Promoter methylation of Wnt5a is associated with microsatellite instability and BRAF V600E mutation in two large populations of colorectal cancer patients.

BACKGROUND: In colorectal cancer (CRC), tumour microsatellite instability (MSI) status and CpG island methylator phenotype (CIMP) status are indicators of patient outcome, but the molecular events that give rise to these outcomes remain largely unknown. Wnt5a is a critical regulator of non-canonical Wnt activity and promoter hypermethylation of this gene has emerging prognostic roles in CRC; however the frequency and prognostic significance of this epigenetic event have not been explored in the context of colorectal tumour subtype. Consequently, we investigated the frequency and prognostic significance of Wnt5a methylation in a large cohort of MSI-stratified CRCs. METHODS: Methylation was quantified in a large cohort of 1232 colorectal carcinomas from two clinically distinct populations from Canada. Associations were examined between methylation status and clinicopathlogical features, including tumour MSI status, BRAF V600E mutation, and patient survival. RESULTS: In Ontario, Wnt5a methylation was strongly associated with MSI tumours after adjustment for age, sex, and tumour location (odds ratio (OR)=4.2, 95% confidence interval (CI)=2.4-7.4, P<10(-6)) and with BRAF V600E mutation, a marker of CIMP (OR=12.3, 95% CI=6.9-21.7, P<10(-17)), but was not associated with patient survival. Concordant results were obtained in Newfoundland. CONCLUSION: Methylation of Wnt5a is associated with distinct tumour subtypes, strengthening the evidence of an epigenetic-mediated Wnt bias in CRC.

The genetic basis of colorectal cancer in a population-based incident cohort with a high rate of familial disease.

BACKGROUND AND AIMS: Colorectal cancer (CRC) is the second most frequent cancer in developed countries. Newfoundland has the highest incidence of CRC in Canada and the highest rate of familial CRC yet reported in the world. To determine the impact of mutations in known CRC susceptibility genes and the contribution of the known pathways to the development of hereditary CRC, an incident cohort of 750 patients with CRC (708 different families) from the Newfoundland population was studied. METHODS: Microsatellite instability (MSI) testing was performed on tumours, together with immunohistochemistry analysis for mismatch repair (MMR) genes. Where indicated, DNA sequencing and multiplex ligation-dependent probe amplifications of MMR genes and APC was undertaken. DNA from all patients was screened for MUTYH mutations. The presence of the BRAF variant, p.V600E, and of MLH1 promoter methylation was also tested in tumours. RESULTS: 4.6% of patients fulfilled the Amsterdam criteria (AC), and an additional 44.6% fulfilled the revised Bethesda criteria. MSI-high (MSI-H) was observed in 10.7% (n=78) of 732 tumours. In 3.6% (n=27) of patients, CRC was attributed to 12 different inherited mutations in six known CRC-related genes associated with chromosomal instability or MSI pathways. Seven patients (0.9%) carried a mutation in APC or biallelic mutations in MUTYH. Of 20 patients (2.7%) with mutations in MMR genes, 14 (70%) had one of two MSH2 founder mutations. 17 of 28 (61%) AC families did not have a genetic cause identified, of which 15 kindreds fulfilled the criteria for familial CRC type X (FCCTX). CONCLUSIONS: Founder mutations accounted for only 2.1% of cases and this was insufficient to explain the high rate of familial CRC. Many of the families classified as FCCTX may have highly penetrant mutations segregating in a Mendelian-like manner. These families will be important for identifying additional CRC susceptibility loci.

Loss-of-function mutations in the Nav1.7 gene underlie congenital indifference to pain in multiple human populations.

Congenital indifference to pain (CIP) is a rare condition in which patients have severely impaired pain perception, but are otherwise essentially normal. We identified and collected DNA from individuals from nine families of seven different nationalities in which the affected individuals meet the diagnostic criteria for CIP. Using homozygosity mapping and haplotype sharing methods, we narrowed the CIP locus to chromosome 2q24-q31, a region known to contain a cluster of voltage-gated sodium channel genes. From these prioritized candidate sodium channels, we identified 10 mutations in the SCN9A gene encoding the sodium channel protein Nav1.7. The mutations completely co-segregated with the disease phenotype, and nine of these SCN9A mutations resulted in truncation and loss-of-function of the Nav1.7 channel. These genetic data further support the evidence that Nav1.7 plays an essential role in mediating pain in humans, and that SCN9A mutations identified in multiple different populations underlie CIP.

