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.

Phase I trial of cremophor EL with bolus doxorubicin.

Cremophor EL (cremophor), a component of the paclitaxel formulation, can potentially reverse P-glycoprotein-associated multidrug resistance. A Phase I trial of cremophor as a 6-h infusion every 3 weeks was performed with bolus doxorubicin (50 mg/m2). The cremophor dose was escalated from 1 to 60 ml/m2. A standard paclitaxel premedication was given before cremophor. Using a bioassay, potentially active cremophor levels (> or = 1 microl/ml) were measured in plasma from patients receiving cremophor doses of 30, 45, and 60 ml/m2. A cross-over design was used to assess the influence of cremophor 30 ml/m2 on the pharmacokinetics of doxorubicin and doxorubicinol. The plasma area under the concentration versus time curve (AUC) of doxorubicin increased from 1448 +/- 350 to 1786 +/- 264 ng/ml x h (P = 0.02) in the presence of cremophor, whereas the AUC of doxorubicinol increased from 252 +/- 104 to 486 +/- 107 ng/ml x h (P = 0.02). This pharmacokinetic interaction was associated with significantly increased neutropenia. With reduction of the doxorubicin dose to 35 mg/m2, the cremophor dose was increased to 60 ml/m2. Dose-limiting toxicities occurred in two of six patients after 45 ml/m2 and two of four patients after 60 ml/m2, which included febrile neutropenia and grade III cremophor-related toxicities of rash, pruritus, headache, and hypotension. All patients who received 45 ml/m2 cremophor reached plasma levels > or = 1.5 microl/ml, but at 60 ml/m2, only two of four reached this level, and the calculated plasma clearance of cremophor was significantly faster at this dose. One patient with hepatoma resistant to epirubicin achieved a near-complete response. Cremophor 45 ml/m2 over 6 h with 35 mg/m2 doxorubicin is recommended for further studies. The pharmacokinetic interaction between cremophor and doxorubicin is quantitatively similar to that described in trials of paclitaxel with doxorubicin and suggests that the cremophor in the paclitaxel formulation is responsible.

DNA methylation in mouse A-repeats in DNA methyltransferase-knockout ES cells and in normal cells determined by bisulfite genomic sequencing.

Mouse ES cells with a null mutation of the known DNA methyltransferase retain some residual DNA methylation and can methylate foreign sequences de novo. We have used bisulfite genomic sequencing to examine the sequence specificity and distributions of methylation of a hypermethylated CG island sequence, mouse A-repeats. There were 13 CG dinucleotides in the region examined, 12 of which were methylated to variable extents in all DNAs. We found that: (1) there is considerable residual DNA methylation in ES cells lacking the known DNA methyltransferase (29% of normal methylation in the complete knockout ES DNA); (2) this other activity methylates at exactly the same CG sites as the major methyltransferase; and (3) differences in the distribution of methylated sites between A-repeats in these DNAs are consistent with this other activity methylating in a random de novo fashion. Also, the lack of any methylation in non-CG sites argues that, in other studies where non-CG methylation sites have been found by bisulfite sequencing, detection of such sites of non-CG methylation is not an inherent artifact in this methodology.

Asymmetric methylation in the hypermethylated CpG promoter region of the human L1 retrotransposon.

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.

Plasma concentrations of polysorbate 80 measured in patients following administration of docetaxel or etoposide.

