PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs.

PPARB was identified as a target of APC through the analysis of global gene expression profiles in human colorectal cancer (CRC) cells. PPARdelta expression was elevated in CRCs and repressed by APC in CRC cells. This repression was mediated by beta-catenin/Tcf-4-responsive elements in the PPARdelta promotor. The ability of PPARs to bind eicosanoids suggested that PPARdelta might be a target of chemopreventive non-steroidal anti-inflammatory drugs (NSAIDs). Reporters containing PPARdelta-responsive elements were repressed by the NSAID sulindac. Furthermore, sulindac was able to disrupt the ability of PPARdelta to bind its recognition sequences. These findings suggest that NSAIDs inhibit tumorigenesis through inhibition of PPARdelta, the gene for which is normally regulated by APC.     

A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia.

Cell cycle checkpoints can enhance cell survival and limit mutagenic events following DNA damage. Primary murine fibroblasts became deficient in a G1 checkpoint activated by ionizing radiation (IR) when both wild-type p53 alleles were disrupted. In addition, cells from patients with the radiosensitive, cancer-prone disease ataxia-telangiectasia (AT) lacked the IR-induced increase in p53 protein levels seen in normal cells. Finally, IR induction of the human GADD45 gene, an induction that is also defective in AT cells, was dependent on wild-type p53 function. Wild-type but not mutant p53 bound strongly to a conserved element in the GADD45 gene, and a p53-containing nuclear factor, which bound this element, was detected in extracts from irradiated cells. Thus, we identified three participants (AT gene(s), p53, and GADD45) in a signal transduction pathway that controls cell cycle arrest following DNA damage; abnormalities in this pathway probably contribute to tumor development.     

Rapid purification of DNA from agarose gels by centrifugation through a disposable plastic column

A simple and rapid method for purifying DNA from agarose gels is described. Agarose slices containing DNA are placed in a disposable plastic column and the DNA is separated from the agarose by centrifugation in a microfuge. Recoveries averaging 25% are obtained for DNA of 14 kb or less. The recovered DNA can be labeled to high specific activity, cleaved with restriction endonucleases, and ligated efficiently using standard cloning vectors.     

Characterization of MAD2B and other mitotic spindle checkpoint genes.

Aneuploidy is a characteristic of the majority of human cancers, and recent work has suggested that mitotic checkpoint defects play a role in its development. To further explore this issue, we isolated a novel human gene, MAD2B (MAD2L2), which is homologous to the spindle checkpoint gene MAD2 (MAD2L1). We determined the chromosomal localization of it and other spindle checkpoint genes, including MAD1L1, MAD2, BUB3, TTK (MPS1L1), and CDC20. In addition, we resolved the genomic intron-exon structure of the human BUB1 gene. We then searched for mutations in these genes in a panel of 19 aneuploid colorectal tumors. No new mutations were identified, suggesting that genes yet to be discovered are responsible for most of the checkpoint defects observed in aneuploid cancers.     

A subset of small nuclear ribonucleoprotein particle antigens is a component of the nuclear matrix

We have assessed whether antigenic proteins associated with small nuclear ribonucleoprotein particles (snRNP) are associated with the nuclear matrix. Immunofluorescence studies showed that a subset of these particles (those reactive with anti-Sm antisera) were associated with the nuclear matrix, while a different set of particles (those reactive with anti-La antisera) were not associated with the nuclear matrix. Immunoprecipitation experiments showed that three specific polypeptide components of the snRNP reactive with the anti-Sm antisera were significantly enriched in nuclear matrix proteins.     

Purification of DNA from formaldehyde fixed and paraffin embedded human tissue

The ability to isolate DNA from preserved human tissues would provide numerous experimental opportunities. In this report it is shown that DNA can be extracted from tissues prepared for routine histopathological examination (i.e., fixed with formaldehyde and embedded in paraffin). Although the extracted DNA is not intact, it is double stranded, cleavable with restriction endonucleases, and suitable for a variety of standard techniques used in molecular biology.     

Localization of SV40 genes within supercoiled loop domains

Recent studies indicate that eukaryotic DNA is organized into supercoiled loop domains. These loops appear to be anchored at their bases to an insoluble nuclear skeleton or matrix. Most of the DNA in the loops can be released from the matrix by nuclease digestion; the residual DNA remaining with the nuclear matrix represents sequences at the base of the loops, and possibly other sequences which are intimately associated with the nuclear matrix for other reasons. Using a quantitative application of the Southern blotting technique, we have found this residual DNA from SV40 infected 3T3 cells to be enriched in SV40 sequences, indicating that they reside near matrix-DNA attachment points. An enrichment of 3-7 fold relative to total cellular DNA, was found in each of three different lines of SV40 infected 3T3 cells. Control experiments with globin genes showed no such enrichment in this residual matrix DNA. This sequence specificity suggests that the spatial organization of DNA sequences within loops may be related to the functionality of these sequences within the cell.