Very high incidence of familial colorectal cancer in Newfoundland: a comparison with Ontario and 13 other population-based studies.

Newfoundland has the highest rate of colorectal cancer (CRC) of any Canadian province. In order to investigate the factors, especially genetic components, responsible for CRC we established the Newfoundland Colorectal Cancer Registry. In a 5-year period we examined every case of CRC diagnosed under the age of 75 years and obtained consent from 730 cases. Careful analysis of family history was used to assign a familial cancer risk, based on established criteria. We observed that 3.7% of CRC cases came from families meeting the Amsterdam II criteria and a further 0.9% of cases involved familial adenomatous polyposis (FAP). An additional 43% of cases met one or more of the revised Bethesda criteria and 31% of all cases had a first-degree relative affected with CRC. We compared the Newfoundland data with data from the province of Ontario, where the same recruitment and risk-assessment criteria were used. In all categories, the indicators of familial risk were significantly higher in Newfoundland. These data were also compared to results published from 13 other population-based studies worldwide. In every category the proportion of Newfoundland cases meeting the criteria was higher than in any other population. The mean differences were: 3.5-fold greater for FAP, 2.8-fold higher for Amsterdam criteria, 2.0-fold higher for Bethesda criteria and 1.9-fold higher for the number of affected first-degree relatives. We conclude that the high incidence of CRC in Newfoundland may be attributable to genetic, or at least familial, factors. In the high-risk families we provide evidence for the involvement of founder mutations in the APC and MSH2 genes.

SISE matters: the sum of information on seventy-yr-old equivalents measures pedigree information content when assessing the risk of HNPCC in a family.

Hereditary non-polyposis colon cancer (HNPCC) is a significant cause of colorectal and other malignancies. Due to the lack of features that reliably differentiate between a sporadic case and an inherited case of colon cancer, it is likely that HNPCC is under reported. The diagnosis of HNPCC relies heavily on finding multiple cases of colorectal or other specific cancers within a family. In the absence of a significant family history, a diagnosis of HNPCC is seldom considered. We postulate that small kinships--or, more specifically, kinships with a low information content--are more likely to be designated as having a low risk of an inherited cancer predisposition than are large kinships. This has the potential to exacerbate the under-diagnosis of HNPCC in small families, leading to inadequate treatment, follow-up and family counselling. We have developed an objective measure of the information content of individual pedigrees called the Sum of Information on Seventy-yr-old Equivalents (SISE) coefficient. The SISE coefficient is a function of the number of relatives in a kinship and their relationship to the proband, of their ages and of the age-dependent penetrance of HNPCC mutations. A population-based series of colorectal cancer cases was assessed, by currently accepted methods, for the likelihood of there being an HNPCC mutation segregating in each family. We observed that families with a low SISE coefficient were significantly more likely to be designated at low risk of HNPCC (P< or =0.001). Using a cumulative binomial distribution function, we estimated the likelihood of observing multiple cancers in families of different SISE coefficients.

HLA-DP epitope typing using monoclonal antibodies.

We have made a panel of murine anti-DP monoclonal antibodies for serological typing of HLA-DP polymorphisms; they can be used in microcytotoxicity (for 7 epitopes) and binding assays (for 8 epitopes). The antibodies detect polymorphic differences in both alpha and beta chains. As immunogens we sometimes used B-lymphoblastoid lines or purified DP molecules but mostly used mouse fibroblast transfectants expressing DP molecules. The DP beta genes were made from a cloned DPB1*0201 gene by replacing its major area of polymorphism with matching stretches of DNA amplified from other alleles; cloned DPA1*01 and DPA1*02 genes were used for transfection along with the beta chain genes. The monoclonal antibodies showed reaction patterns that correlated with the presence of particular amino-acid sequence motifs; thus none of the antibodies is allele-specific. They bind instead to epitopes which are found on a number of different HLA-DP types. We have constructed frequency tables so that the epitope (motif) data can be interpreted as the most likely genotype in each case. The basic assumption to justify this work is that HLA-DP matching or mismatching will likely influence transplant outcome, particularly in bone marrow transplantation. The present challenge is to define permissive and nonpermissive combinations of HLA-DP; it may be that matching for epitopes, rather than for full alleles, will help to resolve this issue.

