ABSTRACT
Alterations in DNA methylation patterns are implicated in playing a major role in the development of cancer, thus highlighting the need to continually develop new technologies to analyze epigenetic marks. Methylated-CpG Island Recovery Assay (MIRA), based on the high affinity of the MBD2b/MDB3L1 complex for double-stranded methylated DNA, allows for the recovery of methylated DNA without the use of bisulfite conversion or antibody recognition. MIRA is capable of detecting low-density methylation of a single methylated CpG nucleotide. This technique can be used in conjunction with microarrays or next-generation sequencing to analyze recovered methylated DNA on a genome-wide scale.
Subject(s)
CpG Islands/genetics , DNA Methylation/genetics , Genetic Techniques , DNA/genetics , DNA/isolation & purification , Epigenesis, Genetic/genetics , Genetic Techniques/instrumentation , MicrospheresABSTRACT
Cell culture models of oncogenesis that use cellular reprogramming to generate a neoplastic cell from a normal cell provide one of the few opportunities to study the early stages of breast cancer development. Human mammary epithelial cells (HMECs) were induced to undergo a neoplastic transformation using defined genetic elements to generate transformed HMECs (THMECs). To identify proteins that displayed significantly different levels of abundance at three consecutive time points in oncogenesis over an 80 day period, protein extracts were analyzed by two-dimensional difference gel electrophoresis (2D-DIGE). Nine proteins were found to be significantly different in abundance: keratin 1, keratin 7, heat shock protein 4A-like, t-complex protein 1, stathmin, gelsolin, FK506 binding protein 5, ribosomal protein P0, and maspin. Keratin 7 and maspin displayed a linear down-regulation over 80 days. All of these proteins have been shown to be involved in the maintenance of a metastatic state including cytoskeletal modifications and motility. We conclude that, following neoplastic induction, THMECs display an early and progressive increase in metastatic potential. Further investigations into the function and regulatory mechanisms of these proteins will provide an unparalleled understanding of the initial states through which a breast cancer cell transitions following acquisition of the genetic abnormalities required for oncogenesis.
Subject(s)
Breast Neoplasms/metabolism , Cell Transformation, Neoplastic/metabolism , Electrophoresis, Gel, Two-Dimensional/methods , Mammary Glands, Human/metabolism , Animals , Cell Line, Transformed , Cells, Cultured , Female , Gene Transfer Techniques , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Models, Biological , Neoplasm Metastasis , Proteome/analysis , Proteome/metabolism , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Serpins/genetics , Serpins/metabolism , Signal Transduction , Transplantation, HeterologousABSTRACT
Telomerase, a ribonucleoprotein important to neoplastic immortality, is up-regulated in approximately 85% of cancers, including leukemias. In this study, 9cUAB30, a novel retinoic acid, resulted in differentiation of HL60 leukemia cells as indicated by morphologic changes characteristic of granulocytes. It also caused a down-regulation of hTERT gene expression and a decrease in telomerase activity. Telomerase inhibition was followed by loss of proliferative capacity, induction of apoptosis, and partial differentiation. These findings demonstrate the effectiveness of 9cUAB30 at inhibiting telomerase activity by down-regulating hTERT gene expression in human leukemic cells.
ABSTRACT
A significant portion of ongoing epigenetic research involves the investigation of DNA methylation and chromatin modification patterns seen throughout many biological processes. Over the last few years, epigenetic research has undergone a gradual shift and recent studies have been directed toward a genome-wide assessment. DNA methylation and chromatin modifications are essential components of the regulation of gene activity. DNA methylation effectively down-regulates gene activity by addition of a methyl group to the five-carbon of a cytosine base. Less specifically, modification of the chromatin structure can be carried out by multiple mechanisms leading to either the upregulation or down-regulation of the associated gene. Of the many assays used to assess the effects of epigenetic modifications, chromatin immunoprecipitation (ChIP), which serves to monitor changes in chromatin structure, and bisulfite modification, which tracks changes in DNA methylation, are the two most commonly used techniques.