Docetaxel (Taxotere, Rhone-Poulenc Rorer) and etoposide are water-insoluble drugs formulated with polysorbate 80 for intravenous administration. We have previously reported that surfactants, including polysorbate 80 and Cremophor EL, can reverse the multidrug resistance (MDR) phenotype in an experimental system and that plasma Cremophor EL concentrations measured following a 3-h infusion of paclitaxel were > or = 1 microliter/ml, sufficient to modulate MDR in vitro. The purpose of this study was to measure polysorbate 80 plasma concentrations in patients following intravenous administration of etoposide or docetaxel using a bioassay in which MDR-expressing cells are incubated with daunorubicin (DNR) plus 50/50 growth medium/plasma and equilibrium intracellular DNR fluorescence is measured by flow cytometry. In vitro experiments show maximal reversal of MDR at concentrations of 1.0-2.0 microliters/ml and 50% reversal at 0.2-0.3 microliter/ml. Patients received docetaxel at 75 mg/m2 (five patients) or 100 mg/m2 (four patients) (total dose 125-178 mg, containing 3.12-4.45 ml polysorbate 80) over 60 min. The median end-infusion polysorbate 80 concentration was 0.1 microliter/ml (range 0.07-0.41 microliter/ml). Only one patient had a level of > 0.2 microliter/ml. Five patients received intravenous etoposide at 120 mg/m2 over 45-120 min (total dose 180-250 mg, containing 0.67-0.93 ml polysorbate 80). In the end-infusion plasma sample, polysorbate 80 was not detectable (< 0.06 microliter/ml) in any patient. Plasma polysorbate 80 levels following an intravenous infusion of 120 mg/m2 etoposide or of docetaxel at doses used in Phase II trials, are insufficient to show modulation of MDR in vitro.

Response to cardiac sympathetic activation in transgenic mice overexpressing beta 2-adrenergic receptor.

Transgenic mice have been created with 200-fold overexpression of beta 2-adrenergic receptors specifically in the heart. Cardiac function was studied in these transgenic mice and their controls at baseline and during isoproterenol perfusion or sympathetic nerve stimulation. The model used was an in situ buffer-perfused, innervated heart, and the left ventricle maximal derivative of pressure over time (dP/dtmax) and heart rate (HR) were measured. Basal HR and dP/dtmax were 30-40% higher in hearts from transgenic mice than controls. Electrical stimulation of sympathetic nerves (2, 4, and 8 Hz) or infusion of isoproterenol markedly increased HR and dP/dtmax in control hearts. Hearts from transgenic mice did not respond to isoproterenol. However, hearts from transgenic mice retained the HR response to nerve stimulation, and a small increase in dP/dtmax was also detected. Atenolol inhibited the response to nerve stimulation in control hearts but not that in hearts from transgenic mice. ICI-118551 inhibited the response in transgenic hearts. Basal HR and dP/dtmax were decreased by ICI-118551 only in transgenic hearts. Thus overexpression of cardiac beta 2-receptors modifies beta-adrenergic activity, but the responses to endogenous and exogenous adrenergic stimulation are affected differently.

A sensitive RNase protection assay to detect transcripts from potentially functional human endogenous L1 retrotransposons.

A high background of read-through transcripts from degenerate human L1 retrotransposons is present in almost all human cell types. This prevents the detection of RNA transcripts from potentially functional elements. To overcome this, we have developed an RNase protection assay based on the reconstructed consensus sequence for the 5' end of the major L1 family. In the human Ntera2D1 teratocarcinoma cell line, this assay readily detected L1 transcripts that were located primarily in the cytoplasm and where 20% were in filterable particles. By this assay, potentially functional L1 elements are also transcriptionally active in lymphocytes from some but not all normal individuals. Together with the full length protection product, there were three other discrete L1 RNAs, two of which (305 and 275 bases) were transcribed from the 5' end of the L1 element. These smaller L1 RNAs do not appear to be derived from transcripts from divergent L1 families but are either discrete shorter transcripts or specifically processed products from longer initial transcripts.

Cross contamination of the genomes in human/hamster cell hybrids by multiple short recombination events.

We have isolated sites of de novo rearrangements from interspecific cell hybrids. Of 147,000 clones screened from a human/hamster hybrid genomic library, 14 clones were found with homology to both human and hamster repetitive DNA sequences. Five of these clones contained recombination events involving less than 13 kb of DNA, three with human DNA recombined into a section of hamster DNA, and two with hamster DNA recombined into human DNA. None of the clones involving human LI sequences were found to be de novo transposition events, but simply short recombination or insertion events. Considering the apparent random nature of these events, they are likely to involve unique as well as repetitive sequences and also involve integration into the homologous as well as heterologous chromosome sets. These results suggest that the chromosome sets in somatic cell hybrids may be randomly contaminated with small DNA segments derived from either set of chromosomes.

