An improved PCR-based amplification of unknown homologous DNA sequences

The PCR primers used for cloning of evolutionary conserved genes or homologous DNA sequences are usually guessmer oligonucleotides. We introduce a simple way using Pfu polymerase to overcome possible PCR amplification failure because of 3'-end mismatches of guessed primers with the target DNA.     

An Integrative CGH,MSI and Candidate Genes Methylation Analysis of Colorectal Tumors

Background

Different DNA aberrations processes can cause colorectal cancer (CRC). Herein, we conducted a comprehensive molecular characterization of 27 CRCs from Iranian patients.

Materials and Methods

Array CGH was performed. The MSI phenotype and the methylation status of 15 genes was established using MSP. The CGH data was compared to two established lists of 41 and 68 cancer genes, respectively, and to CGH data from African Americans. A maximum parsimony cladogram based on global aberrations was established.

Results

The number of aberrations seem to depend on the MSI status. MSI-H tumors displayed the lowest number of aberrations. MSP revealed that most markers were methylated, except RNF182 gene. P16 and MLH1 genes were primarily methylated in MSI-H tumors. Seven markers with moderate to high frequency of methylation (SYNE1, MMP2, CD109, EVL, RET, LGR and PTPRD) had very low levels of chromosomal aberrations. All chromosomes were targeted by aberrations with deletions more frequent than amplifications. The most amplified markers were CD248, ERCC6, ERGIC3, GNAS, MMP2, NF1, P2RX7, SFRS6, SLC29A1 and TBX22. Most deletions were noted for ADAM29, CHL1, CSMD3, FBXW7, GALNS, MMP2, NF1, PRKD1, SMAD4 and TP53. Aberrations targeting chromosome X were primarily amplifications in male patients and deletions in female patients. A finding similar to what we reported for African American CRC patients.

Conclusion

This first comprehensive analysis of CRC Iranian tumors reveals a high MSI rate. The MSI tumors displayed the lowest level of chromosomal aberrations but high frequency of methylation. The MSI-L were predominantly targeted with chromosomal instability in a way similar to the MSS tumors. The global chromosomal aberration profiles showed many similarities with other populations but also differences that might allow a better understanding of CRC''s clinico-pathological specifics in this population.     

Epigenetic and genetic analysis of WNT signaling pathway in sporadic colorectal cancer patients from Iran

The WNT signaling is deregulated in most human colorectal cancers (CRC). Promoter methylation has been proposed as an alternative mechanism to inactivate genes in tumors. To gain insight into the methylation silencing of the WNT pathway during colorectal carcinogenesis, we examined the aberrant methylation profile of four genes, APC, Axin1, Axin2, and GSK3β in an unselected series of 112 sporadic colorectal tumors by methylation specific PCR. It has been suggested that the Axin2 C148T SNP is associated with the risk of developing certain types of cancers. To assess the contribution of Axin2 SNP to CRC susceptibility, we examined the Axin2 C148T genotype in CRC patients and 170 healthy controls by PCR-RFLP. The frequency of CRCs with at least one gene methylated was 18.75%. Promoter methylation of Axin2 and APC genes was detected in 7.1 and 11.9% of tumors, respectively. No aberrant methylation was found in Gsk3β and Axin1 gene in these tumor series. The methylation status of APC had no significant association with clinical parameters. But, promoter methylation of Axin2 was sex-related, occurring more frequently in females (P = 0.002). The frequency of Axin2 C148T genotypes were similar in patients and controls. Moreover, we observed no association between the Axin2 SNP and risk of CRC in patients stratified by age, sex, and smoking status. However, the heterozygote CT genotype was associated with a reduced CRC risk in distal patients compared with proximal patients (OR = 0.3; 95% CI 0.1–0.9, P = 0.04). Our findings indicate that Axin1 and GSK3β methylation play a minor role in colorectal carcinogenesis.     

Evolution of a tumorigenic property conferred by glycophosphatidyl-inositol membrane anchors of carcinoembryonic antigen gene family members during the primate radiation

GPI membrane anchors of cell surface glycoproteins have been shown to confer functional properties that are different from their transmembrane (TM)-anchored counterparts. For the human carcinoembryonic antigen (CEA) family, a subfamily of the immunoglobulin superfamily, conversion of the mode of membrane linkage from TM to GPI confers radical changes in function: from tumor suppression or neutrality toward inhibition of differentiation and anoikis and distortion of tissue architecture, thereby contributing to tumorigenesis. We show here that GPI anchorage in the CEA family evolved twice independently in primates, very likely from more primitive TM anchors, by different packages of mutations. Both mutational packages, one package found in many primates, including humans, and a second, novel package found only in the Cebidae radiation of New World monkeys, give rise to efficiently processed GPI-linked proteins. Both types of GPI anchors mediate inhibition of cell differentiation. The estimated rate of nonsynonymous mutations (Ka) in the anchor-determining domain for conversion from TM to GPI anchorage in the CEA family that were fixed during evolution in these primates is 7 times higher than the average Ka in primates, indicating positive selection. These results suggest therefore that the functional changes mediated by CEA GPI anchors, including the inhibition of differentiation and anoikis, could be adaptive and advantageous.     

Recombinant production of native human α-1-antitrypsin protein in the liver HepG2 cells

Objectives

Alpha-1 antitrypsin (A1AT) deficiency is associated with emphysema and liver disease. Only plasma-derived A1AT protein is available for augmentation therapy. Recombinant A1AT (recA1AT) protein expressed in various types of available hosts are either non-glycosylated or aberrantly glycosylated resulting into reduced stability and biological activity. To overcome these limitations, we have used the human liver HepG2 cell line to produce recA1AT protein.

