A randomised, phase II trial of the DNA-hypomethylating agent 5-aza-2'-deoxycytidine (decitabine) in combination with carboplatin vs carboplatin alone in patients with recurrent, partially platinum-sensitive ovarian cancer.

BACKGROUND: Our previous laboratory and clinical data suggested that one mechanism underlying the development of platinum resistance in ovarian cancer is the acquisition of DNA methylation. We therefore tested the hypothesis that the DNA hypomethylating agent 5-aza-2'-deoxycytodine (decitabine) can reverse resistance to carboplatin in women with relapsed ovarian cancer. METHODS: Patients progressing 6-12 months after previous platinum therapy were randomised to decitabine on day 1 and carboplatin (AUC 6) on day 8, every 28 days or carboplatin alone. The primary objective was response rate in patients with methylated hMLH1 tumour DNA in plasma. RESULTS: After a pre-defined interim analysis, the study closed due to lack of efficacy and poor treatment deliverability in 15 patients treated with the combination. Responses by GCIG criteria were 9 out of 14 vs 3 out of 15 and by RECIST were 6 out of 13 vs 1 out of 12 for carboplatin and carboplatin/decitabine, respectively. Grade 3/4 neutropenia was more common with the combination (60% vs 15.4%) as was G2/3 carboplatin hypersensitivity (47% vs 21%). CONCLUSIONS: With this schedule, the addition of decitabine appears to reduce rather than increase the efficacy of carboplatin in partially platinum-sensitive ovarian cancer and is difficult to deliver. Patient-selection strategies, different schedules and other demethylating agents should be considered in future combination studies.

Most low-level microsatellite instability in colorectal cancers can be explained without an elevated slippage rate.

Many cancers show a low level of microsatellite slippage and are labelled MSI-L (microsatellite instability--low). However, it is unclear whether this slippage can be attributed to some underlying genetic change that results in a mutator phenotype, analogous to mismatch repair deficiency in MSI-H cancers, or whether the apparent instability is the result of relatively frequent normal somatic slippage. Here, we have used a mathematical model of microsatellite slippage during cancer growth to estimate the degree of microsatellite slippage expected in a cancer due to normal somatic slippage. We compared the model to the slippage observed in 42 non-MSI-H cancers that were macro-dissected into four distinct regions and genotyped at N = 9 microsatellite loci. When the slippage rate was set at mu = 10(-5) per locus per division, ten cancers showed a level of slippage in at least one region that was too severe to be expected from normal somatic slippage alone, suggesting that these cancers had acquired MSI-L. Only one of these ten cancers had putative MSI-L in all four regions. When we considered a slightly higher slippage rate of mu = 5 x 10(-5), none of the cancers showed a degree of slippage that could not be reasonably explained by normal somatic slippage. Counting the number of 'unstable' loci was a poor indicator of putative MSI-L status. We conclude that most low-level microsatellite instability in colorectal cancers can be explained without requiring an elevated slippage rate during neoplastic development, and hence there is little evidence for a discrete MSI-L group of cancers. Putative MSI-L status is indicated by the presence of at least one locus that has multiple alleles that differ by at least five motif repeats from the germline. If an underlying genetic change does cause MSI-L, it appears to be a relatively uncommon event that occurs late in oncogenesis.

The identification of chemical intermediates in enzyme catalysis by the rapid quench-flow technique.

Traditionally, enzyme transient kinetics have been studied by the stopped-flow and rapid quench-flow (QF) methods. Whereas stopped-flow is the more convenient, it suffers from two weaknesses: optically silent systems cannot be studied, and when there is a signal it cannot always be assigned to a particular step in the reaction pathway. QF is a chemical sampling method; reaction mixtures are aged for a few milliseconds or longer, 'stopped' by a quenching agent and the product or the intermediate is measured by a specific analytical method. Here we show that by exploiting the array of current analytical methods and different quenching agents, the QF method is a key technique for identifying, and for characterising kinetically, intermediates in enzyme reaction pathways and for determining the order by which bonds are formed or cleaved by enzymes acting on polymer substrates such as DNA.

