sodium-bisulfite and Cell-Transformation--Neoplastic

Topics: Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosome Disorders; Chromosome Mapping; Gene Amplification; Gene Expression Regulation; Humans; Karyotyping; Mutation; Neoplasms; Nucleic Acid Hybridization; Proto-Oncogenes; Sulfites; Translocation, Genetic

TREX (transcription/export) is a multiprotein complex that plays a key role in the transcriptional elongation and transport of mRNA from the nucleus to the cytoplasm. We previously reported the purification of the human TREX protein and found that expression of a member of this complex, p84N5 (referred to as hTREX84 or hHPR1), a RB binding protein, correlated with breast tumor size and metastasis. Here we examine the mechanisms of aberrant expression of hTREX84 in breast and ovarian cancer cells and evaluate its role in tumorigenesis. We show that ovarian tumor cells over-express hTREX84 4-fold and 10-fold compared to immortal, non-tumorigenic and primary ovarian surface epithelial cells, respectively. Reduction of hTREX84 levels by small interfering RNA result in inhibition of cellular proliferation and G(2/M) arrest. Even though we observed that hTREX84 expression was induced by treatment with a demethylation agent, 5-aza-2'-deoxycytidine (5-aza-dC), sodium bisulfite DNA sequencing and methylation specific PCR found no evidence of changes in DNA methylation in the CpG islands in the regulator region of hTREX84. We subsequently identify several transcriptional factors, including NF-κB binding sites in the hTREX84 gene promoter and demonstrate by chromatin immunoprecipation (ChIP) and site directed mutagenesis that RelA/p65 binds the NF-kB binding sites and induces hTREX84 expression. Finally, we show by immunohistochemistry (IHC) that RelA/p65 is abundantly expressed in malignant cells that aberrantly express hTREX84 indicating that RelA/p65 might play a pivotal role in regulating hTREX84 expression in cancer. Our results indicate that overexpression of hTREX84 is associated with cancer cell transformation, proliferation and may be regulated by RelA/p65.

Topics: Azacitidine; Base Sequence; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cell Transformation, Neoplastic; Decitabine; DNA-Binding Proteins; Exons; Female; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Nuclear Proteins; Ovarian Neoplasms; Promoter Regions, Genetic; RNA-Binding Proteins; Sequence Analysis, DNA; Sulfites; Transcription Factor RelA; Transcription, Genetic

Epigenetic alterations have been reported in colorectal neoplasia which can either complement or in some cases be predisposed to genetic alterations such as K-ras mutations. We examined the promoter methylation status of the CDKN2A and O6-methylguanine-DNA methyltransferase (MGMT) genes, after sodium bisulfite conversion and DNA amplification with methylation specific PCR. Moreover, we searched for G to A transitions in codons 12 and 13 of the K-ras oncogene in normal colorectal mucosae, aberrant crypt foci (ACF, early premalignant lesions) and carcinomas. CDKN2A hypermethylation was an infrequent event in ACF (2 of 26, 7.7%). On the contrary, MGMT hypermethylation was found in the normal mucosae (3 of the 12 samples, 25%), in 14 of the 26 ACF (53.8%) and in 7 of the 9 (77.8%) carcinomas examined. K-ras mutations were evident in 6 ACF (23%) and in 3 carcinomas (33.3%), mostly associated with MGMT promoter hypermethylation. These findings strongly support the hypothesis that epigenetic mechanisms play an important role in the early steps of colorectal carcinogenesis.

Topics: Aged; Aged, 80 and over; Carcinoma; Cell Transformation, Neoplastic; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p16; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Intestinal Mucosa; Male; Middle Aged; Mutation; O(6)-Methylguanine-DNA Methyltransferase; Promoter Regions, Genetic; Sequence Analysis, DNA; Sulfites

Accurate assessment of gene methylation in formalin-fixed, paraffin-embedded archived tissue (FF-PEAT) by microdissection remains challenging because the tissue volume is small and DNA is damaged. In addition, methods for methylation assessment, such as methylation-specific PCR (MSP), require sodium bisulfite modification (SBM) on purified DNA, which causes major loss of DNA. On-slide SBM, in which DNA is modified in situ before isolation of tumor cells, eliminates DNA purification steps and allows histology-oriented assessment of gene methylation. This study describes a protocol and use of on-slide SBM using 20 FF-PEAT of colorectal cancers with intratumoral adenoma components to detect accumulation of gene methylation during colorectal malignant transformation. Deparaffinized tissue sections were incubated in sodium bisulfite solution for 8 hours at 60 degrees C, stained with hematoxylin, and then microdissected. Proteinase K lysate was directly used as a template in subsequent PCR. Using on-slide SBM, 282-bp-long bisulfite direct sequencing was possible. Yield of modified DNA was 2.6-fold greater than standard SBM on average. The mean conversion rate was 97%, and false-positive or false-negative results were not observed in subsequent MSP. Intratumoral heterogeneity by accumulation of p16 and Ras association domain family protein 1a methylation during malignant transformation were shown by MSP comparing cancer with adenoma parts within a single section. On-slide SBM is applicable in most methylation studies using FF-PEAT. It allows detailed, intratumoral analysis of methylation heterogeneity within solid tumors. On-slide SBM will significantly improve our approach and understanding of epigenetic events in minimal disease and the carcinogenic process.

