell nucleus and causing promutagenic damage that includes DNA base modifications, inter- and intramolecular crosslinking of DNA and proteins, DNA strand breaks, rearrangements, and depurination. The chemistry of that damage and the resulting mutations observed in vitro and in metal-induced tumors are both characteristic for oxidative attack on DNA. The underlying mechanism involves various kinds of active oxygen and other radical species arising from metal-catalyzed redox reactions of O2, H2O2, lipid peroxides, and others, with certain amino acids, peptides, and proteins. Other metal-mediated pathogenic effects, such as enhancement of lipid peroxidation, stimulation of inflammation, inhibition of cellular antioxidant defenses, and inhibition of DNA repair, may also contribute to that mechanism. Thus far, published data revealing the oxidative character of metal-induced promutagenic DNA alterations are particularly strong for two of the most powerful human metal carcinogens, chromium and nickel. However, without excluding contribution of other effects, the promotion of oxidative damage tends to take the leading role in explaining mechanisms of carcinogenicity and acute toxicity of certain other metals as well.

Animals↗
PubMed1996

Chemistry and biology of DNA methyltransferases.

Recognition of a specific DNA sequence by a protein is probably the best example of macromolecular interactions leading to various events. It is a prerequisite to understanding the basis of protein-DNA interactions to obtain a better insight into fundamental processes such as transcription, replication, repair, and recombination. DNA methyltransferases with varying sequence specificities provide an excellent model system for understanding the molecular mechanism of specific DNA recognition. Sequence comparison of cloned genes, along with mutational analyses and recent crystallographic studies, have clearly defined the functions of various conserved motifs. These enzymes access their target base in an elegant manner by flipping it out of the DNA double helix. The drastic protein-induced DNA distortion, first reported for HhaI DNA methyltransferase, appears to be a common mechanism employed by various proteins that need to act on bases. A remarkable feature of the catalytic mechanism of DNA (cytosine-5) methyltransferases is the ability of these enzymes to induce deamination of the target cytosine in the absence of S-adenosyl-L-methionine or its analogs. The enzyme-catalyzed deamination reaction is postulated to be the major cause of mutational hotspots at CpG islands responsible for various human genetic disorders. Methylation of adenine residues in Escherichia coli is known to regulate various processes such as transcription, replication, repair, recombination, transposition, and phage packaging.

DNA↗
PubMed2005

Increased heparanase expression is caused by promoter hypomethylation and up-regulation of transcriptional factor early growth response-1 in human prostate cancer.

PURPOSE: Heparanase degrades heparan sulfate and has been implicated in tumor invasion and metastasis. The transcription factor, early growth response 1 (EGR1), is associated with the inducible transcription of the heparanase gene. We hypothesize that CpG hypomethylation in the heparanase promoter coupled with up-regulation of EGR1 levels may induce heparanase expression in human prostate cancer. EXPERIMENTAL DESIGN: Cultured prostate cancer cell lines (Du145, DuPro, LNCaP, and PC-3) with and without 5'-aza-2-deoxycytidine treatment, 177 prostate cancer samples, and 69 benign prostatic hyperplasia (BPH) samples were used. The frequency and level of heparanase promoter methylation were analyzed by methylation-specific primers which covered the core binding motif of EGR1 (GGCG) or SP1 (GGGCGG) or both. RESULTS: In cultured Du145, DuPro, LNCaP, and PC-3 cell lines, mRNA transcripts of heparanase were significantly increased after 5'-aza-2-deoxycytidine treatment, suggesting that promoter methylation was involved in the regulation of heparanase mRNA transcript. Significantly higher methylation was found in BPH samples than in prostate cancer samples (P < 0.0001), whereas mRNA transcripts of the heparanase gene were inversely lower in BPH samples than in prostate cancer samples (P < 0.01). EGR1 expression in prostate cancer tissues was significantly higher than in BPH tissues (P < 0.001) and correlated with heparanase expression (P < 0.0001). Moreover, multiple regression analysis revealed that up-regulation of EGR1 contributed significantly more to heparanase expression than did promoter CpG hypomethylation in prostate cancer samples (P < 0.0001). CONCLUSIONS: To our knowledge this is the first comprehensive study demonstrating that increased heparanase expression in prostate cancer tissues is due to promoter hypomethylation and up-regulation of transcription factor EGR1.

Aged↗
PubMed1988

Do tightly-bound chromatin proteins play a role in DNA methylation?

When chromatin matrix, "stripped" from its loosely-bound components by extraction with 3 M NaCl, is extensively digested with DNAase I, a fraction is obtained, which carries no endogenous DNA methyltransferase activity but which is a good substrate for externally added enzyme. Under the same conditions, protein-free DNA isolated from this fraction can instead hardly be methylated, this different behaviour pointing to a role of DNA-tightly-bound proteins in favoring or promoting the catalytic action of the enzyme. A similar stimulation of enzymatic methylation could also be shown when, in the presence of this same fraction, single stranded Micrococcus luteus DNA was incubated with placental methyltransferase, using S-adenosylmethionine as a methyl donor. This finding can be correlated to the existence, in chromatin loops, of small regions which resist digestion by DNAase I also after high-salt removal of their loosely-bound components (presumably because of the presence of tightly-bound proteins) and whose DNA is characterized by high methylation levels and, at the same time, by high relative content of thymine.

Binding Sites↗
PubMed1987

[Effectiveness of the modification of a single-stranded DNA fragment by alkylating derivatives of oligonucleotides].

Modification of a single-stranded DNA fragment 303 nucleotides long with addressed reagents d(pTGACCCTCTTCCC) A greater than CHRCl (I), d(pACCCTCTTCCC) A greater than CHRCl (II), d(CCTCTTCCC) A greater than CHRCl (III) and d(TCTTCCC) A greater than CHRCl (IV) complementary to the sequence 261-274 has been studied. It was shown that not only G258 residue, located near to the above sequence, but also G179 residue is modified. The latter can be explained by the vicinity of G179 and the alkylating group in the three-dimensional structure of the complex. Some modification of fragment 19-24 was observed due to non-complementary binding of the reagent. Association constants of the reagents (I)-(IV) with 261-274 sequence of the fragment were calculated using the dependence of the modification extent of G258 and G179 on the reagent concentration. The constants at 25 and 35 degrees C were found to be 260 and 31 (I), 0,5 and 2 (II), 0,46 and 0,13 (III), 0,0020 and 0,0023 (IV) microM-1.

Alkylating Agents↗