Haematopoietic radioprotection by Cremophor EL: a polyethoxylated castor oil.

The polyethoxylated castor oil, Cremophor EL (Cremophor) is approved for human use as a vehicle for oral and intravenous administration of water-insoluble compounds. Cremophor has also previously been shown to reverse the multidrug resistance phenotype at clinically acceptable doses. This study demonstrates that doses of Cremophor in the range of 25-50 microliters/kg intravenously (i.v.) administered 1 day prior to near-lethal irradiation protected the regenerative capacity of the marrow, resulting in haematopoietic radioprotection and long-term survival of near-lethally-irradiated mice. In normal mice, Cremophor administration (1) markedly reduced the level of serum haematopoietic inhibitory activity 4-8 h following injection; (2) resulted in a transient decrease in femoral bone marrow cellularity and upregulated B220 (B cells), and 7/4 (neutrophils and activated macrophages), but not Thy-1 (T-cells) surface antigen expression in bone marrow cells within 24 h of injection; and (3) transiently elevated the incidence of both primitive and committed haematopoietic progenitor cells detected in clonal agar culture within 48 h of injection. Bone marrow progenitor cell content, and peripheral blood white cell, platelet and reticulocyte counts were unaffected. This suggests that the haematopoietic radioprotection and recovery observed in irradiated mice pretreated with Cremophor may be the result of accessory cell activation and/or modulation of accessory factors regulating haematopoietic progenitor cells. Our data suggest a potential clinical use of Cremophor as an adjunct to, or as a substitute for, cytokines to minimize myelosuppression following cytotoxic therapy.

A low-affinity, low-molecular-mass endothelin-A receptor in neonatal rat heart.

Endothelin receptors with endothelin-A (ETa) specificity were present in neonatal rat ventricle. However, in both receptor-binding studies and studies of inositol phosphate accumulation, these receptors had lower affinity for endothelin-1 than ETa receptors on isolated neonatal cardiomyocytes or adult left atria. Receptors in the three myocardial preparations were cross-linked to 125I-endothelin-1 and their molecular masses measured using SDS/PAGE. Receptors on left atria and neonatal cardiomyocytes had the expected molecular mass of 48 kDa, whereas the receptors in neonatal ventricle were smaller (38 kDa). Despite this, neonatal ventricles contained ETa receptor mRNA which was not different in size from that in the isolated cells (4.5 kb). Thus the 38 kDa ETa receptor present in neonatal ventricle appears to be transcribed from full-length ETa receptor mRNA and is possibly formed by processing of the 48 kDa receptor.

Modified-cytosine restriction-system-induced recombinant cloning artefacts in Escherichia coli.

We have tested whether, and to what extent, recombinant clones from DNA segments with 5-methylation of cytosines recovered in methylation-restrictive (mcr+) hosts contain mutations. We constructed a model system in which the tetracycline-resistance-encoding gene (tet) from pBR322 was cloned into the plasmid pGEM3Zf+. The central region of tet was removed from the construct, methylated in vitro and then religated back into the unmethylated remainder of the construct. The central region of tet was either (1) methylated with a combination of four bacterial methyltransferases (M.AluI, M.HaeIII, M.HpaII plus M.HhaI) or (2) methylated with M.SssI which methylates at all CpG dinucleotides. These two protocols generated theoretical levels of DNA methylation in the central fragment of 10.5% and 33%, respectively. The construct was transformed into a series of isogenic (recA+) bacterial strains that were mcrA+ mcrB+C+, mcrA+ mcrB-C+, mcrA- mcrB+C+, mcrA- mcrB-C+ or mcrA- delta mcrBC, and also into a set of isogenic recA- derivatives of these strains. With the two methylation protocols, there was an average 48- and 141-fold reduction, respectively, in the number of transformants recovered from the recA+ mcr+ hosts compared with a methylation-tolerant host (mcr-). Of the clones recovered in recA+mcr+ hosts, > 20% of clones had an inactivating mutation in tet. The majority of such mutant clones contained deletions that frequently extended into the unmethylated portion of tet and even into the plasmid sequences beyond the end of the polylinker. With the recA- mcr+ hosts, effective restriction was much more stringent, rendering the plasmid containing the methylated segment effectively unclonable.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli host strains SURE and SRB fail to preserve a palindrome cloned in lambda phage: improved alternate host strains.