Large-scale studies of the association between variation at the TNF/LTA locus and susceptibility to type 2 diabetes.

AIMS/HYPOTHESIS: The proinflammatory cytokine TNF-alpha has been implicated in the pathogenesis of insulin resistance and type 2 diabetes, and variation in the gene encoding TNF-alpha (TNF) has shown inconsistent associations with susceptibility to both conditions. Additionally, the coding non-synonymous variant T60N in the neighbouring LTA gene has been reported to be associated with type 2 diabetes. The present study aimed to obtain a robust assessment of the role of variation in the tightly linked TNF/LTA region in diabetes susceptibility by genotyping TNF and LTA variants in large case-control resources. MATERIALS AND METHODS: The G-308A and G-238A TNF promoter variants and the LTA T60N polymorphism were genotyped in two UK case samples that were ascertained for positive family history and/or early onset of type 2 diabetes (combined n=858) and in 1,257 ethnically matched controls. RESULTS: There were no significant associations between the T60N, G-308A or G-238A genotype and type 2 diabetes in the combined analysis (exact Cochran-Mantel-Haenszel statistic for ordered genotypes for T60N, p=0.69; for G-308A, p=0.51; for G-238A, p=0.16). CONCLUSIONS/INTERPRETATION: The present study, one of the largest association analyses yet reported at this locus, provides no evidence that the specific TNF or LTA variants examined influence susceptibility to type 2 diabetes. More comprehensive studies of the TNF/LTA locus in substantially larger sample sets are required to establish whether genome sequence variation at this locus truly influences susceptibility to type 2 diabetes.

O(6)-methylguanine methyltransferase in colorectal cancers: detection of mutations, loss of expression, and weak association with G:C>A:T transitions.

BACKGROUND AND AIMS: O(6)-methylguanine methyltransferase (MGMT) repairs inappropriately methylated guanine in DNA. MGMT mutations have not previously been reported in cancers, but in colorectal tumours MGMT promoter methylation is common and has been associated with increased G:C>A:T transitions, a high frequency of K-ras mutations, and low level microsatellite instability (MSI low). However, some have suggested that MGMT changes are background or secondary events, with little importance for tumorigenesis. METHODS: We have analysed fresh frozen colorectal cancers and colorectal cancer cell lines for MGMT changes: mutations, allelic loss, and protein expression. RESULTS: Six of 113 cancers harboured somatic missense MGMT mutations, at least three of which probably caused reduced MGMT function and were accompanied by silencing or loss of the wild-type allele. Cancers with pathogenic MGMT mutations tended to harbour G:C>A:T somatic mutations at other loci. Overall, MGMT expression was reduced or lost in more than half of the cancers. We found no association between MGMT expression and the somatic mutation spectrum at APC, beta-catenin, K-ras, or p53, but decreased MGMT expression was weakly associated with the presence of a G:C>A:T change at any one of these loci. Reduced MGMT expression was not however associated with an increased frequency of K-ras mutations or with MSI low. CONCLUSION: In summary, we found that mutation of MGMT contributes to decreased protein function. Our findings provide good evidence to show that MGMT changes, including methylation, are selected rather than background events, at least in some cases. Decreased MGMT expression or function probably has a weak or moderate effect on the mutation spectrum in colorectal cancers.

Chromosomal localization, genomic organization and evolution of the genes encoding human phosphatidylinositol transfer protein membrane-associated (PITPNM) 1, 2 and 3.

Eukaryotic proteins containing a phosphatidylinositol transfer (PITP) domain can be divided into two groups, one consisting of small soluble 35-kDa proteins and the other those that are membrane-associated and show sequence similarities to the Drosophila retinal degeneration B (rdgB) protein. The rdgB protein consists of four domains, an amino terminal PITP domain, a Ca2+-binding domain, a transmembrane domain and a carboxyl terminal domain that interacts with the protein tyrosine kinase PYK2. Three mammalian phosphatidylinositol transfer protein membrane-associated genes (PITPNM1, 2 and 3) with homology to Drosophila rdgB have previously been described and shown to be expressed in the mammalian retina. These findings and the demonstration that the rdgB gene plays a critical role in the invertebrate phototransduction pathway have led to the mammalian genes being considered as candidate genes for human eye diseases. In order to facilitate the analysis of these genes we have used radiation hybrid mapping and fluorescence in situ hybridization to localize the PITPNM2 and 3 genes to human chromosomes 12p24 and 17p13 respectively and hybrid mapping to confirm the localization of PITPNM1 to chromosome 11q13. We have also determined the genomic organization of both the soluble and membrane-associated Drosophila and human PITP domain-containing genes. Phylogenetic analysis indicates that the two groups arose by gene duplication that occurred very early in animal evolution.

