D.M. Woodcock

Many strains of E. coli K12 restrict DNA containing cytosine methylation such as that present in plant and animal genomes. Such restriction can severely inhibit the efficiency of cloning genomic DNAs. We have quantitatively evaluated a total of 39 E. coli strains for their tolerance to cytosine methylation in phage and plasmid cloning systems. Quantitative estimations of relative tolerance to methylation for these strains are presented, together with the evaluation of the most promising strains in practical recombinant cloning situations. Host strains are recommended for different recombinant cloning requirements. These data also provide a rational basis for future construction of 'ideal' hosts combining optimal methylation tolerance with additional advantageous mutations.

A polyethoxylated castor oil, Cremophor EL, which is used as a vehicle for p.o. and i.v. administration of water-insoluble compounds in humans, can reverse the multidrug resistance (MDR) phenotype at doses which are likely to be readily achievable clinically. Using flow cytofluorometric analysis of daunorubicin (DNR) uptake as a measure of the expression of the MDR phenotype, Cremophor EL (1:10(3] in the growth medium increased intracellular DNR in an MDR cell line, R100 cells, to levels similar to that observed in the drug-sensitive parental cells, CCRF-CEM. A similar Cremophor EL-induced increase in DNR uptake was also observed in an unrelated MDR cell line derived from K562 cells. Cremophor EL (less than or equal to 3:10(4] did not inhibit the growth of CCRF-CEM cells or its vinblastine-resistant derivative, R100 cells, but would significantly increase the sensitivity of R100 cells to both vinblastine and DNR. Also Cremophor EL did not increase the sensitivity of normal bone marrow progenitor cells cultured in vitro to high concentrations of vinblastine. Cremophor EL may prove to be a relatively pharmacologically inactive addition to chemotherapeutic protocols which may be able to reverse the MDR phenotype in tumors and also help to prevent the selection of MDR cell variants from within a tumor cell population during chemotherapy.

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 hemi-methylated 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.

BACKGROUND: Paclitaxel (Taxol) is the first of a new class of cytotoxic agents with activity against tumors resistant to other drugs. For clinical use, paclitaxel is currently formulated in a vehicle of 50% ethanol and 50% polyethoxylated surfactant Cremophor EL (Cremophor). We have previously shown that Cremophor will block the P-glycoprotein drug efflux pump responsible for the multidrug-resistant phenotype. Overexpression of P-glycoprotein is one mechanism of in vitro resistance to a number of currently used cytotoxic agents including paclitaxel. PURPOSE: Our aim was to develop a bioassay to measure plasma levels of Cremophor and to determine whether or not plasma levels of Cremophor achieved during paclitaxel therapy are sufficient to inhibit the activity of the P-glycoprotein. METHODS: All patients studied had histologically proven, advanced ovarian carcinoma with measurable or evaluable disease and had received at least one prior platinum-containing regimen. The bioassay used flow cytometry to measure the increase in equilibrium intracellular daunorubicin levels in multidrug-resistant human T-cell leukemia cells (CEM/VLB100) in the presence of a series of concentrations of Cremophor. Levels of Cremophor were measured in plasma from 21 patients after a 3-hour infusion of 135 or 175 mg/m2 paclitaxel. Both dose levels were given following premedication with oral dexamethasone, intravenous promethazine hydrochloride, and intravenous cimetidine. The Cremophor bioassay involved incubation of CEM/VLB100 cells (5 x 10(5)) for 1 hour with 2 micrograms/mL daunorubicin in 0.5 mL HL-1 medium plus 0.5 mL plasma prior to flow cytometric analysis. Pretreatment plasma was used to derive a standard curve for the effect of Cremophor on equilibrium daunorubicin levels. All measurements were done in triplicate. RESULTS: In vitro experiments indicated that, for maximal inhibition of P-glycoprotein activity, concentrations of Cremophor of 0.1% (vol/vol) were required. At the end of a 3-hour infusion of paclitaxel, plasma levels of Cremophor in 19 of 21 patients were 0.1% or higher and 0.09% in the remaining two. Concentrations of 5-20 microM paclitaxel dissolved in ethanol without Cremophor did not inhibit P-glycoprotein in this assay. CONCLUSION: The concentrations of Cremophor measured in plasma drawn from patients after a 3-hour infusion of paclitaxel at 135 or 175 mg/m2 were found to be sufficient to inhibit P-glycoprotein activity in vitro. IMPLICATIONS: 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.