Cloning Of Fractaline And Study Of Its Antitumor Fuction

Objective: Tumor infiltrating leukocytes are involved in development and growth of tumors and their metastasizes. The origin of these leukocytes may be associated with residual evidence of the host's ineffective immune response to reject the tumor; thus, enhancing the amount of infiltrating leukocytes might lead to an improved host-antitumor effect.Fractalkine, which is also called neuroactin, is the sole member of the CX3C chemokine subfamily, known to induce both adhesion and migration of leukocytes. The membrane-bound version of fractalkine rapidly induces firm adhesion of CX3CR1-expressing cells under static and flow conditions without requiring selectin-mediated rolling or activation of integrins. In addition, soluble fractalkine released from the cell surface by proteolytic cleavage induces migration of CX3CR1-expressing cells in a similar way to other soluble chemokines.In this study, mouse FKN was cloned and NXS2 neuroblastoma cells was genetically engineered to express FKN by transfection with FKN mammalian expression vector. The antitumor effect of FKN was monitored in tumor-bearing mouse model which was established by NXS2-FKN in A/J mice. With these data, we tried to test the hypothesis that fractalkine secreted by tumor cells may influence trafficking of leukocytes and subsequently, tumor growth and may open possibilities for designing novel interventions in antitumor and antivirus gene therapy.Method: 1.Clone and identification of mouse FKN: The coding sequence of mouse FKN was amplified from total RNA which was extracted from D2F2, a mouse breast cancer cell line, by RT-PCR and then cloned into PCR2.1-TOPO vector through A-T clone. The size and the sequence of this insert were verified by restriction enzyme digestion and molecular sequencing analysis.2.Construction of FKN mammalian expression vector and expression of FKN in tumor cells: To express FKN protein, FKN cDNA was subcloned from pCR2.1TOPO-FKN to a mammalian expression vector pIRES (pIRES-FKN). The length and location of FKN cDNA in this vector was verified by restriction enzyme digestion. Expression of FKN was selected in the presence of G418 selection media and two successive rounds of serial dilution and expansion after pIRES-FKN was transfected into NXS2 neuroblatoma cells with superfect. Transcription of FKN in three different NXS2 cell lines (NXS2-wild type, NXS2-mock, NXS2-FKN) was detected by RT-PCR. The soluble and the membrane-bound forms of FKN weredemonstrated by ELISA and flow cytometry, respectively.3.Analysis of anti-neuroblastoma function of FKN in tumor-bearing mouse model:On syngeneic female A/J mice, primary tumors were induced by subcutaneous injection of three kinds of NXS2 cells (NXS2-wild type, NXS2-mock, NXS2-FKN) at the dosage of 2×106 in the left flank and tumor growth was monitored by microcaliper measurement. Tumors were removed on the 14th days after their inoculation. Tumors were weighed and transcription as well as expression of FKN in these three groups of tumors were detected by RT-PCR and immunohistochemistry. To characterize the recruiting lymphocytes at the tumor sites, cryopreserved tumor samples were stained by anti-CD4, anti-CD8 and anti-CD45 through immunohistochemistry. 3 weeks after surgery, livers were harvested and spontaneous liver metastatic colonies were counted. For histologic evaluation, 5um sections from paraffin-embedded liver tissues were stained with hematoxylin and eosin. Results:1.Clone and identification of mouse FKN: Total RNA was extracted by Rneasy Minikit (Qiagen) from D2F2 cells. The high concentration and purity were determined by measuring the absorbance at 260 nm and 280 nm (A260/A280) in a spectrophotometer. On an agarose gel, DNA contamination is not visible; meanwhile the integrity and size distribution were qualified. By RT-PCR from this tRNA, a fragment around 1200bp long was got which was then cloned into pCR2.1TOPO vector by A-T clone technique. Restriction enzyme digestion by MluⅠand NheⅠ verified the right size of the insert.