Topics: Alu Elements; Base Sequence; Cell Transformation, Neoplastic; Colorectal Neoplasms; DNA Methylation; DNA-Cytosine Methylases; Estrogen Receptor alpha; Female; Genes, p16; Humans; Male; Molecular Sequence Data; Polymerase Chain Reaction; Promoter Regions, Genetic; Sulfites; Thymine DNA Glycosylase; Tumor Suppressor Proteins

The ability of sodium bisulfite to induce morphological transformation and mutagenesis of cultured Syrian hamster embryo cells was examined. Treatment of the cells at neutral pH for 15 min with 5-20 mM sodium bisulfite resulted in a dose-dependent increase in cell transformation but no induction of gene mutations measured at two genetic loci. Treatment of the cells for 24 h increased the level of transformation, but also failed to induce chromosome aberrations, aneuploidy or DNA strand breaks in the cells. The only positive response for a DNA alteration was an increase in sister chromatid exchanges, but this effect was observed only with the longer exposures and not with the 15 min exposure, which also transformed the cells. Possible mechanisms for bisulfite-induced cell transformation are discussed.

Topics: Animals; Benzo(a)pyrene; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Chromosomes; Cricetinae; DNA Damage; Embryo, Mammalian; Genes; Mesocricetus; Mutation; Sulfites

2 types of chromatin compaction were studied by cytochemical and ultrastructural approach. The 1st type was induced by cultivating transformed cells with dimethylsulfoxide. It caused cell transition into a more differentiated state coupled with the appearance of multiple small chromatin condensates. The 2nd type was induced by nonionic detergent Triton X-100 and/or saline penetration inside the nuclei. It was found to be connected with hypercompaction of chromocenters with their integral areas left unincreased. Both types of chromatin compaction differ in their capacity to induce toluidine blue polymerization on DNA substrate and in stability of DNA towards an early step of acid hydrolysis. The relation of the 2 mechanisms of chromatin compaction to different ways of DNA folding is discussed.

Topics: Animals; Antioxidants; Cell Line; Cell Transformation, Neoplastic; Chromatin; Coloring Agents; Cricetinae; Dimethyl Sulfoxide; DNA; Fibroblasts; Liver; Mice; Microscopy, Electron; Phenylhydrazines; Rats; Simian virus 40; Sulfites

Sodium bisulfite, a nonmutagen at neutral pH, induces neoplastic transformation of cultured Syrian hamster fetal cells. Morphologically transformed fibroblast colonies were isolated, and derived cell lines formed anchorage-independent colonies in agarose and progressively growing s.c. fibrosarcomas in nu/nu mice. Five tumorigenic cell lines analyzed by G- and C-banding were chromosomally abnormal with numerical deviations and structural alterations. Three tumors that developed in nude mice had the chromosome constitution of the inoculated transformed cell as well as secondary changes associated with tumor progression. Transformed cell lines had either a predominantly near-diploid or a near-tetraploid population with consistent chromosome gain and loss. Monosomy of the chromosome 13 observed in three cell lines was a nonrandom numerical alteration. Four lines had abnormal chromosomes resulting from deletions, unbalanced translocations, or centric fusions, and one cell line had a chromosome with a homogeneously staining region. Changes of chromosomes 1 and X were observed in three lines. The breakpoints on X chromosome nonrandomly involved the region qa5 which is frequently affected in hamster cells transformed by other carcinogens and may result in loss of genes essential for the maintenance of a normal phenotype. The formation of abnormal chromosomes cannot be directly attributed to the initial DNA damage as bisulfite concentrations effective in causing neoplastic transformation induced a significant but minimal increase in sister chromatid exchanges and failed to cause chromosome aberrations. Bisulfite inhibition of DNA replication might be a contributing factor in the occurrence of abnormal chromosomes. This cytogenetic analysis provides the first evidence that neoplastically transformed cells by a nonclastogenic carcinogen exhibit persistent chromosome rearrangements, a genetic alteration essential to the process of malignant transformation.

Topics: Animals; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Chromosome Banding; Cricetinae; Karyotyping; Mesocricetus; Mice; Mice, Nude; Sister Chromatid Exchange; Sulfites; X Chromosome

Pretreatment of hamster embryo cells or mouse C3H/10T-1/2 cells with sodium bisulfite (0.5, 2.5, 5.0 and 100 ppm) inhibits the oncogenic transformation of the cells following their exposure to x-rays or benzo(a)pyrene. The results suggest that low doses of bisulfite, a widely used food additive, can serve as a radioprotective and chemopreventive agent with anticarcinogenic potential.

Topics: Animals; Benzo(a)pyrene; Cell Transformation, Neoplastic; Cells, Cultured; Chemical Phenomena; Chemistry; Cricetinae; Mice; Mice, Inbred C3H; Radiation-Protective Agents; Sulfites