cytidylyl-3--5--guanosine and Lymphoma

Codelivery nanovaccines of antigens and adjuvants have achieved positive therapy for cancer immunotherapy. The insufficient immunogenicity of these vaccines leads to the difficulty of eliciting robust immune effects for immune clearance due to the inadequate loading efficiency, complex preparation processes, low safety concerns, and weak immune responses. Herein, a visible codelivery nanovaccine of an antigen and adjuvant based on self-cross-linked antigen nanoparticles (ovalbumin nanoparticles (ONPs)) combined with the adjuvant (CpG) for cancer immunotherapy was prepared using antigens themselves as carriers. ONPs not only provide sufficient antigens for continuous simulation of the immune response with high antigen loading efficiency but also serve as natural carriers of CpG. In vitro and in vivo experiments proved that ONPs-CpG can elicit a robust immune response including DC maturity, T cell activation, and IFN-γ production. ONPs-CpG induced strong tumor-specific immunity and exhibited remarkable antitumor immunotherapy effects in vivo using mouse models of lymphoma. Furthermore, to perform the precise vaccine delivery, the dual fluorescent codelivery nanovaccine was monitored in real time in vivo by the visible imaging method. With regard to migration tracking, fluorescence imaging allowed for both high resolution and sensitivity of visible detection based on the fluorescence of ONPs and CpG. The multifunctional nanovaccine could function as a robust platform for cancer immunotherapy and a visible system for antigen-adjuvant tracking.

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; Cells, Cultured; Dinucleoside Phosphates; Disease Models, Animal; Drug Delivery Systems; Fluorescent Dyes; Immunotherapy; Lymphoma; Mice; Nanoparticles; Optical Imaging; Ovalbumin; Particle Size

Methyl-CpG binding domain (MBD) proteins have been shown to couple DNA methylation to transcriptional repression. This biological property suggests a role for MBD proteins in the silencing of tumor suppressor genes that are hypermethylated at their promoter CpG islands in cancer cells. Despite the demonstration of the presence of MBDs in the methylated promoter of several genes, we still ignore how general and specific is this association. Here, we investigate the profile of MBD occupancy in a large panel of tumor suppressor gene promoters and cancer cell lines. Our study shows that most hypermethylated promoters are occupied by MBD proteins, whereas unmethylated promoters are generally devoid of MBDs, with the exception of MBD1. Treatment of cancer cells with the demethylating agent 5-aza-2'-deoxycytidine results in CpG island hypomethylation, MBD release, and gene reexpression, reinforcing the notion that association of MBDs with methylated promoters is methylation-dependent. Whereas several promoters are highly specific in recruiting a particular set of MBDs, other promoters seem to be less exclusive. Our results indicate that MBDs have a great affinity in vivo for binding hypermethylated promoter CpG islands of tumor suppressor genes, with a specific profile of MBD occupancy that it is gene and tumor type specific.

Topics: Binding Sites; Blotting, Western; Cell Line, Tumor; Dinucleoside Phosphates; DNA Methylation; DNA-Binding Proteins; Genes, Tumor Suppressor; HeLa Cells; Humans; Lung Neoplasms; Lymphoma; Neoplasms; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction

The frequent occurrence of human mutations at CpG dinucleotide sites has been attributed to cytosine methylation and hydrolytic deamination of the resulting 5-methylcytosine residue. Previously, we reported an unusually strong hotspot for spontaneous transitions at a CpG site in the gene for regulatory (R) subunit of protein kinase A in S49 mouse lymphoma cells. Now, using polymerase chain reaction-based methods to screen mutant populations for mutations at particular CpG sites, we show that two methylated CpG sites in the gene for hypoxanthine phosphoribosyltransferase are much less mutable than the R subunit hotspot site, suggesting that different methylated CpG sites are differentially susceptible to spontaneous mutation. We also present data on spontaneous R subunit mutations in cloned populations of 5-azacytidine-treated S49 cells that had been demethylated at the hotspot site in both R subunit alleles. Of 13 independent mutants isolated from populations grown from fully demethylated cells, seven had the hotspot mutation. We conclude that CG-->TA mutations at strong CpG hotspots do not require prior methylation of CpG sites.

Topics: 5-Methylcytosine; Animals; Arginine; Azacitidine; Cytosine; Dinucleoside Phosphates; DNA Methylation; DNA, Neoplasm; Hypoxanthine Phosphoribosyltransferase; Lymphoma; Mice; Mutagenesis; Tumor Cells, Cultured

Mutants of S49 mouse lymphoma cells resistant to cytolysis by analogs of cyclic AMP (cAMP) generally have missense mutations in the gene encoding the regulatory subunit of cAMP-dependent protein kinase. We have compared the mutations in 95 spontaneous isolates with those in 60 mutagen-induced isolates by sequence analysis of amplified cDNAs. Twenty-nine single basepair substitutions in 19 codons produced selectable phenotypes. The spontaneous mutant spectrum was dominated by a CpG transition hotspot in the codon for Arg334. This and other nearby CpG sites were found to be methylated in genomic S49 cell DNA by restriction enzyme analyses. Most of the remaining spontaneous mutants had either G-C-->C-G or T-A-->G-C transversions, which have been associated with damage caused by oxygen radicals. In contrast, the majority of mutants induced with the alkylating mutagens ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine had G-C-->A-T mutations at non-CpG sites; in addition, EMS induced several A-T-->G-C, A-T-->T-A, and G-C-->T-A substitutions. A single ICR191-induced mutant analyzed had a unique A-T-->G-C lesion. A number of spontaneous and mutagen-induced isolates had closely linked double or triple substitutions, and two isolates had tandem triple substitutions.

Topics: Alkylating Agents; Animals; Base Sequence; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dinucleoside Phosphates; Drug Resistance; Lymphoma; Methylation; Mice; Molecular Sequence Data; Mutagens; Point Mutation; Tumor Cells, Cultured