Mismatch repair deficiency: a temozolomide resistance factor in medulloblastoma cell lines that is uncommon in primary medulloblastoma tumours.

BACKGROUND: Tumours are responsive to temozolomide (TMZ) if they are deficient in O(6)-methylguanine-DNA methyltransferase (MGMT), and mismatch repair (MMR) proficient. METHODS: The effect of TMZ on medulloblastoma (MB) cell killing was analysed with clonogenic survival assays. Expression of DNA repair genes and enzymes was investigated using microarrays, western blot, and immunohistochemistry. DNA sequencing and promoter methylation analysis were employed to investigate the cause of loss of the expression of MMR gene MLH1. RESULTS: Temozolomide exhibited potent cytotoxic activity in D425Med (MGMT deficient, MLH1 proficient; IC(50)=1.7 µM), moderate activity against D341Med (MGMT proficient, MLH1 deficient), and DAOY MB cells (MGMT proficient, MLH1 proficient). MGMT inhibitor O(6)-benzylguanine sensitised DAOY, but not D341Med cells to TMZ. Of 12 MB cell lines, D341Med, D283Med, and 1580WÜ cells exhibited MMR deficiency due to MLH1 promoter hypermethylation. DNA sequencing of these cells provided no evidence for somatic genetic alterations in MLH1. Expression analyses of MMR and MGMT in MB revealed that all patient specimens (n=74; expression array, n=61; immunostaining, n=13) are most likely MMR proficient, whereas some tumours had low MGMT expression levels (according to expression array) or were totally MGMT deficient (3 out of 13 according to immunohistochemistry). CONCLUSION: A subset of MB may respond to TMZ as some patient specimens are MGMT deficient, and tumours appear to be MMR proficient.

The gene expression profiles of medulloblastoma cell lines resistant to preactivated cyclophosphamide.

The total expression profiles of two medulloblastoma cell lines resistant to the preactivated form of cyclophosphamide (4-hydroperoxycyclophosphamide, 4-HC) were examined using the Affymetrix GeneChip U133A array. Our primary objective was to look for possible genes, other than the well-studied aldehyde dehydrogenases (ALDH) that may be involved in cyclophosphamide (CP) resistance in medulloblastomas. We present here the lists of the most highly upregulated [30 for D341 MED (4-HCR); 20 for D283 MED (4-HCR)] and downregulated [19 for D341 MED (4-HCR); 15 for D283 MED (4-HCR)] genes which may be involved in conferring CP-resistance to the two medullobalstoma cell lines. The lists of genes from the two sublines almost had no overlap, suggesting different mechanisms of CP-resistance. One of the most noteworthy upregulated gene is TAP1 [90-fold increase in D341 MED (4-HCR) relative to D341 MED]. TAP1, a protein belonging to the ABC transporter family is normally involved in major histocompatibility class I (MHC I) antigen processing. This suggests the possible role of multidrug resistance (MDR), albeit atypical (which means it does not involve the usual MDR1 and MRP glycoproteins), in medulloblastoma's CP-resistance. Apart from TAP1, a number of other genes involved in MHC1 processing were upregulated in D341 MED (4HCR). D341 MED (4-HCR) also had a 20-fold increase in the expression of the aldo-keto reductase gene, AKR1B10, which may deactivate the reactive cyclophosphamide metabolite, aldophosphamide. For D283 MED (4-HCR), the most notable increase in expression is that of ALDH1B1, a member of the aldehyde dehydrogenase (ALDH) family of proteins.

Mutagenicity of a single 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene in Escherichia coli located in a GGC sequence.