Exon 3 beta-catenin mutations are specifically associated with colorectal carcinomas in hereditary non-polyposis colorectal cancer syndrome.

BACKGROUND AND AIMS: Activating beta-catenin mutations in exon 3 have been implicated in colorectal tumorigenesis. Although reports to the contrary exist, it has been suggested that beta-catenin mutations occur more often in microsatellite unstable (MSI+) colorectal carcinomas, including hereditary non-polyposis colorectal cancer (HNPCC), as a consequence of defective DNA mismatch repair. We have analysed 337 colorectal carcinomas and adenomas, from both sporadic cases and HNPCC families, to provide an accurate assessment of beta-catenin mutation frequency in each tumour type. METHODS: Direct sequencing of exon 3 of beta-catenin. RESULTS: Mutations were rare in sporadic (1/83, 1.2%) and HNPCC adenomas (1/37, 2.7%). Most of the sporadic adenomas analysed (80%) were small (<1 cm), and our data therefore differ from a previous report of a much higher mutation frequency in small adenomas. No oncogenic beta-catenin mutations were identified in 34 MSI+ and 78 microsatellite stable (MSI-) sporadic colorectal cancers but a raised mutation frequency (8/44, 18.2%) was found in HNPCC cancers; this frequency was significantly higher than that in HNPCC adenomas (p=0.035) and in both MSI- (p<0.0001) and MSI+ (p=0.008) sporadic cancers. Mutations were more common in higher stage (Dukes' stages C and D) cancers (p=0.001). CONCLUSION: Exon 3 beta-catenin mutations are associated specifically with malignant colorectal tumours in HNPCC; mutations appear not to result directly from deficient mismatch repair. Our data provide evidence that the genetic pathways of sporadic MSI+ and HNPCC cancers may be divergent, and indicate that mutations in the HNPCC pathway of colorectal tumorigenesis may be determined by selection, not simply by hypermutation.

Examining the relationships between the Pro12Ala variant in PPARG and Type 2 diabetes-related traits in UK samples.

AIMS: The Pro12Ala polymorphism in the PPARG gene alters amino acid sequence and has shown consistent association with susceptibility to Type 2 diabetes in several populations. The present study makes use of large, well-characterized case-control resources to enhance understanding of this susceptibility effect by examining related traits, such as body mass index (BMI), waist-hip ratio and age at diagnosis. METHODS: The Pro12Ala variant was genotyped in two UK case samples, ascertained for positive family history and/or early onset of Type 2 diabetes (combined n=971); and in 1257 ethnically matched control subjects. RESULTS: There were significant associations of the Pro12Ala single nucleotide polymorphism (SNP) genotypes with diabetes in both case-control comparisons (P=0.025 and P=0.039). Comparing individuals homozygous for the Pro allele, with those carrying an Ala allele, the combined odds ratio for diabetes was 1.40 (95% CIs, 1.12-1.76, P=0.0031). There was no association between the variant and either waist-hip ratio or age at diagnosis. Proline homozygosity was associated with increased BMI in one patient group (P=0.013) and decreased BMI in the other (P=0.038). CONCLUSIONS: This study confirms that variation within PPARG influences susceptibility to Type 2 diabetes in UK samples. However, the relationship between PPARG variation and BMI is more complex, and studies in much larger sample sets will be required to more precisely characterize the effect of this variant on adiposity.

Ocular coloboma: a reassessment in the age of molecular neuroscience.