We have attempted to produce Escherichia coli strains with the optimal combination of host mutations required for the construction of genomic libraries in lambda and cosmid vectors. For lambda vectors, we defined this as a strain that combined high efficiency of phage plating with optimal tolerance to DNA methylation and the ability to propagate recombinants containing regions of potential secondary structure. To optimize this latter property, we have tested a series of strains for the ability to propagate a lambda phage containing a palindromic sequence. These included an mcr- derivative of a strain shown by Ishiura et al. [J. Bacteriol. 171 (1989) 1068-1074] to allow optimal stability of inserts in cosmid clones. All the sbcC strains allowed plaque formation of the palindrome-containing lambda phage. However, while the palindrome-containing phage plated with reasonable efficiency on SURE (recB sbcC recJ umuC uvrC) and SRB (sbcC recJ umuC uvrC), the majority of phage recovered from these strains no longer required an sbcC host for subsequent plating. These two strains also gave poorer titres with a low-yielding phage clone from the human Prader-Willi chromosome region. Optimal phage hosts appear to be those that are mcrA delta(mcrBC-hsd-mrr) combined with mutations in sbcC plus recBC or recD and without mutations in additional recombination functions such as recJ or recJ umuC uvrC (all of our E. coli strains are available on request).

The human Pi class glutathione transferase sequence at 12q13-q14 is a reverse-transcribed pseudogene.

A previous in situ hybridization study with a Pi class glutathione S-transferase cDNA probe revealed the presence of hybridizing sequences on the long arms of chromosomes 11 and 12. Since the GSTP1 gene is known to be on chromosome 11 and since it is thought that chromosomes 11 and 12 arose from an ancient tetraploidization event, it was of interest to determine if the gene on chromosome 12 encoded a closely related Pi class glutathione S-transferase isoenzyme. This gene has now been cloned and sequenced. The results are surprising and indicate that the gene is a partial reverse-transcribed pseudogene that has been inserted into the genome at 12q by chance and has not resulted from the prior tetraploidization of the human genome.

Reversal of multidrug resistance by surfactants.

Cremophor EL, a pharmacologically inactive solubilising agent, has been shown to reverse multidrug resistance (MDR). Using flow cytometric evaluation of equilibrium intracellular levels of daunorubicin (DNR), we found that eight other surface active agents will also reverse MDR. All the active detergents contain polyethoxylated moieties but have no similarities in their hydrophobic components. The properties of three polyethoxylated surfactants that showed the lowest toxicities, Cremophor, Tween 80 and Solutol HS15, were examined in more detail. The concentrations of Tween 80 and Solutol required to reverse DNR exclusion were 10-fold lower than for Cremophor. However while concentrations greater than or equal to 1:10(2) of the former two surfactants resulted in breakdown of cells, even 1:10 of Cremophor did not lyse cells. Studies of the effects of Cremophor on the uptake and efflux of DNR in normal and MDR cell types showed that Cremophor increases intracellular DNR primarily by locking the rapid efflux from the cells. This blockage of drug efflux may be mediated by a substantial alteration in the fluidity of cell membranes induced by Cremophor, as shown by decreased fluorescence anisotropy of a membrane probe. Consistent with these data, coinjection of adriamycin plus Cremophor into mice carrying a multidrug resistant P388 transplantable tumour significantly increased the survival time of the mice compared with adriamycin treatment alone.

Levels of expression of the mdr1 gene and glutathione S-transferase genes 2 and 3 and response to chemotherapy in multiple myeloma.

We have quantitated the levels of mRNAs in bone marrow samples from patients with multiple myeloma of the mdr1 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 mdr1 than patients who responded (P = 0.01). In the total myeloma patient data set, mRNA levels for mdr1 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 mdr1 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 mdr1 gene together with other genes whose products will generate additional mechanisms of resistance to chemotherapeutic agents.