Characterization of a cell type-specific enhancer found in the human papilloma virus type 18 genome.

We have previously isolated long-range acting enhancer elements from the HeLa genome by functional selection. In this report, the structural and functional characteristics of one (GA1) of the enhancers are reported. By cloning various restriction fragments and by deletion mutagenesis, the activity of GA1 was located in a 230-bp region. The nucleotide sequence of GA1 and genomic Southern blot analysis indicated that GA1 is derived from human papilloma virus (HPV) 18 DNA that had been integrated into the HeLa genome. The enhancer is located in the non-coding region of the HPV 18 genome. The HPV 18 enhancer consists of two functional domains, both of which have full enhancer activity in HeLa cells. The enhancer does not contain enhancer core sequences but contains several blocks of potential Z-DNA sequence. Like SV40 and polyoma virus enhancers, the activity of the HPV 18 enhancer was repressed by adenovirus E1a products. The HPV 18 enhancer shows a narrow cell type specificity: it is active in some cervical carcinoma cell lines, but inactive in all non-cervical cells except for one neuroblastoma cell line. These results suggest that the HPV 18 enhancer plays an important role in regulation of the viral genes.

A possible role for K+ channels in tumor cell injury. Membrane vesicle shedding and nuclear DNA fragmentation.

Mastocytoma P815 tumor cells subjected to low temperature (O degrees C/l h) and shifted to 22 degrees or 37 degrees C undergo morphological, physiological and biochemical changes which are analogous to those induced by immune effector cells, i.e., changes in cell-surface morphology and membrane permeability, elevated O2 consumption rates and nuclear DNA fragmentation [18-21]. Utilizing this low-temperature shift method for the induction of cell injury, we investigated the possible role of K+ channels in this process. Results show that the two classical K+ channel blockers, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), inhibited the low temperature-induced cell-surface membrane vesicle shedding as well as the nuclear DNA-fragmentation process. These results indicate that K+ channel function is required for tumor-cell injury as manifested by nuclear DNA fragmentation and cell-surface membrane vesicle (MV) shedding.

Chicken histone genes retain nuclear matrix association throughout the cell cycle.

The association between histone genes and the nuclear matrix (NM) during periods of high (S-phase) and low (non-S-phase) transcriptional activity has been investigated with synchronized cells from a chicken erythroid cell line (abbreviated ts34). By DNase I and restriction enzyme analysis, these studies reveal that both core and linker histone genes (represented by H2A and H1 genes respectively) are attached to the NM independent of their transcriptional activity during the cell-cycle. The tissue-specific histone gene H5, expressed constitutively, is nuclear matrix (NM)-associated in ts34 cells but is found in the supernatant (S/N) fractions of a non-erythroid T-cell line. Furthermore, we show that DNA sequences necessary for NM-attachment of the H5 gene lie within a 780 base pair region spanning part of the coding and 5' non-translated region. Of the three non-histone genes investigated, beta-actin sequences are expressed and are NM-attached, feather keratin genes are not expressed and predominate in the S/N, and beta-globin genes although not expressed in the ts34 cell line used were found in the NM fraction. In this case the association may be fortuitous or may reflect an early event prior to transcription of globin genes in differentiating erythroid cells. These results generally support the notion that actively transcribed genes are NM-attached, but that attachment per se is not synonymous with transcription.

Mutagenesis of conserved 5' elements and transcription of a chicken H1 histone gene.

In addition to readily identifiable TATA and CAAT boxes, chicken H1 histone genes contain a highly conserved G-rich region at about minus 80 and an A-rich H1 histone gene-specific motif some 120 bases upstream from the H1 mRNA cap site. The level of transcription from wild-type, 'G-box' and 'A-box' H1 deletion mutant templates was tested in Xenopus oocytes and in HeLa cells. Removal of the H1 gene-specific motif had no effect on H1 gene transcription in either assay system, whereas deletion of the G-rich sequence decreased H1 mRNA levels by about ten-fold in oocytes and in HeLa cells. At least in heterologous systems, the H1-gene specific A-rich region does not appear to influence the level of H1 gene transcription.

