Breast Cancer Selective Gene Expression And Kill Mediated By PGL3-DF3-DTA

Diphtheria toxin is a bacterial exotoxin secreted by pathogenic strains of Corynebacterium diphtheriae as a single polypeptide chain, which can lead to severe toxic reaction. It is made up by 535 amino acids,and the molecular weight is 58000 single polypeptide. Under the effect of trypsinase,it changes into two subunits: fragments A(amino acids 193) and B (amino acids 342). Fragment A(21 kDa)has ADP dependent ribose acyl enzymatic activity. Fragment B(37 kDa) consists of two separate and structurally different domains: cross the plasma membrane and receptor-bound. Fragment A can be activiated unless that the receptor-bound domain of B-fragment bind with specific cell surface receptors ,followed by endocytosis, and induces A-fragment to translocate to the cytosol. The integrity toxin is noxious, because the activate site is between the juncture of cross the plasma membrane and receptor-bound domains. The entrance of activate site is wrapped by loop CL2 which is made up by 18 residues and across the plasma membrane domain, the passages elongation factor 2 reach the activate site are interrupted, therefore, Diphtheria toxin without separation is not toxicant. To be active, chain separation by cell-mediated reduction is required for release of fragment A to the cytosol. Fragment A catalyze dihydrouracil dehydrogenase to make elongation factor 2 form Adenosinediphosphoribosylate, thus elongation factor 2 replace the Nicotinic Acid Amide of dihydrouracil dehydrogenase, and then form inactive ADPR-EF-â…¡. When the elongation factor 2 miss the activity, peptide chain could not to be extended and protein synthesis is inhibited, which damage the cell function and lead to degeneration and necrosis. The high weight mucin-like glycoprotein, DF3(MUC1)is overexpressed in the majority of human breast cancers. Research indicated that DF3(MUC1)tumor antigen expressions of human breast cancer in protein and RNA was almost 30 times to breast normal tissues and benign lesion. With regard to the gene therapy of breast cancer, making use of the characteristic of Diphtheria toxin and recombinant plasmid vector, it will be important to investigate the plasmid vector selective gene expression and eradicating effect on breast cancer.The experiment is to demonstrate the selective expression and eradicating effect of recombinant plasmid vector DTA containing transcriptional regulation sequence of human breast cancer DF3 /MUC1 promoter (PGL3-DF3-DTA) on DF3 positive breast carcinoma cell lines. The MCF-7 and MDA-MB231 breast carcinoma cell lines were transplanted into the dorsal subcutaneous tissue of nude mice in different groups. The mice were subcutaneously injected with recombinant plasmid PGL3-DF3-DTA 7 days after transplantation and the animals were sacrificed 29 days after transplantation. The dimensions of the tumor were measured and tumors were excised and their ex-vivo weight and volume were determined. The expression of DTA was detected by reverse transcription-polymerase chain reaction (RT-PCR).The results suggest that recombinant plasmid vector PGL3-DF3-DTA was expressed in xenograft of MCF-7 breast carcinoma cell lines, and it inhibited the growth of tumor significantly. Recombinant plasmid vector PGL3-DF3-DTA has the selective expression and eradicating effect on breast cancer in DF3-positive.