DNA methylation in the promoter region of the p16 (CDKN2/MTS-1/INK4A) gene in human breast tumours

The p16 (CDKN2/MTS-1/INK4A) gene is one of several tumour-suppressor genes that have been shown to be inactivated by DNA methylation in various human cancers including breast tumours. We have used bisulphite genomic sequencing to examine the detailed sequence specificity of DNA methylation in the CpG island promoter/exon 1 region in the p16 gene in DNA from a series of human breast cancer specimens and normal human breast tissue (from reductive mammaplasty). The p16 region examined was unmethylated in the four normal human breast specimens and in four out of nine breast tumours. In the other five independent breast tumour specimens, a uniform pattern of DNA methylation was observed. Of the nine major sites of DNA methylation in the amplified region from these tumour DNAs, four were in non-CG sequences. This unusual concentration of non-CG methylation sites was not a general phenomenon present throughout the genome of these tumour cells because the methylated CpG island regions of interspersed L1 repeats had a pattern of (almost exclusively) CG methylation similar to that found in normal breast tissue DNA and in DNA from tumours with unmethylated p16 genes. These data suggest that DNA methylation of the p16 gene in some breast tumours could be the result of an active process that generates a discrete methylation pattern and, hence, could ultimately be amenable to therapeutic manipulation.     

Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells

Abnormal regional increases in DNA methylation, which have potential for causing gene inactivation and chromosomal instability, are consistently found in immortalized and tumorigenic cells. Increased DNA methyltransferase activity, which is also a characteristic of such cells, is a candidate to mediate these abnormal DNA methylation patterns. We now show that, in NIH 3T3 mouse fibroblasts, constitutive overexpression of an exogenous mouse DNA methyltransferase gene results in a marked increase in overall DNA methylation which is accompanied by tumorigenic transformation. These transformation changes can also be elicited by dexamethasone-inducible expression of an exogenous DNA methyltransferase gene. Our findings provide strong evidence that the increase in DNA methyltransferase activity associated with tumor progression could be a key step in carcinogenesis and provide a model system that can be used to further study this possibility.     

Effect of cytosine methylation on DNA-DNA recognition at CpG steps

Although DNA methylation is a fundamental mechanism for repressing genetic activity, the influence of methyl groups on DNA conformation is found to be small. In this study, the role of cytosine methylation is analysed in the context of DNA condensation by examining its influence on DNA-DNA recognition processes. Previously CpG sites were found to act as sequence determinants for the close and specific self-fit of B-DNA helices into cross-overs. In the present study, the crystal structure of the B-DNA dodecamer d(ACCGCCGGCGCC) methylated at its central CpG sequence shows that the methyl groups do not interfere with DNA self-fitting. In contrast, the two methyl groups form a clamp, which traps the incoming phosphate in the groove-backbone interaction. This geometry allows the formation of two new C-H...O hydrogen bonds between the methyl groups and the anionic oxygen atoms of the phosphate, which may further stabilize the interaction. This finding relates cytosine methylation to the formation of higher-order DNA structures and could provide new insights for understanding the mode of action of DNA methylation in genetic inactivation.     

DNA-PKcs: a T-cell tumour suppressor encoded at the mouse scid locus

Severe combined immunodeficiency (SCID) mice are defective in their ability to rearrange their variable (V), diversity (D) and joining (J) genetic elements to generate functional immunoglobulin (Ig) and T-cell receptor (TCR) molecules; as a result, they lack mature B and T cells. These mice are highly sensitive to ionizing radiation, suggesting that the product of the scid gene plays a critical role in both V(D)J recombination and DNA double-strand break repair. Recent studies suggest that the SCID defect lies in the gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PK; refs 6-8), a nuclear protein made up of the Ku 70 and Ku 86 subunits as well as the large catalytic subunit, DNA-PKcs. Other reports have implied that the SCID phenotype correlates with nonsense mutations at the extreme 3' end of Prkdc, the DNA-PKcs gene. The identity of the gene remains in doubt, however, because the consequences of genetic inactivation of Prkdc have not been determined. This study shows that complete inactivation of Prkdc in a novel insertional mouse mutant recapitulates the SCID phenotype and that Prkdc and scid are alleic. Significantly, DNA-PKcs null mice demonstrate complete penetrance of thymic lymphoblastic lymphomas, strongly suggesting that Prkdc functions in mice as a T-cell tumour suppressor and, by virtue of its association with DNA repair and recombination, belongs to the 'caretaker' class of tumour-suppressor genes that includes ATM, BRCA1 and BRCA2 (ref. 15).     

Receptor imaging: competitive or complementary to antibody imaging?