Low temperature-induced cell surface membrane vesicle shedding is associated with DNA fragmentation.

Temperature shift conditions of 0 degree to 22 degrees C or 0 degree to 37 degrees C induce the formation and shedding of membrane vesicles (MV) from P815 tumor cell surfaces. When the MV shedding process takes place at 22 degrees C it occurs without changes in cell surface membrane permeability, whereas at 37 degrees C, changes in permeability to 51Cr and trypan blue do occur, thus mimicking the lymphocyte-mediated lytic process of tumor cells [1]. The present studies demonstrate that nuclear DNA fragmentation also occurs in both 0 degree to 22 degrees C and 0 degree to 37 degrees C temperature shifts but cell surface membrane permeability to DNA fragments occurs only in the latter condition, i.e., 0 degree to 37 degrees C. The microtubule-stabilizing agent deuterium oxide (D2O) inhibited the MV shedding process, the changes in membrane permeability, and DNA fragmentation. When P815 cells which had been induced to shed MV by the 0 degree to 22 degrees C temperature shift were labeled with 51Cr and used as targets for alloimmune lymphocytes, they were found to be as susceptible to T-cell lysis as control P815 cells. This result indicates that the lytic effect of alloimmune T lymphocytes can be exerted at the target cell surface membrane level independently of nuclear DNA fragmentation.

A mouse monoclonal antibody with HLA-DR4 associated specificity.

A mouse monoclonal antibody is described which reacts with HLA-DR4 positive cells. Gel analysis of the immunoprecipitated antigen is consistent with it being a DR molecule.

An association between replicating adenovirus DNA and the nuclear matrix of infected HeLa cells.

An association between newly synthesized human adenovirus type 5 DNA and the nuclear matrix of infected HeLa cells is described. Adenovirus-infected cells were pulsed labeled with [3H]thymidine late in infection and the nuclear matrix was prepared. After a 1-min pulse more than 95% of the labeled viral DNA was matrix associated and, when compared with total cell DNA, was resistant to DNase I digestion. When the pulse is longer or is followed by a chase period, the viral DNA remains nuclear matrix associated and less nuclease sensitive than bulk cellular DNA. The resistance to nuclease digestion may result from the close association of viral DNA with the nuclear matrix or could be due to a number of viral-specific proteins which are nuclear matrix associated. It is concluded that viral DNA synthesis occurs in association with the nuclear matrix and the newly synthesized DNA remains matrix associated until it is incorporated into a mature virus particle.

Adenovirus DNA is associated with the nuclear matrix of infected cells.

Viral DNA was found to be tightly associated with the nuclear matrix from HeLa cells lytically infected with human adenovirus type 5. The bound viral DNA, like cell DNA, was resistant to nonionic detergent and to extraction with high-salt (2 M NaCl) solution. However, whereas over 95% of the cell DNA was recovered in the matrix fraction, the amount of associated viral DNA varied during infection. Throughout the lytic cycle, the amount of matrix-associated adenovirus type 5 DNA increased until it reached a plateau level at 20 to 24 h after infection. At this stage, the matrix-bound DNA represented 87% of the total viral DNA; after this stage, additional newly synthesized viral DNA accumulated as non-matrix-associated DNA. DNase digestion studies revealed that all viral DNA sequences were equally represented in the matrix-bound DNA both early and late in infection; thus, unlike cell DNA, there seem to be no preferred attachment sites on the viral genome. An enrichment of viral DNA relative to cell DNA was found in the matrix-associated DNA after extensive DNase I digestion. This finding, together with an in situ hybridization study, suggests that the viral DNA is more intimately associated with the nuclear matrix than is cell DNA and probably does not exist in extended loops.

Replication and interaction of virus DNA and cellular DNA in mouse cells infected by a human adenovirus.