Congenital colobomata of the eye are important causes of childhood visual impairment and blindness. Ocular coloboma can be seen in isolation and in an impressive number of multisystem syndromes, where the eye phenotype is often seen in association with severe neurological or craniofacial anomalies or other systemic developmental defects. Several studies have shown that, in addition to inheritance, environmental influences may be causative factors. Through work to identify genes underlying inherited coloboma, significant inroads are being made into understanding the molecular events controlling closure of the optic fissure. In general, severity of disease can be linked to the temporal expression of the gene, but this is modified by factors such as tissue specificity of gene expression and genetic redundancy.

In patients with head injuries who undergo rapid sequence intubation using succinylcholine, does pretreatment with a competitive neuromuscular blocking agent improve outcome? A literature review.

A literature search was undertaken for evidence of the effect of succinylcholine (SCH) on the intracranial pressure (ICP) of patients with acute brain injury and whether pretreatment with a defasciculating dose of competitive neuromuscular blocker is beneficial in this patient group. The authors could find no definitive evidence that SCH caused a rise in ICP in patients with brain injury. However, these studies were often weak and small. For those patients suffering acute traumatic brain injury the authors could find no studies that investigated the issue of pretreatment with defasciculating doses of competitive neuromuscular blockers and their effect on ICP in patients given SCH. There is level 2 evidence that SCH caused an increase in ICP for patients undergoing neurosurgery for brain tumours with elective anaesthesia and that pretreatment with defasciculating doses of neuromuscular blockers reduced such increases. It is unknown if this affects neurological outcome for this patient group.

Hopping, jumping and looping by restriction enzymes.

Type II restriction endonucleases recognize specific DNA sequences and cleave both strands of the DNA at fixed locations at or near their recognition sites. Many of these enzymes are dimeric proteins that recognize, in symmetrical fashion, palindromic DNA sequences. They generally catalyse independent reactions at each recognition site on the DNA, although in some cases they act processively; cutting the DNA first at one site, then translocating along the DNA to another site and cutting that before leaving the DNA. The way in which the degree of processivity varies with the length of DNA between the sites can reveal the mechanism of translocation. In contrast with the common view that proteins move along DNA by 'sliding', the principal mode of transfer of the EcoRV endonuclease is by 'hopping' and 'jumping', i.e. the dissociation of the protein from one site followed by its re-association with another site in the same DNA molecule, either close to or distant from the original site. Other type II restriction enzymes require two copies of their recognition sites for their DNA cleavage reactions. Many of these enzymes, such as SfiI, are tetramers with two DNA-binding surfaces. SfiI has no activity when bound to just one recognition site, and instead both DNA-binding surfaces have to be filled before it becomes active. Although the two sites can be on separate DNA molecules, SfiI acts optimally with two sites on the same DNA, where it traps the DNA between the sites in a loop. SfiI thus constitutes a test system for the analysis of DNA looping.

DNA cleavage reactions by type II restriction enzymes that require two copies of their recognition sites.

Several type II restriction endonucleases interact with two copies of their target sequence before they cleave DNA. Three such enzymes, NgoMIV, Cfr10I and NaeI, were tested on plasmids with one or two copies of their recognition sites, and on catenanes containing two interlinked rings of DNA with one site in each ring. The enzymes showed distinct patterns of behaviour. NgoMIV and NaeI cleaved the plasmid with two sites faster than that with one site and the catenanes at an intermediate rate, while Cfr10I gave similar steady-state rates on all three substrates. Both Cfr10I and NgoMIV converted the majority of the substrates with two sites directly to the products cut at both sites, while NaeI cleaved just one site at a time. All three enzymes thus synapse two DNA sites through three-dimensional space before cleaving DNA. With Cfr10I and NgoMIV, both sites are cleaved in one turnover, in a manner consistent with their tetrameric structures, while the cleavage of a single site by NaeI indicates that the second site acts not as a substrate but as an activator, as reported previously. The complexes spanning two sites have longer lifetimes on catenanes with one site in each ring than on circular DNA with two sites, which indicates that the catenanes have more freedom for site juxtaposition than plasmids with sites in cis.

