Trevor Duhig

The p21WAF1/CIP1 Promoter Is Methylated in Rat-1 Cells: Stable Restoration of p53-Dependent p21WAF1/CIP1 Expression after Transfection of a Genomic Clone Containing the p21WAF1/CIP1 Gene

ABSTRACT Rat-1 cells are used in many studies on transformation, cell cycle, and apoptosis. Whereas UV treatment of Rat-1 cells results in apoptosis, X-ray treatment does not induce either apoptosis or a cell cycle block. X-ray treatment of Rat-1 cells results in both an increase of p53 protein and expression of the p53-inducible geneMDM2 but not the protein or mRNA of the p53-inducible p21WAF1/CIP1 gene, which in other cells plays an important role in p53-mediated cell cycle block. The lack of p21WAF1/CIP1 expression appears to be the result of hypermethylation of the p21WAF1/CIP1 promoter region, as p21WAF1/CIP1 protein expression could be induced by growth of Rat-1 cells in the presence of 5-aza-2-deoxycytidine. Furthermore, sequence analysis of bisulfite-treated DNA demonstrated extensive methylation of cytosine residues in CpG dinucleotides in a CpG-rich island in the promoter region of the p21WAF1/CIP1 gene. Stable X-ray-induced p53-dependent p21WAF1/CIP1 expression and cell cycle block were restored to a Rat-1 clone after transfection with a P1 artificial chromosome (PAC) DNA clone containing a rat genomic copy of the p21WAF1/CIP1 gene. The absence of expression of the p21WAF1/CIP1 gene may contribute to the suitability of Rat-1 cells for transformation, cell cycle, and apoptosis studies.

Chimeric amplicons containing the c- myc gene in HL60 cells

The major amplicon present in HL60 cells is chimeric in nature being composed of 70 kb of DNA sequence derived from the MYC locus linked to 80 kb of novel DNA sequence derived from a non contiguous region located telomeric to the c-myc gene at 8q24 (). Here we show by fluorescence in situ hybridization (FISH) that these coamplified sequences, MCR (Myc Coamplified Region), are derived from a locus located 3–4 Mb telomeric to the c-myc gene in the q24.2-24.3 region of chromosome 8. Genomic cloning and Southern blot analysis indicate the arrangement of chimeric amplicons are in tandem arrays. Analysis of the DNA sequences at the juncture of the MYC locus and the MCR suggest that these non syntenic regions were joined by nonhomologous recombination events. Visualization of the organization of the amplified DNA by fiber-FISH analysis illustrates we have cloned the complete amplicon. This is the first complete mammalian amplicon to be cloned and have its structure visualized. In addition to the major class of tandemly repeated amplicons, a second class of amplicons was detected by fiber-FISH in which the extent of the MCR component is about twice the size of the MCR component in the major amplicon. These longer amplicons most likely contain inverted repeats of MCR and MYC region sequences. Whether the amplicons contain mixtures of these two types of structures or separate amplicons only contain one type of structure has not yet been resolved. Properties of the MCR sequences responsible for retention in the chimeric HL60 amplicons upon long term passage are discussed.

Epithelial Mucin Antibodies and Their Epitopes: Core Protein Epitopes of a Polymorphic Epithelial Mucin (PEM)

Largely because of their complexity, the detailed structure of the mucins has been difficult to analyze. This group of compounds is categorized mainly by the fact that they contain a high level of carbohydrate which is attached in O-linkage to serine and/or threonine via the linkage sugar N-acetylgalactosamine. The mucous secretions produced by some epithelial cells, particularly those lining the gastro-intestinal tract and the lungs, contain mucins along with other products and these components have been studied for some time at the biochemical level. However, other glandular epithelial cells, such as the salivary gland, breast, ovary, endometrium, and sweat glands, also produce mucins, and some of these simpler mucins have recently received much attention. This is because many antibodies selected for epithelial or tumor specificity have been found to react with high molecular weight glycoproteins which are produced by simple epithelial cells and have the properties of mucins.1–9

Characterization and Evolution of an Expressed Hypervariable Gene for a Tumor-Associated Mucin, MUC-1

Mucins, present on highly polarized, secretory epithelial cells, have gained prominence in recent years as many monoclonal antibodies selected for their reactivity on differentiated or normal tissues react with epitopes present on these molecules. Although difficult to analyze biochemically because of their large size and large amount of O-linked carbohydrate, recent cloning studies from a number of different laboratories have produced structural information of the core protein. In most cases cDNA clones were obtained from λgt11 expression libraries following the development of antibodies to the stripped core protein. Thus far, three human mucin partial or full-length cDNA clones have been characterized as well as the porcine submaxillary mucin and a Xenopus integumentary mucin. In each case a domain of the core protein was found to consist of tandem repeats of a defined length. Although no homology exists between the tandem repeats of the various mucin genes at either the DNA or protein level, the repeated sequences in each case code for molecules which could be highly O-glycosylated. One characteristic feature of mucins is the presence of between 50 and 90% carbohydrate which is linked to serines or threonines via an O-glycosidic linkage to N-acetylgalactosamine. A second characteristic is the presence of prolines which along with glycosylation help to provide the extended core protein structure characteristic of mucins. All of the predicted proteins coded for by the mucin clones contain these features.