C57 black mouse cells infected with human adenovirus type 5 (Ad5) produced large amounts of early virus proteins, small amounts of late virus proteins and less than 0.2 infectious units (i.u.)/cell of infectious virus. Many cells died but the cultures recovered. Virus DNA and cellular DNA were synthesized. Some Ad5 DNA sedimented with cell DNA in alkaline sucrose, but virus DNA was rapidly lost from the culture after recovery and none of 28 unselected cloned survivors contained detectable amounts of virus DNA or antigens. Ad5 ts36 was temperature-sensitive for virus DNA replication in mouse cells, but ts125 was detective at 32.5 degrees C as well as at 39.9 degrees C. No difference was detected in the percentage of virus DNA that sedimented in alkali with cell DNA, in mouse cells infected by Ad5 ts+, ts36 or ts125 at 32.5 or 39.9 degrees C. All parts of the virus genome were equally represented in virus DNA that sedimented with cell DNA, in mouse cells infected by Ad5 ts+ or ts36 at either temperature.

Spontaneous, mutagen-induced and adenovirus-induced anchorage independent tumorigenic variants of mouse cells.

Normal C57 Black mouse embryo cells did not form colonies in agarose, but rare variant (ar+) cells able to grow in agarose were detected. Fluctuation analysis showed that ar+ variants arose by spontaneous mutation in the cultured cells. The frequency of ar+ variants was increased by treating cells with N-methyl-N'nitro-N-nitrosoguanidine or ethyl methane sulphonate, or by abortive infection by human adenovirus type 5. Induced ar+ cells were fibroblastic; most grew slowly and had slightly reduced saturation density and increased serum requirement, but formed colonies in agarose. Fourteen of twenty ar+ clones induced by Ad5 were T antigen negative and two of these were also negative when tested for viral DNA. Six clones contained a few cells that were T antigen positive when first tested, but were negative when retested later. The ar+ variants were tumorigenic in athymic and in normal syngeneic mice. The results suggest that the ar+ phenotype can arise by spontaneous or chemically-induced mutation, and can be induced by adenovirus by a process different from classical transformation.

Some adenovirus DNA is associated with the DNA of permissive cells during productive or restricted growth.

We have investigated the association of viral DNA with cell DNA in chicken embryo kidney (CEK) cells productively infected with chicken embryo lethal orphan (CELO) virus and in human (HEK) cells infected with mutants ts36 and ts125 of human adenovirus type 5 under permissive and restrictive conditions. Cell and viral DNA molecules were separated after CELO virus infection of CEK cells by alkaline sucrose gradient centrifugation, network formation, and CsCl density gradient centrifugation, methods that rely on different properties of the DNA. The cell DNA was then tested for viral sequences by DNA reannealing kinetics. Between 500 and 1,000 viral genome equivalents per cell were found at 36 h postinfection associated with cell DNA purified by each method. These values greatly exceeded the amount of free viral DNA found contaminating cell DNA prepared by the same methods from uninfected cells to which CELO virus DNA had been added. Quantitative agreement in the amounts of viral DNA found associated with cell DNA purified by these different methods suggests that CELO virus DNA is integrated into chick cell DNA during lytic infection. Similar experiments in HEK cells using mutants ts36 and ts125 of adenovirus type 5 at both restrictive and permissive temperatures showed that the same proportion of viral DNA is associated with cell DNA in the absence of viral DNA replication, and this suggests that the difference in the frequency with which cells are transformed by these mutants is not due to a difference in the frequency integration.

Characterization of the DNA from bacteriophage P2-186 hybrids and physical mapping of the 186 chromosome.

The DNA from two P2-186 hybrid phages and three 186 Insertion mutants have been characterized by heteroduplex analysis and denaturation mapping. The results allow the orientation of the physical and genetic maps of bacteriophage 186 DNA and put physical limits on the chromosomal locations of the phage attachment sites, immunity genes and tail genees.

Physical mapping of bacteriophage P2 mutations and their relation to the genetic map.

Three new deletion mutants and an insertion mutant of E. coli bacteriophage P2, del2, vir79, del4 and sig5, were mapped by the electron microscope heteroduplex method. The deletions were found to cover 45.5-51.6%, 75.6-76.7% and 92.3-99.3% respectively of P2 DNA while sig5 represented a 3.7% insertion at 78.6% from the left end. The region covering 75.9-76.7% of P2 DNA is also deleted in the two previously characterized immunity insensitive variants of P2, vir22 and Hy dis. This region may identify the portion of the genome responsible for immunity. The physical and genetic maps of P2 were previously found to be colinear with respect to the two mutations vir22 and vir37. This relationship is confirmed by the position of del2.