Analysis of DNA looping interactions by type II restriction enzymes that require two copies of their recognition sites.

Before cleaving DNA substrates with two recognition sites, the Cfr10I, NgoMIV, NaeI and SfiI restriction endonucleases bridge the two sites through 3D space, looping out the intervening DNA. To characterise their looping interactions, the enzymes were added to plasmids with two recognition sites interspersed with two res sites for site-specific recombination by Tn21 resolvase, in buffers that contained either EDTA or CaCl2 so as to preclude DNA cleavage by the endonuclease; the extent to which the res sites were sequestered into separate loops was evaluated from the degree of inhibition of resolvase. With Cfr10I, a looped complex was detected in the presence but not in the absence of Ca(2+); it had a lifetime of about 90 seconds. Neither NgoMIV nor NaeI gave looped complexes of sufficient stability to be detected by this method. In contrast, SfiI with Ca(2+) produced a looped complex that survived for more than seven hours, whereas its looping interaction in EDTA lasts for about four minutes. When resolvase was added to a SfiI binding reaction in EDTA followed immediately by CaCl2, the looped DNA was blocked from recombination while the unlooped DNA underwent recombination. By measuring the distribution between looped and unlooped DNA at various SfiI concentrations, and by fitting the data to a model for DNA binding by a tetrameric protein to two sites in cis, an equilibrium constant for the looping interaction was determined. The equilibrium constant was essentially independent of the length of DNA between the SfiI sites.

SMAD4 mutations in colorectal cancer probably occur before chromosomal instability, but after divergence of the microsatellite instability pathway.

Loss of chromosome 18q21 is well documented in colorectal cancer, and it has been suggested that this loss targets the DCC, DPC4/SMAD4, and SMAD2 genes. Recently, the importance of SMAD4, a downstream regulator in the TGF-beta signaling pathway, in colorectal cancer has been highlighted, although the frequency of SMAD4 mutations appears much lower than that of 18q21 loss. We set out to investigate allele loss, mutations, protein expression, and cytogenetics of chromosome 18 copy number in a collection of 44 colorectal cancer cell lines of known status with respect to microsatellite instability (MSI). Fourteen of thirty-two MSI(-) lines showed loss of SMAD4 protein expression; usually, one allele was lost and the other was mutated in one of a number of ways, including deletions of various sizes, splice site changes, and missense and nonsense point mutations (although no frameshifts). Of the 18 MSI(-) cancers with retained SMAD4 expression, four harbored missense mutations in the 3' part of the gene and showed allele loss. The remaining 14 MSI(-) lines had no detectable SMAD4 mutation, but all showed allele loss at SMAD4 and/or DCC. SMAD4 mutations can therefore account for about 50-60% of the 18q21 allele loss in colorectal cancer. No MSI(+) cancer showed loss of SMAD4 protein or SMAD4 mutation, and very few had allelic loss at SMAD4 or DCC, although many of these MSI(+) lines did carry TGFBIIR changes. Although SMAD4 mutations have been associated with late-stage or metastatic disease, our combined molecular and cytogenetic data best fit a model in which SMAD4 mutations occur before colorectal cancers become aneuploid/polyploid, but after the MSI(+) and MSI(-) pathways diverge. Thus, MSI(+) cancers may diverge first, followed by CIN(+) (chromosomal instability) cancers, leaving other cancers to follow a CIN(-)MSI(-) pathway.

Characterization of a novel human opsin gene with wide tissue expression and identification of embedded and flanking genes on chromosome 1q43.

As part of an ongoing search to identify novel mammalian photopigments that may mediate nonvisual tasks such as circadian entrainment and acute suppression of pineal melatonin levels, a number of recently cloned nonvisual opsin sequences were used to search dbEST. panopsin (OPN3) was one of the clones identified using this approach. Expression analysis detects two transcripts of approximately 2.1 and 2.5 kb, in a wide range of tissues including brain, liver, and retina, which encode a predicted protein of 403 amino acids. The gene was localized to the region of chromosome 1q43 also encompassing the kynurenine monooxygenase (KMO) and choroideremia-like Rab escort protein 2 (CHML) genes. KMO and panopsin overlap at their 3' ends but are transcribed in opposite directions. CHML, an intronless gene, lies in intron 1 of panopsin.

