The faulty regulation of imprinting gene lead to the abnormal development of reconstructed embryo after nuclear transfer. However, the correlation between the imprinting status of donor cell and preimplantation stage of embryo development is not yet clear. In this study, to determine this correlation, we used the porcine spermatogonial stem cell (pSSC) and fetal fibroblast (pFF) as donor cells. As the results, the isolated cells with laminin matrix selection strongly expressed the GFRα-1 and PLZF genes of SSCs specific markers. The pSSCs were maintained to 12 passages and positive for the pluripotent marker including OCT4, SSEA1 and NANOG. The methylation analysis of H19 DMR of pSSCs revealed that the zinc finger protein binding sites CTCF3 of H19 DMRs displayed an androgenic imprinting pattern (92.7%). Also, to investigate the reprogramming potential of pSSCs as donor cell, we compared the development rate and methylation status of H19 gene between the reconstructed embryos from pFF and pSSC. This result showed no significant differences of the development rate between the pFFs (11.2±0.8%) and SSCs (13.3±1.1%). However, interestingly, while the CTCF3 methylation status of pFF-NT blastocyst was decreased (36.3%), and the CTCF3 methylation status of pSSC-NT blastocyst was maintained. Therefore, this result suggested that the genomic imprinting status of pSSCs is more effective than that of normal somatic cells for the normal development because the maintenance of imprinting pattern is very important in early embryo stage.

Expression of invasion/metastasis suppressor, E-cadherin, is reduced in many types of human carcinomas. Although somatic and germline mutations in the CDH1, which encodes the human E-cadherin, have frequently been reported in cases with diffuse gastric and lobular breast cancers, irreversible genetic inactivations are rare in other human carcinomas. Recently, it has been well documented that some genes in human cancers may be inactivated by altered CpG methylation. Herein, we determined the expression and methylation status of E-cadherin in oral squamous cell carcinoma(SCC) by immunohistochemistry and methylation-specific PCR. The expression of E-cadherin was significantly higher in the well-differentiated oral SCCs than the moderately or poorly differentiated ones. None of eight tested benign epithelial hyperplasias showed aberrant methylation, whereas five of 12 oral squamous cell carcinomas showed aberrant methylation. When we compared E-cadherin expression with methylation status, oral SCCs with normal methylation showed a higher expression of E-cadherin than those with methylation. These findings suggest that aberrant CpG methylation of CDH1 promoter region is closely associated with transcriptional inactivation and might be involved in tumor progression of the oral mucosa.

p16INK4A and p15INK4B tumor suppressor genes are frequently altered in various human tumors. Hypermethylation of the promoter region of p16INK4A and p15INK4B seem to be the major mechanism of inactivation. To determine whether the change in p16INK4A and p15INK4B methylation status occur in oral squamous cell carcinomas (OSCCs) and benign oral epithelial hyperplasias, we analyzed 46 OSCCs and 20 benign oral epithelial hyperplasias by methylation-specific PCR. We also analyzed a subset of the samples for p16INK4A and p15INK4B protein expression by immunohistochemistry. The promoter region of p15INK4B was hypermethylated in 13 specimens of the 15 finally analyzed OSCCs and three specimens of the five analyzed benign oral epithelial hyperplasias. By immunohistochemical analysis, we confirmed the loss of p15INK4B expression of all hypermethylated specimens. The promoter region of p16INK4A was amplified by both an unmethylated- and a methylated-specific primers in just one OSCCs. The remaining specimens including 11 OSCCs and four benign oral epithelial hyperplasias were normally methylated. By immunohistochemistry, we analyzed the loss of p16INK4A expression in seven specimens of the 12 OSCCs and two specimens of the four benign oral epithelial hyperplasias. Except for one OSCC, however, all specimens showing loss of expression were normally methylated. These results suggest that loss of p16INK4A and p15INK4B protein expression play an important role in the development of both OSCCs and benign oral epithelial hyperplasias.