Results

HepG2 cells were transfected by A1AT cDNA and cell populations were generated that stably overexpressed A1AT protein. Real-time RT-PCR and rocket immunoelectrophoresis of cell culture supernatants indicated that the transfection resulted more than two-fold increase in A1AT production compared to that of control parental cells. Immunoblot analysis showed that both plasma and HepG2-produced A1AT proteins have identical molecular weight in either glycosylated or deglycosylated form. Partial digestion with PNGase F indicated that the three N-glycosylation sites of recA1AT, like the native A1AT protein in plasma, are occupied. Recombinant A1AT also like the native A1AT was thermostable and could efficiently inhibit trypsin proteolytic activity against BSA and BAPNA chromogenic substrate. The recombinant HepG2 cells cultured in media containing B27 serum free supplement released recA1AT at the same level as in the serum containing media.

Conclusions

RecA1AT production in HepG2 cells grown under serum free condition at a large scale could provide a reliable source of the native protein suitable for therapeutic use in human.

     

Distinct High-Profile Methylated Genes in Colorectal Cancer

Background

Mutations and promoters'' methylation of a set of candidate cancer genes (CAN genes) are associated with progression of colorectal cancer (CRC). We hypothesized that these genes'' promoters are inactivated through epigenetic silencing and may show a different profile in high-risk populations. We investigated the status of CAN gene methylation and CHD5 protein expression in African American CRC tissue microarrays (TMA) using immunohistochemical staining.

Methodology/Principal Findings

The promoter methylation status of the CAN genes was studied by methylation-specific PCR (MSP) in 51 Iranians (a white population) and 51 African Americans (AA). Microsatellite instability (MSI) was analyzed as well. The differential frequency of methylation for each gene was tested by chi-square analysis between the two groups based on matched age and sex. CHD5 protein expression was evaluated in moderate to well differentiated and poorly differentiated carcinomas compared to matched normal tissue using TMA. In addition, the correlation between these epigenetic biomarkers and various clinicopathological factors, including, age, location, and stage of the disease were analyzed.Seventy-seven and 34% of tumors were distal in Iranian and African American patients, respectively. In both populations, the percentage of methylation was >65% for SYNE1, MMP2, APC2, GPNMB, EVL, PTPRD, and STARD8, whereas methylation was <50% for LGR6, RET, CD109, and RNF. The difference in methylation between the two populations was statistically significant for CHD5, ICAM5 and GPNMB. Thirty-one percent AA tumors showed MSI-H, compared to 28% in Iranians.

Conclusions/Significance

A significantly higher methylation rate was found for GPNMB, ICAM5, and CHD5 genes in AA patients compared to Iranians. These genes might play a role in the high incidence and aggressiveness of CRC in the AA population. The hypermethylation of the CAN genes can be considered as a marker of colon carcinogenesis.     

Effect of Oral Resveratrol on the BDNF Gene Expression in the Hippocampus of the Rat Brain

Resveratrol is a plant polyphenolic compound. Evidence indicates that resveratrol has beneficial effects against aging and neurodegenerative diseases. The goal of our study was in vivo examination of the effects of resveratrol on the abundance of mRNA encoding Brain Derived Neurotrophic Factor (BDNF) in the hippocampus of rat brain. Rats were administrated orally by different doses (2.5–20 mg/kg bwt) of resveratrol for 3, 10 and 30 days. Saline was used as control and 10% ethanol in saline was used as vehicle for resveratrol. Measurement of BDNF mRNA by Real-time RT–PCR showed that levels of the mRNA for BDNF were significantly and dose dependently elevated in the hippocampal tissues of rats. The findings suggest that the neuroprotective effects of resveratrol may be at least partly due to its inducing effects on the expression levels of the BDNF mRNA.     

Carcinoembryonic antigen expression level as a predictive factor for response to 5-fluorouracil in colorectal cancer

Carcinoembryonic antigen (CEA) expression has been shown to protect cancer cell lines from apoptosis and anoikis. The aim of this study was to further elucidate the role of CEA expression on resistance to anticancer drugs in human colorectal cancer (CRC). We transfected CEA negative CRC cell line SW742 as well as CHO cells to overexpress CEA and their chemoresistance were assessed by MTT assay. In comparison to the parental cell lines, transfected cells had significantly increased resistance to 5-fluorouracil (5-FU). The results also showed a direct correlation between the amount of cellular CEA protein and 5-FU resistance in CEA expressing cells. We found no significant difference in sensitivity to cisplatin and methotrexate between CEA-transfected cells and their counter parental cells. We also compared the association between CEA expression and chemoresistance of 4 CRC cell lines which differed in the levels of CEA production. The CEA expression levels in monolayer cultures of these cell lines did not correlate with the 5-FU resistance. However, 5-FU treatment resulted in the selection of sub-populations of resistant cells that displayed increased CEA expression levels by increasing drug concentration. We analyzed the effect of 5-FU in a 3D multicellular culture generated from the two CRC cell lines, LS180 and HT29/219. Compared with monolayer culture, CEA production and 5-FU resistance in both cell lines were stimulated by 3D growth. In comparison to the 3D spheroids of parental CHO, we observed a significantly elevated 5-FU resistance in 3D culture of the CEA-expressing CHO transfectants. Our findings suggest that the CEA level may be a suitable biomarker for predicting tumor response to 5-FU-based chemotherapy in CRC.