CDX2 mutations do not account for juvenile polyposis or Peutz-Jeghers syndrome and occur infrequently in sporadic colorectal cancers.

Peutz-Jeghers syndrome (PJS) and juvenile polyposis (JPS) are both characterized by the presence of hamartomatous polyps and increased risk of malignancy in the gastrointestinal tract. Mutations of the LKB1 and SMAD4 genes have been shown recently to cause a number of PJS and JPS cases respectively, but there remains considerable uncharacterized genetic heterogeneity in these syndromes, particularly JPS. The mouse homologue of CDX2 has been shown to give rise to a phenotype which includes hamartomatous-like polyps in the colon and is therefore a good candidate for JPS and PJS cases which are not accounted for by the SMAD4 and LKB1 genes. By analogy with SMAD4, CDX2 is also a candidate for somatic mutation in sporadic colorectal cancer. We have screened 37 JPS families/cases without known SMAD4 mutations, 10 Peutz-Jeghers cases without known LKB1 mutations and 49 sporadic colorectal cancers for mutations in CDX2. Although polymorphic variants and rare variants of unlikely significance were detected, no pathogenic CDX2 mutations were found in any case of JPS or PJS, or in any of the sporadic cancers.

SfiI endonuclease activity is strongly influenced by the non-specific sequence in the middle of its recognition site.

The SfiI endonuclease cleaves DNA at the sequence GGCCNNNN NGGCC, where N is any base and downward arrow is the point of cleavage. Proteins that recognise discontinuous sequences in DNA can be affected by the unspecified sequence between the specified base pairs of the target site. To examine whether this applies to SFII, a series of DNA duplexes were made with identical sequences apart from discrete variations in the 5 bp spacer. The rates at which SFII cleaved each duplex were measured under steady-state conditions: the steady-state rates were determined by the DNA cleavage step in the reaction pathway. SFII cleaved some of these substrates at faster rates than other substrates. For example, the change in spacer sequence from AACAA to AAACA caused a 70-fold increase in reaction rate. In general, the extrapolated values for k(cat) and K(m) were both higher on substrates with inflexible spacers than those with flexible structures. The dinucleotide at the site of cleavage was largely immaterial. SFII activity is thus highly dependent on conformational variations in the spacer DNA.

A phase II study evaluating the tolerability and efficacy of CAELYX (liposomal doxorubicin, Doxil) in the treatment of unresectable pancreatic carcinoma.

BACKGROUND: Preclinical studies of liposomal doxorubicin (CAELYX) have demonstrated significant inhibition of growth of human pancreatic cancer explants in nude mice. This study evaluated the efficacy of CAELYX in chemotherapy-naïve patients with unresectable, histologically confirmed pancreatic carcinoma. Secondary endpoints were quality of life (QOL). time to progression and overall survival. PATIENTS AND METHODS: Twenty-two patients (median age 65) were enrolled. CAELYX was administered to the first five patients at a dose of 30 mg/m2 three-weekly. Two of these patients were dose escalated to 50 mg/m2 four-weekly. Subsequent patients were all treated on the latter schedule. RESULTS: Two patients died after consenting to enter the study but before treatment was commenced and are not included in the analysis. Sixteen patients were evaluable for response. No objective responses were seen. Six patients had stable disease. One patient experienced grade 4 toxicity with palmar plantar dysaesthesia (PPE), but continued treatment after dose reduction and delay. Four patients experienced grade 3 stomatitis and two grade 3 nausea. Median survival from time of starting chemotherapy was 3.2 months (range 21 days to 19 months) and one year survival was 10%. Eight patients completed at least two EORTC QLQ C-30 questionnaires. There was no significant change in either global QOL or in any functional or symptom subscale score. CONCLUSION: No objective responses were seen with CAELYX in this study. CAELYX was however associated with stable disease, but data were inconclusive with regard to clinical benefit. It warrants further investigation in the context of combination trials.