1-Nitropyrene, a common environmental pollutant, forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-amino- pyrene (dG(AP)). Mutational spectra of randomly introduced dG(AP) in Escherichia coli included different types of mutations, which depended on the base sequence surrounding the adduct. In earlier works we investigated the DNA sequence context effects of the adduct in repetitive CpG and non-repetitive CpGpC sequences. In the current work this adduct was incorporated into a non-repetitive GpGpC sequence in single-stranded M13mp7L2 DNA with the adduct located at either the 5' or 3' G. Potent genotoxicity of dG(AP) was evident from a significant reduction in the population of progeny phage following replication of these constructs in repair-competent E.coli cells. However, progeny derived from the 3'-G(AP) construct were much larger than those from the 5'-G(AP) construct. We suspect that a more facile translesion synthesis past the adduct at the 3' G relative to that at the 5' G, presumably due to a difference in conformation of dG(AP) in these two sites, might be responsible for this effect. With both adducted constructs, >95% of the progeny did not show any mutations at or near the adduct site, indicating highly efficient error-free translesion synthesis. However, a small population of mutants with one base deletions and base substitutions were detected. While the adduct induced -1 frameshifts (<1%) in each G site, base substitutions (1-2%), exhibiting predominantly G-->C transversions, were detected only when the adduct was located at the 5' G. A comparison of the data from this study with a prior study in the CpGpC sequence suggests that dG(AP) mutagenesis is highly sensitive to the local DNA sequence and that a 5'-pyrimidine base might be important for targeted base substitutions by this adduct in E.coli.

trans-1-[(2-Phenylcyclopropyl)methyl]-4-arylpiperazines: mixed dopamine D(2)/D(4) receptor antagonists as potential antipsychotic agents.

The dopaminergic receptor profile of a series of trans-1-[(2-phenylcyclopropyl)methyl]-4-arylpiperazines was examined. Aromatic substitution patterns were varied with the goal of identifying a compound having affinities for the D(2) and D(4) receptors in a ratio similar to that observed for the atypical neuroleptic clozapine. The compounds (1S, 2S)-trans-1-[(2-phenylcyclopropyl)methyl]-4-(2, 4-dichlorophenyl)piperazine (5m) and (1S, 2S)-trans-1-[(2-phenylcyclopropyl)methyl]-4-(2, 4-dimethylphenyl)piperazine (5t) were selected for functional antagonists at D(2) and D(4) receptors and had a D(2)/D(4) ratio approximating that of clozapine; they proved inactive in behavioral tests of antipsychotic activity.

Mutagenicity of the 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene in Escherichia coli located in a nonrepetitive CGC sequence.

1-Nitropyrene, a common environmental pollutant, forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene (dG(AP)). Mutational spectra of randomly introduced dG(AP) in Escherichia coli included many different types of mutations. However, a prior site-specific study in a CGCG(AP)CG sequence showed only CpG deletions and +1 frame shifts. To further explore the context effects of dG(AP) in mutagenesis, in this work this adduct was incorporated into a nonrepetitive CGC sequence in single-stranded M13mp7L2 DNA. Upon replication of this construct in repair-competent E. coli, one-base deletions and base substitutions were detected. The -1 frame shifts, whose frequency increased 3-6-fold with SOS (to an average frequency of 1.5%), involved deletion of the adjacent C residues. The base substitutions ( approximately 2.2%) included targeted G-to-T and G-to-C transversions, whose frequencies did not increase with SOS. This suggests that dG(AP) mutagenesis is highly dependent on the local DNA sequence.

Sequence specific mutagenesis of the major (+)-anti-benzo[a]pyrene diol epoxide-DNA adduct at a mutational hot spot in vitro and in Escherichia coli cells.

In the supF gene, most (+)-anti-benzo[a]pyrene diol epoxide ((+)-anti-B[a]PDE) mutagenesis hot spots in Escherichia coli are in 5'-GG sequences [Rodriguez and Loechler (1993) Carcinogenesis 14, 373-383]. A major hot spot was detected at G1 in the sequence 5'-GCG1G2-CCAAAG, whereas G2 yielded very few mutants. In order to investigate the details of such sequence context effects of (+)-anti-B[a]PDE mutagenesis, we have constructed 25-mer oligonucleotides and single-stranded M13 genomes containing the above decamer sequence, in which the trans-N2-dG adduct induced by (+)-anti-B[a]PDE [(+)-trans-anti-B[a]P-N2-dG] at G1 or G2 was introduced. In vitro DNA synthesis on the adducted 25-mers was strongly blocked at each site, although the 3'-->5' exonuclease-deficient Klenow fragment could incorporate a nucleotide opposite the adduct in the presence of Mn2+. For both sites purine nucleotides were preferred. The ratio Vmax/K(m) indicated that the efficiency of incorporation of dGTP opposite these sites was very similar, but dATP incorporation opposite the adduct at G1 was five-fold more efficient than that at G2. For each site, further extension beyond the adducted nucleotide was investigated by annealing four different primers, in which only the nucleotide opposite the adducted deoxyguanosine was altered. Significant extension was only observed when deoxyadenosine was located opposite adducted G1. When the M13 genomes containing the (+)-trans-anti-B[a]P-N2-dG were replicated in E. coli, survival of each adducted genome was less than 1% as compared to the unadducted genome. Upon induction of SOS, viability increased 2-6-fold. DNA sequencing showed no base substitutions in the progeny from SOS-uninduced cells, although small deletions in a quasipalindromic sequence occurred with the adduct being located at either site. However, following SOS induction, up to 40% targeted base substitutions were detected when the adduct was located at G1, while approximately 12% of the progeny were mutants with the adduct at G2. Most base substitutions were targeted G-->T transversions. We conclude that (+)-trans-anti-B[a]P-N2-dG is a highly mutagenic and replication blocking lesion. In addition, the biological consequence of this adduct depends on whether it is located at G1 or G2, suggesting that sequence context plays a major role in the mutagenic processing of this adduct.

