Background

Aberrant DNA methylation leads to loss of heterozygosity (LOH) or loss of imprinting (LOI) as the first hit during human carcinogenesis. Recently we developed a new high-throughput, high-resolution DNA methylation analysis method, bisulphite PCR-Luminex (BPL), using sperm DNA and demonstrated the effectiveness of this novel approach in rapidly identifying methylation errors.

Results

In the current study, we applied the BPL method to the analysis of DNA methylation for identification of prognostic panels of DNA methylation cancer biomarkers of imprinted genes. We found that the BPL method precisely quantified the methylation status of specific DNA regions in somatic cells. We found a higher frequency of LOI than LOH. LOI at IGF2, PEG1 and H19 were frequent alterations, with a tendency to show a more hypermethylated state. We detected changes in DNA methylation as an early event in ovarian cancer. The degree of LOI (LOH) was associated with altered DNA methylation at IGF2/H19 and PEG1.

Conclusions

The relative ease of BPL method provides a practical method for use within a clinical setting. We suggest that DNA methylation of H19 and PEG1 differentially methylated regions (DMRs) may provide novel biomarkers useful for screening, diagnosis and, potentially, for improving the clinical management of women with human ovarian cancer.

There is an increased prevalence of imprinting disorders, such as Beckwith–Wiedemann syndrome, associated with human assisted reproductive technologies (ART). Work on animal models suggests that in vitro culture may be the source of these imprinting errors. However, in this study we report that, in some cases, the errors are inherited from the father. We analyzed DNA methylation at seven autosomal imprinted loci and the XIST locus in 78 paired DNA samples. In seven out of seventeen cases where there was abnormal DNA methylation in the ART sample (41%), the identical alterations were present in the parental sperm. Furthermore, we also identified DNA sequence variations in the gene encoding DNMT3L, which were associated with the abnormal paternal DNA methylation. Both the imprinting errors and the DNA sequence variants were more prevalent in patients with oligospermia. Our data suggest that the increase in the incidence of imprinting disorders in individuals born by ART may be due, in some cases, to the use of sperm with intrinsic imprinting mutations.

There is an increased prevalence of imprinting disorders, such as Beckwith–Wiedemann syndrome, associated with human assisted reproductive technologies (ART). Work on animal models suggests that in vitro culture may be the source of these imprinting errors. However, in this study we report that, in some cases, the errors are inherited from the father. We analyzed DNA methylation at seven autosomal imprinted loci and the XIST locus in 78 paired DNA samples. In seven out of seventeen cases where there was abnormal DNA methylation in the ART sample (41%), the identical alterations were present in the parental sperm. Furthermore, we also identified DNA sequence variations in the gene encoding DNMT3L, which were associated with the abnormal paternal DNA methylation. Both the imprinting errors and the DNA sequence variants were more prevalent in patients with oligospermia. Our data suggest that the increase in the incidence of imprinting disorders in individuals born by ART may be due, in some cases, to the use of sperm with intrinsic imprinting mutations.

Melanogenesis substrate, N-propionyl-cysteaminylphenol (NPrCAP), is selectively incorporated into melanoma cells and inhibits their growth by producing cytotoxic free radicals. Magnetite nanoparticles also disintegrate cancer cells and generate heat shock protein (HSP) upon exposure to an alternating magnetic field (AMF). This study tested if a chemo-thermo-immunotherapy (CTI therapy) strategy can be developed for better management of melanoma by conjugating NPrCAP on the surface of magnetite nanoparticles (NPrCAP/M). We examined the feasibility of this approach in B16 mouse melanoma and evaluated the impact of exposure temperature, frequency, and interval on the inhibition of re-challenged melanoma growth. The therapeutic protocol against the primary transplanted tumor with or without AMF exposure once a day every other day for a total of three treatments not only inhibited the growth of the primary transplant but also prevented the growth of the secondary, re-challenge transplant. The heat-generated therapeutic effect was more significant at a temperature of 43°C than either 41°C or 46°C. NPrCAP/M with AMF exposure, instead of control magnetite alone or without AMF exposure, resulted in the most significant growth inhibition of the re-challenge tumor and increased the life span of the mice. HSP70 production was greatest at 43°C compared to that with 41°C or 46°C. CD8+T cells were infiltrated at the site of the re-challenge melanoma transplant.