A comparative analysis of methylation status of tumor suppressor genes in paired biopsy and serum samples from cervical cancer patients among north indian population

Tumor-specific genetic or epigenetic alterations have been detected in serum DNA in case of various types of cancers. In breast cancer, the detection of tumor suppressor gene hypermethylation has been reported in several body fluids. Promoter hypermethylation of some genes like MYOD1, CALCA, hTERT, etc. has also been detected in serum samples from cervical cancer. The present study is the first report on the comparison of promoter hypermethylation of tumor suppressor genes like p14, p15, p16, p21, p27, p57, p53, p73, RARβ2, FHIT, DAPK, STAT1, and RB1 genes in paired biopsy and serum samples from cervical cancer patients among north Indian population. This is also the first report on the hypermethylation of these genes in serum samples from cervical cancer patients among north Indian population. According to the results of the present study, promoter hypermethylation of these genes can also be detected in serum samples of cervical cancer patients. The sensitivity of detection of promoter hypermethylalion in serum samples of cervical cancer patients as compared to paired biopsy samples was found to be around 83.3%. It was observed that promoter hypermethylation was mainly observed in the serum samples in the higher stages and very rarely in the lower stages. The present study clearly showed that serum of patients with cervical cancer can also be used to study methylated genes as biomarkers.     

Methylation of the <Emphasis Type="Italic">RASSF1A</Emphasis> promoter region and the allelic imbalance frequencies in chromosome 3 critical regions correlate with progression of clear cell renal carcinoma

The short arm of chromosome 3 (3p) contains several critical regions that have increased frequencies of allelic deletions and harbor a set of tumor suppressor genes. In particular, the range of functions performed by RASSF1A (LUCA region, 3p21.31) includes those potentially associated with carcinogenesis. Among 3p genes, RASSF1A has the highest methylation frequency in epithelial tumors of various locations. For the first time, two different methods (methylation-specific PCR and methylation-sensitive restriction analysis) independently showed that the methylation level of the CpG island in the RASSF1A promoter region significantly correlated with grade and clinical stage of clear cell renal cell carcinoma (RCC). An analysis of 23 3p polymorphic markers in a representative set of 80 RCC cases characterized clinically and histologically revealed that RCC progression significantly correlated with the frequency of allelic imbalances in some critical regions of 3p (LUCA and AP20), but not in 3p as a whole. These data suggest that RCC progression is associated with the methylation of the RASSF1A promoter and, possibly, with structural and functional alterations in other 3p genes. In addition, significant correlation between RASSF1A methylation and allelic losses at the nearby polymorphic marker locus suggests the “two hit” model for the inactivation of this tumor suppressor gene in RCC.     

Methylation of the genes for the microRNAs miR-129-2 and miR-9-1, changes in their expression,and activation of their potential target genes in clear cell renal cell carcinoma

Methylation of promoter CpG islands and microRNA (miRNA) interactions with mRNAs of target genes are epigenetic mechanisms that play a crucial role in deregulation of gene expression and signaling pathways in tumors. Altered expression of six chromosome 3p genes (RARB(2), SEMA3B, RHOA, GPX1, NKIRAS1, and CHL1) and two miRNA genes (MIR-129-2 and MIR-9-1) was observed in primary clear cell renal cell carcinomas (ccRCCs, 31–48 samples) by RT-PCR and qPCR. Significant downregulation (p < 0.05, Fisher’s exact test) was observed for SEMA3B, NKIRAS1, and CHL1; and differential expression, for the other chromosome 3p and miRNA genes. Methylation-specific PCR with primers to RARB(2), SEMA3B, MIR-129-2, and MIR-9-1 showed that their methylation frequency was significantly (p < 0.05, Fisher’s exact test) elevated in the ccRCC samples. Significant correlations between promoter methylation and expression were confirmed for SEMA3B and observed for the first time for RARB(2), GPX1, and MIR-129-2 in ccRCC (Spearman’s correlation coefficient r _s ranging 0.31–0.60, p < 0.05). The MIR-129-2 and RARB(2) methylation frequencies significantly correlated with ccRCC progression. MIR-129-2 methylation correlated with upregulation of RARB(2), RHOA, NKIRAS1, and CHL1 (r _s ranging 0.35–0.53, p < 0.05). The findings implicate methylation in regulating RARB(2), SEMA3B, GPX1, and MIR-129-2 and indicate that miR-129-2 and methylation of its gene affect RARB(2), RHOA, NKIRAS1, and CHL1 expression.     

<Emphasis Type="Italic">FAT4</Emphasis> hypermethylation and grade dependent downregulation in gastric adenocarcinoma

Gastric cancer is one of the major causes of death due to cancer in the world. It is a multi-factorial disease with epigenetic factors being also involved in its development. FAT4 is a tumor suppressor gene exerting an important role in cell adhesion. This study aimed at analyzing FAT4 expression and promoter methylation in gastric cancer. FAT4 expression was studied in 30 tumoral tissues and their non-tumoral counterparts using Taqman real time PCR method. Promoter methylation was assessed using bisulfite conversion method followed by sequencing. Tumor tissues showed reduced FAT4 expression (P = 0.04). FAT4 downregulation was associated with tumor grade, with higher repression at advanced grades. Significant increase of promoter methylation was observed in tumoral tissues. Reduced expression of FAT4 and increased methylation of its promoter may be one of the effective processes in turning a healthy stomach tissue into a tumor tissue.     

