Methylation of the Promoter Region of the RASSF1A Candidate Tumor Suppressor Gene in Primary Epithelial Tumors

The methylation of the promoter CpG island of the RASSF1A tumor suppressor gene in primary tumors of 172 Muscovites with renal cell carcinoma (RCC), breast cancer (BC), or ovarian epithelial tumors (OET) was assayed by means of methylation-specific PCR (MSP) and PCR-based methylation-sensitive restriction enzyme analysis (MSRA). The MSP, MSRA, and previous bisulfite sequencing data correlated significantly with each other (P 10^–6 for Spearman's rank correlation coefficients). By MSP and MSRA, the respective methylation frequencies of the RASSF1A promoter were 86% (25/29) and 94% (50/53) in RCC, 64% (18/28) and 78% (32/41) in BC, and 59% (17/29) and 73% (33/45) in OET. Methylation-sensitive restriction enzymes (HpaII, HhaI, Bsh1236I, AciI) increased the analysis sensitivity and made it possible to establish the methylation status for 18 CpG dinucleotides of the RASSF1A promoter region. With the MSRA data, the density of methylation of the CpG island was estimated at 72% in RCC, 63% in BC, and 58% in OET (the product of the number of CpG dinucleotides and the number of specimens with RASSF1A methylation was taken as 100%). Methylation of the RASSF1A promoter region was observed in 11–35% of the DNA specimens from the histologically normal tissue adjacent to the tumor but not in the peripheral blood DNA of 15 healthy subjects. The RASSF1A methylation frequency showed no significant correlation with the stage, grade, and metastatic potential of the tumor. On the other hand, epigenetic modification of RASSF1A was considerably more frequent than hemizygous or homozygous deletions from the RASSF1A region. These results testify that methylation of the RASSF1A promoter region takes place early in carcinogenesis and is a major mechanism inactivating RASSF1A in epithelial tumors.     

Optimization of genetic constructs for high-level expression of the darbepoetin gene in mammalian cells

Genetic constructs were designed in order to optimize darbepoetin production in CHO cells. They are characterized by a higher level of structural optimization of the darbepoetin gene, a higher gene dose, and the selection of promoter elements that have never been used before for this purpose. A transient transfection of CHO cells by the obtained constructs was performed. It was shown that each of the variable factors in the constructs influenced darbepoetin gene expression. A construct containing a doubled dose of the darbepoetin synthetic gene with optimized codon composition under the control of the CMV-EF1α chimerical promoter was proved to be the most efficient.     

Design of a stable cell line producing recombinant darbepoetin alpha based on CHO cells

A stable cell line that is based on CHO cells and produces 100 mg per liter of culture medium of recombinant darbepoetin alpha with a target glycosylated isoforms effective yield of about 30% has been selected. The expression product of the cell line was characterized and compared to the originator (Aranesp, Amgen). It was shown that the obtained preparation contained all isoforms characteristic of the originator. The created cell line can be used for the development of industrial cultivation and a purification scheme for recombinant darbepoetin alpha production.