Adenovirus Mediated Protein Tyrosine Phosphates SHP-1 Gene Transfect On Influence The Growth And Differentiation Of Human Extranodular Nasal Type NK/T Cell Lymphoma

Background and Objective Human extranodar nasal type NK/T cell lymphoma (ENKTCL-N) is a neoplasm with special morphology, immunophenotype and biological behavior, and has been listed as an entity of lymphoma in the updated WHO Classification for Tumors of Haematopoitic and Lymphoid Tissues published in 2001. This tumor shares a different apart of non-Hodgkin lymphomas (NHL) in different country. It occupies a relatively higher incidence in Asia and South America regions than that of in Europe and North America. About more than 60 cases of the tumor are newly diagnosed each year, it occupies about 13.83% of all lymphomas and 16% of all NHL in West China Hospital of Sicuan University. Extensive studies on SHP-1 gene, which is an SH2 domain-containing protein tyrosine phosphatase gene, revealed that the expression of SHP-1 protein was diminished or abolished in most cancer cell lines and tissues examined. On the other hand, the growth of tumor cells was suppressed after introducing the SHP-1 gene into the corresponding cell lines. The findings supported the hypothesis that the SHP-1 gene function as a tumor suppressor. At the first time, SHP-1 mRNA was found strong decrease in NK-YS cell line, which was belonged to ENKTCL-N, and SHP-1 protein was negative in all tumor tissues of ENKTCL-N in 2001. This evidence strongly suggests that loss of SHP-1 gene expression plays an important role in multistep tumorigenesis of ENKTCL-N.SHP-1 is primarily expressed in hematopoietic cells and behaves as a key regulator controlling intracellular phosphotyrosine levels in lymphocytes. SHP-1 protein can bind the immunoreceptor tyrosine based inhibition motif(ITIM) of the inhibitory receptors, such as T cell receptor(TCR), ZAP70, CD3, CD5, B cell receptor(BCR), CD22, killer cell inhibitory receptor(KIR) of NK cell etc, and downregulate the function of the molecular and play a important role in the process of cell growth and differentiation. In the previous study, a cDNA microarray analysis which was used to screen the differential expression genes in 1 case of ENKTCL-N was performed before, the results showed that the expression level of SHP-1 detected were all dramatically down-regulated. The greatly decreased level of SHP-1 mRNA observed may be have been attributed to that dysfunction of SHP-1 likely induced the development of ENKTCL-N. What is happened in ENKTCL-N if the normal SHP-1 cDNA is transfected into the tumor cell and the normal SHP-1 protein is expressed? And what kind of roles it plays for the growth and differentiation of ENKTCL-N? No related report has been found now.Purposes of the study are to observe the proliferation and differentiation in both the cell lines and the nude mice model of ENKTCL-N after transfected with constructed Ad-SHP1, and to discuss the possible functions of SHP-1 in the process of tumor cell proliferation and differentiation.Materials and Methods1. Human SHP-1 cDNA from healthy volunteers' peripheral blood lymphocyte was gotten by RT-PCR method and the recombinant adenovirus (Ad-SHP1) expressing SHP-1 protein was constructed.2. The recombinant adenovirus Ad-SHP1 was used to transfect the two cell lines(SNK-6 and SNT-8) of the ENKTCL-N and the expression level of SHP-1 was deteced by real-time quantity PCR(RQ-PCR) and western blot(including ZAP70 protein). The proliferation and differatiation of the tumor cells in both cell lines before and after transfected was evaluated by growth curve drawing and cell cycle analysis of flow cytometry.3. The recombinant adenovirus Ad-SHP1 was used to transfect the nude mice model of the ENKTCL-N and the expression level of SHP-1 was deteced by real-time quantity PCR(RQ-PCR) and western blot methods. The proliferation and differatiation of the tumor cells before and after transfected was evaluated by growth curve drawing and flow cytometry.ResultsThe major results are as follows:1. The recombinant adenovirus Ad-SHP1 can express SHP-1 protein stably and effectively.2. After transfected with the Ad-SHP1, the expression level of SHP-1 is up-regulated and ZAP70 is down-regulated in both cell lines(SNK-6 and SNT-8)of ENKTCL-N; the proliferation of tumor cells are all inhabited as well, such as the G₁～S arrest of the cell cycle, increased number of cells in G0/G1 phase, decresed number of cells in S phase; the cells show the tendency of high differentiation.3. No statistics difference of the expression level of SHP-1 is observed in the nude mice model of ENKTCL-N after transfected with the Ad-SHP1, but a up-regulated tendency can be observed in Ad-SHP1 group, what's more, the growth speed of tumor is more slowdown. The cells show the tendency of high differentiation.ConclusionsSHP-1 gene maybe play a important role on the growth and differentiation of the ENKTCL-N. The function link of SHP-1 protein is presumed to surpress actived effect protein and inhibited the cell signal pathway. The loss of SHP-1 protein in ENKTCL-N relief the inhibitor effect and the actived cell signal transducted persistently. Finally, cell growth is out of control. No related report is found all over the world until now.