Prognostic potential of <Emphasis Type="Italic">KLOTHO</Emphasis> and <Emphasis Type="Italic">SFRP1</Emphasis> promoter methylation in head and neck squamous cell carcinoma

Hypermethylation in the CpG island promoter regions of tumor suppressors is known to play a significant role in the development of HNSCC and the detection of which can aid the classification and prognosis of HNSCC. This study aims to profile the methylation patterns in a panel of key genes including CDKN2A, CDKN2B, KLOTHO (KL), RASSF1A, RARB, SLIT2, and SFRP1, in a group of HNSCC samples from Saudi Arabia. The extent of methylation in these genes is determined using the MethyLight assay and correlated with known clinicopathological parameters in our samples of 156 formalin-fixed and paraffin-embedded HNSCC tissues. SLIT2 methylation had the highest frequency (64.6%), followed by RASSF1A (41.3%), RARB (40.7%), SFRP1 (34.9), KL (30.7%), CKDN2B (29.6%), and CKDN2A (29.1%). KL and SFRP1 methylation were more predominant in nasopharyngeal tumors (P = 0.001 and P = 0.031 respectively). Kaplan Meier analysis showed that patients with moderately differentiated tumors who display SFRP1 methylation have significantly worse overall survival in comparison with other samples. In contrast, better clinical outcomes were seen in patients with KL methylation. In conclusion, our findings suggest that the detection of frequent methylation in SFRP1 and KL genes’ promoters could serve as prognostic biomarkers for HNSCC.     

<Emphasis Type="Italic">BMP3</Emphasis> promoter hypermethylation in plasma-derived cell-free DNA in colorectal cancer patients

Detecting cfDNA in plasma or serum could serve as a ‘liquid biopsy’, for circulating tumor DNA with aberrant methylation patterns offer a possible method for early detection of several cancers which could avoid the need for tumor tissue biopsies. Bone Morphogenetic Protein 3 (BMP3) was identified as a candidate tumor suppressor gene putatively down-regulated in colorectal cancer (CRC). In this study, we aimed to assess the potential role of BMP3 promoter methylation changes in plasma DNA for detection of colorectal cancerous and precancerous lesions. Plasma DNA samples were extracted from 50 patients with histologically diagnosed polyps or tumor and 50 patients reported negative for polyps or tumors. The procedure consists of bisulfite conversion of the extracted DNA, purification of bis-DNA, and BMP3 methylation status analysis by using the bisulfite specific high resolution melting analysis. This study demonstrated that there was a significantly higher frequency of BMP3 methylated DNA in plasma in patients with polyps versus healthy controls with a sensitivity and specificity of 40 and 94%, respectively. In conclusion, our results demonstrated that BMP3 DNA methylation in plasma had not have sufficient sensitivity and it should be used in combination with other biomarkers for the detection of CRC.     

Functional analysis of the <Emphasis Type="Italic">Malus domestica MdHMGR2</Emphasis> gene promoter in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The first rate-limiting enzyme of the mevalonate pathway during isoprenoid biosynthesis is 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In this study, the expression pattern of the MdHMGR2 gene in Malus domestica suggests that MdHMGR2 was expressed in a tissue-specific manner and was significantly induced by ethephon (ETH), indoleacetic acid (IAA), methyl jasmonate (MeJA), and salicylic acid (SA). The MdHMGR2 promoter was isolated, sequenced, and analyzed through bioinformatics tools, and the results suggest the presence of various putative cis-acting elements responsive to different hormones. Activity of β-glucuronidase (GUS) driven by the full length MdHMGR2 promoter and its 5′deletion fragments was detected in transgenic Arabidopsis thaliana. A strong GUS activity was observed in seedlings, roots, newly growing true leaves, anthers, and stigmas in transgenic Arabidopsis containing the full MdHMGR2 promoter. The results indicate that a region from -1050 to -827 was crucial for promoter activity. In addition, the MdHMGR2 promoter was induced in response to ETH, IAA, MeJA, and SA. The analysis suggests that an ethylene-responsive element in the region from -1050 to -1005 was required for the ethylene inducibility.     

<Emphasis Type="Italic">K-ras,BRCA1/2</Emphasis>, and <Emphasis Type="Italic">CHEK2</Emphasis> mutations and loss of heterozygosity at 9p, 17p,and 18q in sporadic adenocarcinoma of the pancreas

The diagnostic significance of molecular markers was assessed for the most common somatic aberrations at the K-ras, TP53, CDKN2A, and MADH4 loci, as well as less common mutations of BRCA1, BRCA2, and CHEK2, arising in preinvasive stages of sporadic adenocarcinoma of the pancreas. The study was performed on paired primary pancreatic adenocarcinoma and normal pancreatic tissue specimens obtained from 37 Russian patients. Surgical adenocarcinoma specimens were subjected to manual microdissection. Mutations of K-ras codon 12 were found in 24 tumor specimens (0.65), but not in normal pancreatic tissue specimens. Mutations of BRCA1 (185delAG, 300T > G, 4153delA, 4158A > G, 5382insC), BRCA2 (695insT, 6174delT), and CHEK2 (1100delC) were not found. The informativeness of allelic losses did not differ significantly among the three tumor suppressor loci and was 60% for TP53 (GDB186817) and CDKN2A (D9S974 + D9S162) and 65.7% for MADH4 (D18S363 + D18S474) (t = 0.48). The CDKN2A locus had the highest LOH frequency of 0.95. For TP53 and MADH4 the LOH frequency was 0.62 and 0.70, respectively. In 80% of adenocarcinomas, at least one locus was characterized with LOH. The overall informativeness of the combined data on K-ras mutations and loss of heterozygosity at 9p, 17p, and 18q was 85.7%. Only 9% of the tumors were characterized with microsatellite instability.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.