The hypothesis that a low concordance rate in monozygotic (MZ) twins with systemic lupus erythematosus (SLE) may be accounted for by differences in X-chromosome inactivation was examined. Five MZ twin pairs, four discordant and one concordant, were recruited, zygosity confirmed by DNA fingerprinting, and their pattern of X-chromosome inactivation in DNA samples prepared from peripheral blood and buccal cells were examined. X-chromosome inactivation was assessed by the methylation status of the CpG region near trinucleotide repeats in exon 1 of the androgen receptor gene on X-chromosome after digestion with the methylation-sensitive enzyme HpaII or HhaI and PCR amplification. X-chromosome inactivation patterns were found to be the same between affected and non-affected twins in all four discordant twin pairs, with random patterns in two pairs and skewed patterns in the others. The concordant twins demonstrated the same random patterns. X-chromosome inactivation was also examined from buccal smear DNA and shown to have the same pattern as that noted from peripheral blood DNA in one informative twin pair. Differences in X-chromosome inactivation patterns were not observed in these five MZ twin pairs. The results could not support the hypothesis that differences in X-chromosome inactivation is the mechanism accounting for the low concordance rate noted in MZ twins with SLE.     

Infection with human immunodeficiency virus type 1 upregulates DNA methyltransferase, resulting in de novo methylation of the gamma interferon (IFN-gamma) promoter and subsequent downregulation of IFN-gamma production

The immune response to pathogens is regulated by a delicate balance of cytokines. The dysregulation of cytokine gene expression, including interleukin-12, tumor necrosis factor alpha, and gamma interferon (IFN-gamma), following human retrovirus infection is well documented. One process by which such gene expression may be modulated is altered DNA methylation. In subsets of T-helper cells, the expression of IFN-gamma, a cytokine important to the immune response to viral infection, is regulated in part by DNA methylation such that mRNA expression inversely correlates with the methylation status of the promoter. Of the many possible genes whose methylation status could be affected by viral infection, we examined the IFN-gamma gene as a candidate. We show here that acute infection of cells with human immunodeficiency virus type 1 (HIV-1) results in (i) increased DNA methyltransferase expression and activity, (ii) an overall increase in methylation of DNA in infected cells, and (iii) the de novo methylation of a CpG dinucleotide in the IFN-gamma gene promoter, resulting in the subsequent downregulation of expression of this cytokine. The introduction of an antisense methyltransferase construct into lymphoid cells resulted in markedly decreased methyltransferase expression, hypomethylation throughout the IFN-gamma gene, and increased IFN-gamma production, demonstrating a direct link between methyltransferase and IFN-gamma gene expression. The ability of increased DNA methyltransferase activity to downregulate the expression of genes like the IFN-gamma gene may be one of the mechanisms for dysfunction of T cells in HIV-1-infected individuals.     

The DNA methylation machinery as a target for anticancer therapy

DNA methylation is now recognized as an important mechanism regulating different functions of the genome; gene expression, replication, and cancer. Different factors control the formation and maintenance of DNA methylation patterns. The level of activity of DNA methyltransferase (MeTase) is one factor. Recent data suggest that some oncogenic pathways can induce DNA MeTase expression, that DNA MeTase activity is elevated in cancer, and that inhibition of DNA MeTase can reverse the transformed state. What are the pharmacological consequences of our current understanding of DNA methylation patterns formation? This review will discuss the possibility that DNA MeTase inhibitors can serve as important pharmacological and therapeutic tools in cancer and other genetic diseases.     

Effects of SR 48692, a selective non-peptide neurotensin receptor antagonist, on two dopamine-dependent behavioural responses in mice and rats

In this study, two transformation vectors (pMG101 and pMG103) for Phanerochaete chrysosporium were constructed, based on the ble phleomycin-resistance-encoding gene and a homologous histone H4 promoter. Transformation frequencies were 6-10 per micrograms of DNA. Transformed vector DNA could either exist as an unstable replicating plasmid or could be stably integrated. Integrated vector DNA from pMG101, which also contains a histone-encoding H3 gene in the promoter fragment, becomes methylated, resulting in inactivation of ble-dependent resistance. Plasmid pMG103, which lacks the H3, does not show methylation.     

Regulation of the expression of the tissue transglutaminase gene by DNA methylation

We have investigated the role of DNA methylation in the regulation of the expression of the human tissue transglutaminase gene. Studies on the methylation of the transglutaminase promoter in normal and neoplastic human cells demonstrated that the promoter is methylated in vivo and hypomethylation of the promoter is correlated with constitutive gene expression. Demethylation of the promoter in vivo by treatment of the cells with 5-azacytidine increased transglutaminase expression and hypermethylation of the promoter in vitro suppressed its activity. These studies suggest that alternations in DNA methylation may be one of the mechanisms regulating the tissue-specific expression of the tissue transglutaminase gene.