Aberrant Gene Promoter Methylation Associated with Sporadic Multiple Colorectal Cancer

Background

Colorectal cancer (CRC) multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect.

Methodology/Principal Findings

We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals) and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2), RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008) and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047) as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006). Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17), SFRP1 (r = 0.83, 0.06), HPP1 (r = 0.64, p = 0.17), 3OST2 (r = 0.83, p = 0.06) and GATA4 (r = 0.6, p = 0.24). Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant difference in any evaluated gene.

Conclusions

These results provide a proof-of-concept that gene promoter methylation is associated with tumor multiplicity. This underlying epigenetic defect may have noteworthy implications in the prevention of patients with sporadic CRC.     

Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.     

Monoclonal antibodies specific for type 3 and type 4 chain-based blood group determinants: Relationship to the A1 and A2 subgroups

Two monoclonal antibodies, specific for A type 3 and A type 4 blood group determinants, are described. These antibodies recognized A1 but not A2 erythrocytes. A third monoclonal antibody showing specificity for A type 3 and A type 4, and also for H type 3 and H type 4, did not discriminate between A1 and A2 erythrocytes. On red cells these three antibodies recognized glycosphingolipids and binding to glycoproteins could not be demonstrated. On paraffin-embedded tissue sections the three antibodies labelled a supranuclear area, characteristic of the Golgi apparatus, of all cells producing A antigens. This labelling occurred irrespective of the A1, A2 status.The results suggest that glycolipids of erythrocytes and possibly of other cell types bear the A type 3/4 determinant specific for the A1 subgroup and that A type 3/4 determinants of glycoproteins might be present in both A1 and A2 subgroups on short oligosaccharide chains which are only detectable at the level of the Golgi apparatus.     

Expression of ABH and X (Lex) antigens in various cells

Using a panel of reagents specific to the various subtypes of ABH antigens, it could be demonstrated that platelets carry ABH type 2 monofucosylated determinants on intrinsic glycoproteins. The presence of these antigens is controlled by the H gene and correlates with the presence of alpha-2-L-fucosyltransferase and the absence of alpha-3-L-fucosyltransferase. In contrast, intrinsic ABH antigens were not found on mononuclear cells, correlating with the absence of alpha-2-L-fucosyltransferase on these cells. However, after transformation with the Epstein-Barr virus and stimulation with 12-O-tetradecanoylphorbol-13-O-acetate (TPA), B lymphocytes were found to express the H antigen under control of the H gene and not the Se gene. The lymphoblastoid cell lines also expressed the X and sialylated X antigens which are normally markers of the myeloid lineage. These antigens are also normally found in epithelial cells of the digestive tract, kidney proximal convoluted tubules and hepatocytes. The alpha-3-L-fucosyltransferase responsible for the synthesis of this antigen is present in the serum but we report the existence of two individuals, a mother and her daughter, who lack more than 90% of this serum enzyme. The young girl suffers from a congenital kidney anomaly: oligomeganephronic hypoplasia. Her kidney tubules are devoid of X antigen. However, she and her mother have the X antigen on their granulocytes and its sialylated form on their monocytes. It therefore appears that there are distinct genetic controls for the expression of antigen X in different body compartments. This would be quite similar to the H and Se gene controls in tissues of distinct embryological origins.     

Distribution of H type 1 and H type 2 antigenic determinants in human sera and saliva.

A radioimmunoassay specific for the H type 1 antigenic determinant demonstrated that the H type 1 antigen is under the strict control of the Se gene in both serum and saliva. Similar amounts of H type 1 antigenic determinants were found in saliva from Se/-, le/le donors and in saliva from Se/-, Le/- donors. However, sera from Se/-, le/le donors were about 100 times more efficient in inhibiting the H type 1 assay than were sera from Se/-, Le/- donors. A radioimmunoassay, based on the binding of Ulex europaeus with the H type 2 antigenic determinant, showed that all the H type 2 antigen in saliva is under the control of the Se gene, while only one-third of the H type 2 antigen present in serum is under the control of this gene. The remaining two-thirds of H type 2 antigen in sera is independent of the ABH secretor status of the donor. The amount of H type 2 antigen in both serum and saliva is independent of the Le gene. These results are compatible with the existence of two alpha (1 leads to 2) fucosyl-transferases but suggest that the enzyme of epithelial origin, coded by the Se gene, should be able to transform both type 1 and type 2 natural substrates, while the enzyme of mesodermic origin, coded by the H gene, would work preferentially on the natural type 2 substrates.     

Recognition of the blood group H type 2 trisaccharide epitope by 28 monoclonal antibodies and three lectins

The patterns of cross-reaction of 30 monoclonal antibodies and three lectins were determined by ELISA with 21 ABH, Ii or Lewis related synthetic oligosaccharides coupled to bovine serum albumin. At least seven main groups of cross-reactive patterns were identified among the antibodies, plus several intermediate patterns between two of the main antibody groups. The three lectins had different cross-reaction patterns,Galactia tenuiflora was different from all the antibodies,Ulex europaeus lectin 1 andLotus tetragonolobus were similar, but not identical to groups III and V of antibodies respectively. The anti-H antibodies cross-reacting with A type 2 gave similar agglutination scores with all the normal ABO erythrocytes, while the anti-H antibodies not cross-reacting with A type 2 reacted with different scores: O>A₂>A₂B>B>A₁>A₁B>O_h, suggesting that these antibodies react better with the free H epitopes and do not recognize the H in A or B epitopes. Based on the ELISA and agglutination results and the lowest energy conformations of each oligosaccharide obtained by computer modelling, the most probable oligosaccharide surface areas recognized by each antibody main group are illustrated.     

A comparison of altitudinal species richness patterns of bryophytes with other plant groups in Nepal, Central Himalaya

Aim To explore species richness patterns in liverworts and mosses along a central Himalayan altitudinal gradient in Nepal (100–5500 m a.s.l.) and to compare these patterns with patterns observed for ferns and flowering plants. We also evaluate the potential importance of Rapoport’s elevational rule in explaining the observed richness patterns for liverworts and mosses. Location Nepal, Central Himalaya. Methods We used published data on the altitudinal ranges of over 840 Nepalese mosses and liverworts to interpolate presence between maximum and minimum recorded elevations, thereby giving estimates of species richness for 100‐m altitudinal bands. These were compared with previously published patterns for ferns and flowering plants, derived in the same way. Rapoport’s elevational rule was assessed by correlation analyses and the statistical significance of the observed correlations was evaluated by Monte Carlo simulations. Results There are strong correlations between richness of the four groups of plants. A humped, unimodal relationship between species richness and altitude was observed for both liverworts and mosses, with maximum richness at 2800 m and 2500 m, respectively. These peaks contrast with the richness peak of ferns at 1900 m and of vascular plants, which have a plateau in species richness between 1500 and 2500 m. Endemic liverworts have their maximum richness at 3300 m, whereas non‐endemic liverworts show their maximum richness at 2700 m. The proportion of endemic species is highest at about 4250 m. There is no support from Nepalese mosses for Rapoport’s elevational rule. Despite a high correlation between altitude and elevational range for Nepalese liverworts, results from null simulation models suggest that no clear conclusions can be made about whether liverworts support Rapoport’s elevational rule. Main conclusions Different demands for climatic variables such as available energy and water may be the main reason for the differences between the observed patterns for the four plant groups. The mid‐domain effect may explain part of the observed pattern in moss and liverwort richness but it probably only works as a modifier of the main underlying relationship between climate and species richness.