cytidylyl-3--5--guanosine and Adenocarcinoma

Immunostimulatory CpG DNA was self-assembled to form DNA hydrogels for use as a sustained delivery system for both intercalated doxorubicin (DXR) and immunostimulatory CpG motifs for cancer treatment. X-shaped DNA (X-DNA) was designed as a building unit, and underwent ligation to form DNA hydrogels. Two types of X-DNA were constructed using four oligodeoxynucleotides each, one containing six potent CpG motifs (CpG X-DNA) and the other with none (CpG-free X-DNA). CpG X-DNA was more effective than its components or the CpG-free counterpart in terms of the production of tumor necrosis factor-α from murine macrophage-like RAW264.7 cells, as well as maturation of the murine dendritic DC2.4 cells. The cytotoxic effects of X-DNA, DXR and their complexes were examined in a co-culture system of colon26/Luc cells, a murine adenocarcinoma clone stably expressing firefly luciferase, and RAW264.7 cells. DXR/CpG X-DNA showed the highest ability to inhibit the proliferation of colon26/Luc cells. DXR was slowly released from CpG DNA hydrogels. Injections of DXR/CpG DNA hydrogels into a subcutaneous colon26 tumor effectively inhibited tumor growth. These results show that CpG DNA hydrogels are an effective sustained system for delivery of immunostimulatory signals to TLR9-positive immune cells and DXR to cancer cells.

Topics: Adenocarcinoma; Animals; Cell Line; Cell Line, Tumor; Dinucleoside Phosphates; DNA; Doxorubicin; Drug Carriers; Hydrogels; Immunization; Luciferases, Firefly; Macrophages; Male; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Microscopy, Electron, Scanning; Tumor Necrosis Factor-alpha

Aberrant methylation of 5' CpG islands is thought to play an important role in the inactivation of tumor suppressor genes in several types of cancers. In non-small cell lung cancer (NSCLC), several genes are known to be frequently methylated and the correlation of their methylation with clinical features has been studied. We determined the methylation of p16, CDH13 and RAR-beta which were reported to be methylated frequently in NSCLCs and HPP-1 which was known to be methylated in other types of cancers. The correlation between methylation and clinicopathological features were examined. The frequencies of methylation in NSCLCs were 20% for p16, 37% for CDH13, 34% for RAR-beta, and 13% for HPP1. The methylation of p16 is correlated with smoking history and methylation of HPP1 was significantly more frequent in adenocarcinomas than in squamous cell carcinomas. This is the first description of aberrant methylation of the HPP1 gene in lung cancers and our data support the previous reports on methylation in NSCLCs and association with smoking.

Topics: Adenocarcinoma; Aged; Base Sequence; Biomarkers, Tumor; Cadherins; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cyclin-Dependent Kinase Inhibitor p16; Dinucleoside Phosphates; DNA Methylation; DNA Primers; DNA, Neoplasm; Female; Genes, Tumor Suppressor; Humans; Lung Neoplasms; Male; Membrane Proteins; Middle Aged; Neoplasm Proteins; Receptors, Retinoic Acid

Cell cycle progression is monitored by checkpoint mechanisms to ensure the integrity of the genome and the fidelity of sister chromatid separation. Failure of such checkpoint functions results in genomic instability, a condition that predisposes cells to neoplastic transformation and tumor progression. Recently, Scolnick and Halazonetis defined a new mitotic checkpoint that acts at prophase and delays chromosome condensation in response to mitotic stress, and identified a gene, named checkpoint with FHA and ring finger (Chfr), that seems to be required for delaying prophase in human cells. In the present study, we examined human Chfr mRNA expression in 15 human esophageal cancer cell lines and 43 primary esophageal cancers to investigate the potential involvement of Chfr in the pathogenesis of esophageal cancers. We report here that a significant proportion of human esophageal cancer has loss of expression of Chfr gene. Furthermore, we found aberrant hypermethylation of the promoter region of this checkpoint gene in four of 15 (26.7%) esophageal cancer cell lines and in seven of 43 (16.3%) primary cancers.