Pharmacokinetics and pharmacodynamics in copper deficiency. I. Antiinflammatory activity of aspirin.

The effect of nutritional copper (Cu) deficiency on the antiinflammatory activity and pharmacokinetics of aspirin (ASA) was investigated in rats. Male, weanling Sprague-Dawley rats were fed either a Cu-deficient (CuD) or Cu-sufficient (CuS) diet for 49-50 d. The antiinflammatory activity of ASA was studied using the carrageenan-induced paw edema (CPE) test. ANOVA analyses of edema volumes at 2, 3, 4, 5, and 21 h postcarrageenan indicated significant differences between groups. The percent inhibition of edema due to ASA treatment in CuS was lower than that in CuD rats at 5 h, AUC5h, and AUC21h. ASA was found to be significantly more effective in inhibiting the CPE in CuD rats when compared to the CuS rats. Thus, we hypothesized that the increase in ASA's antiinflammatory activity in CuD rats was a result of a decrement in its elimination during nutritional Cu deficiency. The elimination of ASA in CuD and CuS rats was studied using an iv dose of 200 mg/kg. Concentrations of ASA and salicylic acid (SA) were determined in blood; whereas the concentrations of SA, salicylic phenol-glucuronide (SPG), and salicyluric acid (SUA) were determined in urine by HPLC. The results of the pharmacokinetic analyses from blood and urinary data indicated no significant differences in the disposition of ASA between CuD and CuS rats. For instance, the total body clearance for ASA (mean +/- SD, mL/min/kg) was 37.9 +/- 9.4 and 38.5 +/- 13.9 (p > 0.05); and the volume of distribution (Vd) for ASA (mean +/- SD, mL/kg) was 385.5 +/- 110.3 and 397.1.1 +/- 137.9 (p > 0.05) for CuD and CuS groups, respectively. Thus, contrary to our hypothesis, the enhanced antiinflammatory activity of ASA in CuD rats does not appear to be mediated via a decrement in the elimination of the drug. In addition, plasma ASA-esterase activity was found to be independent of Cu nutritional status.

Binary and ternary salt gradients in hydrophobic-interaction chromatography of proteins.

Hydrophobic-interaction chromatography of mixtures of acidic and basic proteins having a wide range of molecular weights and hydrophobic character was carried out by using binary and ternary salt gradients. Chaotropic and antichaotropic salts as well as organic salts were incorporated in the eluents. The stationary phase consisted of macroporous silica with surface-bound polyether moieties. At constant eluent surface tension, gradient elution with two or three aqueous salt solutions was found to be superior to single-salt gradients in modulating hydrophobic-interaction chromatography retention and selectivity. The effect was attributed to the competitive salt-specific binding to the protein molecule and/or the stationary phase surface. Chaotropic/antichaotropic salt gradient systems exhibited vastly different selectivities upon changing the nature and concentrations of salts in the eluents. In general, the retention of basic proteins increased while that of acidic proteins either decreased or remained unchanged with the use of chaotropic salts. At the same surface tension of the eluent, KSCN and KC1O4 yielded different selectivities. The addition of organic salts, such as tetrabutylammonium bromide was found to be suitable for the separation of proteins having a wide range of isoelectric points.