A sizing artifact of DNA restriction fragments with unusually long single-stranded termini.

The restriction endonucleases (ENases) BstNI (CCATGG) and EcoRII (CCATGG) both cleave DNA at the same time sequences, but only EcoRII produces 5-nucleotide (nt) cohesive ends and is inhibited by 5-methylation of the inner cytosine. The low-Mr fragments in digests of mouse DNA made with these two ENases exhibit different mobilities during agarose-gel electrophoresis. The difference in the mobilities of the BstNI and EcoRII fragments from mouse DNA was not due to closely spaced, differentially methylated sites, or to alternate mechanisms such as circularization of the long cohesive ends of the EcoRII fragments, or to residual bound protein. Rather, it was due to the unusually long 5-nt single-stranded (ss) ends of fragments produced by EcoRII digestion, since the slower mobility of the EcoRII fragments was abolished by treatment with ss-specific nuclease. Similar mobility differences between BstNI and EcoRII fragments which could be removed by ss nuclease were also observed in digests of simian virus 40 DNA.

Assessing risk factors for transmission of infection.

Commonly used measures of effect, such as risk ratios and odds ratios, may be quite biased when used to assess the effect of factors that alter transmission risks given exposure to infected individuals. This is demonstrated in a simulation model involving a higher-risk behavior and a lower-risk behavior affecting the sexual transmission of human immunodeficiency virus. The bias arises because population contact patterns between higher-risk and lower-risk persons change their relative probabilities of exposure to an infected individual as an epidemic progresses. The assessment of contact patterns is thus central to risk assessment for contagious diseases. A new formulation of selective mixing presented here, together with a structured mixing specification of the social settings of contact, provides a theoretic framework for the investigation of contact pattern determinants.

Revised genomic consensus for the hypermethylated CpG island region of the human L1 transposon and integration sites of full length L1 elements from recombinant clones made using methylation-tolerant host strains.

Efficient recovery of clones from the 5' end of the human L1 dispersed repetitive elements necessitates the use of deletion mcr- host strains since this region contains a CpG island which is hypermethylated in vivo. Clones recovered with conventional mcr+ hosts seem to have been derived preferentially from L1 members which have accumulated mutations that have removed sites of methylation. We present a revised consensus from the 5' presumptive control region of these elements. This revised consensus contains a consensus RNA polymerase III promoter which would permit the synthesis of transcripts from the 5' end of full length L1 elements. Such potential transcripts are likely to exhibit a high degree of secondary structure. In addition, we have determined the flanking sequences for 6 full length L1 elements. The majority of full length L1 clones show no convincing evidence for target site duplication in the insertion site as commonly observed with truncated L1 elements. These data would be consistent with two mechanisms of integration of transposing L1 elements with different mechanisms predominating for full length and truncated elements.

Effects of mcr restriction of methylated CpG islands of the L1 transposons during packaging and plating stages of mammalian genomic library construction.

The use of optimally methylation-tolerant mcrA- mcrB- strains has been shown to produce an over tenfold increase in the plating efficiencies of mammalian genomic libraries, compared to a superior conventional phage host strain LE392 which is mcrB+. However, there is an even more significant effect of mcr restriction. Amongst the recombinants recovered with an mcrB+ host, we have found that there is an additional 30-fold reduction in the frequencies of clones containing the heavily methylated 5'-CpG island sequences of both the human and rat L1 repetitive elements. The mcrA product was also found to restrict clones of these methylated genomic segments, but not as strongly as mcrB. However, the use of packaging extracts made from mcrA+ lysogens did not result in convincing reductions in the recoveries of these dispersed methylated elements. The magnitude of mcr restriction during plating due to methylated dispersed elements is sufficient to make a significant proportion of mammalian genomes unclonable from genomic libraries constructed previously using conventional mcr+ hosts.

Reversal of the multidrug resistance phenotype with cremophor EL, a common vehicle for water-insoluble vitamins and drugs.

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.