Topics: Adenocarcinoma; Aged; Base Sequence; Carcinoma, Squamous Cell; Cell Cycle Proteins; Dinucleoside Phosphates; DNA Methylation; DNA Primers; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Proteins; Poly-ADP-Ribose Binding Proteins; Polymerase Chain Reaction; Tumor Cells, Cultured; Ubiquitin-Protein Ligases

Methylation of 5' CpG islands in promoter and upstream coding regions has been identified as a mechanism for transcriptional inactivation of tumor suppressor genes. The purpose of this study was to determine whether hypermethylation of the adenomatous polyposis coli (APC) gene promoter occurs in primary non-small cell lung cancer (NSCLC), and whether hypermethylated APC has any relationship with survival. APC promoter 1A methylation was determined in normal and corresponding tumor tissue from 91 NSCLC patients and in a control group of 10 patients without cancer, using a quantitative fluorogenic real-time PCR (Taqman) system. APC promoter methylation was detectable in 86 (95%) of 91 tumor samples, but also in 80 (88%) of 91 normal samples of NSCLC patients, and in only two (20%) of 10 normal lung tissues of the control group. The median level of APC promoter methylation was 4.75 in tumor compared to 1.57 in normal lung tissue (P<0.001). Patients with low methylation status showed significantly longer survival than did patients with high methylation status (P=0.041). In a multivariate analysis of prognostic factors, APC methylation was a significant independent prognostic factor (P=0.044), as were pT (P=0.050) and pN (P<0.001) classifications. This investigation shows that APC gene promoter methylation occurs in the majority of primary NSCLCs. High APC promoter methylation is significantly associated with inferior survival, showing promise as a biomarker of biologically aggressive disease in NSCLC.

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Aged, 80 and over; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Dinucleoside Phosphates; DNA Methylation; DNA, Neoplasm; Female; Follow-Up Studies; Genes, APC; Humans; Lung; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Multivariate Analysis; Neoplasm Staging; Promoter Regions, Genetic; Survival Rate; Time Factors

CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.

Topics: Adenocarcinoma; Base Sequence; Brain Neoplasms; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Lobular; Colonic Neoplasms; Dinucleoside Phosphates; DNA Methylation; Female; Genome, Human; Humans; Male; Molecular Sequence Data; Neoplasms; Restriction Mapping

Hypermethylation of CpG islands is a common mechanism by which tumor suppressor genes are inactivated. We studied 45 pancreatic carcinomas and 14 normal pancreata for aberrant DNA methylation of CpG islands of multiple genes and clones using methylation-specific PCR (MSP) and bisulfite-modified sequencing. Using MSP, we detected aberrant methylation of at least one locus in 60% of carcinomas. The genes analyzed included RARbeta (methylated in 20%), p16 (18%), CACNA1G (16%), TIMP-3 (11%), E-cad (7%), THBS1 (7%), hMLH1 (4%), DAP kinase (2%), and MGMT (0%). In addition, aberrant methylation was found in three CpG islands (MINT31, -1, and -2) in 38, 38, and 14% of carcinomas, respectively. Hypermethylation was largely confined to the carcinomas with only three loci (E-cad, DAP kinase, and MINT2) harboring methylation in some normal pancreata (36, 21, and 14%, respectively). Simultaneous methylation of at least four loci was observed in 5 of 36 (14%) pancreatic adenocarcinomas. We defined this subgroup of pancreatic adenocarcinomas as "CpG island-methylator-phenotype positive (CIMP+)." Two of four carcinomas with microsatellite instability harbored promoter hypermethylation of hMLH1, and both cases were CIMP+. Thus, we conclude that many pancreatic carcinomas hypermethylate a small percentage of genes, whereas a subset displays a CIMP+ phenotype.

Topics: Adenocarcinoma; Apoptosis Regulatory Proteins; Calcium Channels, T-Type; Calcium-Calmodulin-Dependent Protein Kinases; Death-Associated Protein Kinases; Dinucleoside Phosphates; DNA Methylation; Humans; O(6)-Methylguanine-DNA Methyltransferase; Pancreas; Pancreatic Neoplasms; Promoter Regions, Genetic; Receptors, Retinoic Acid; Thrombospondins; Tissue Inhibitor of Metalloproteinase-3; Transplantation, Heterologous