We have investigated the function and sequence specificity of DNA methylation in the hypermethylated CpG island promoter region of the endogenous human LINE-1 (L1) retrotransposon family. In nontransformed human embryonic fibroblasts, inhibition of DNA methylation with 5-azadeoxycytidine induced a greater than 4-fold increase in transcription from potentially functional L1 elements without increasing the transcription level of the majority of degenerate elements, implicating hypermethylation in the repression of L1 activity. Using bisulfite genomic sequencing to assess the pattern of methylation in a subset of nondegenerate L1 elements, we found 29 sites within a 460-base pair region of the noncoding (top) DNA strand of the L1 promoter in which cytosine methylation was maintained with high efficiency. Of these, 25 were at CG dinucleotides and four were in non-CG sites. When the methylation sites were analyzed for the complementary (bottom) strand, the only highly conserved sites of methylation were in CG dinucleotides. Several of these sites of CG methylation in the bottom (coding) strand were at positions where top (noncoding) strand-derived sequences were unmethylated, suggesting that these sites might be maintained in a hemimethylated state. Hence, there is a subset of human L1 elements in which methylation is efficiently maintained in asymmetric non-CG sites and further that this non-CG methylation may be part of a wider phenomenon involving hemi-methylation at CG dinucleotides. Maintenance of asymmetric methylation at non-CG sites (and possibly at hemi-methylated CG dinucleotides) could be through a novel DNA methyltransferase activity. Alternatively, the promoter region of L1 elements may be induced by factor binding to form some type of secondary structure that presents as a highly efficient substrate for de novo methylation.Five to ten percent of the human genome is derived from one transposable element family, the L1 or LINE-1 family (1, 2), which belongs to the non-LTR retrotransposon class of elements that are spread widely among eukaryotes. Although the majority of human L1 elements are inactive degenerate remnants, some are clearly functional, as de novo insertion of L1 elements have been documented in the germ line of both humans (3-5) and mice (6) as well as in somatic (tumor) cells (7). Nondegenerate full-length mammalian L1 elements are some 6 kilobases in length and contain two evolutionarily conserved open reading frames, the second of which encodes a reverse transcriptase (8, 9) with intrinsic RNase-H and AP endonuclease-like activity (10, 11). The promoter responsible for the full-length L1 transcript has been shown to be located within the 5Ј end of the element and downstream from the transcriptional start site (12). This promoter region also contains a strong binding site for the ubiquitous transcription factor, YY1, which has recently been shown to be the nuclear matrix-associated protein, NMP-1 (13). In contrast to regions normally associated with the nuclear matrix that are high in AT b...

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The efficacy of paclitaxel against some tumors may be aided by its administration in a vehicle solution containing Cremophor in quantities that reach concentrations in the plasma sufficient to reverse multidrug resistance of neoplastic cells.

Summary We have quantitated the levels of mRNAs in bone marrow samples from patients with multiple myeloma of the mdrl gene (responsible for the Multidrug Resistance phenotype) and for two of the glutathione S-transferase gene. GST-2 and GST-3 (which can also inactivate a wide variety of cytotoxic drugs) and examined the relationship between the levels of expression of these genes and response to subsequent chemotherapy. From a total of 47 patients. 37 were treated with chemotherapy with 34 evaluable for response. Twenty-nine of the patients treated had not received any treatment prior to the marrow sampling while eight had previously received chemotherapy. Patients who failed to respond to initial chemotherapy had significantly higher levels of mdrl than patients who responded (P = 0.01). In the total myeloma patient data set. mRNA levels for mdrl and GST-2 were significantly correlated (Spearman rank correlation coefficient (r) = 0.54. P = 0.0004) as were expression levels of GST-2 with GST-3 (r = 0.43. P = 0.017). GST-3 and mdrl levels were more weekly associated (r = 0.16. P = 0.4). These data would suggest a significant relationship between failure of chemotherapy in multiple myeloma patients and increases in expression of the mdrl gene together with other genes whose products will generate additional mechanisms of resistance to chemotherapeutic agents.

SummaryThe 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 theraputic manipulation.