| Background: One kind of cell surface proteins in eukaryote has been found to be anchored to the plasma membrane by glycosylphosphatidylinositol (GPI). These GPI-anchored proteins are functionally diverse. In human, the only purified and well-characterized GPI-specific phospholipase is a D-type phospholipase (GPI-PLD) which can specifically hydrolyze GPI, release GPI-anchored proteins and regulate their expression and biological functions. GPI-PLD-catalyzed anchor cleavage can generate biologically active lipids such as phosphatidic acid which is an important second messenger. GPI-PLD activity can be induced by insulin or other molecules. It is confirmed by molecular biology that liver, pancreatic and bone marrow are the main origins of plasma GPI-PLD. We have cloned a GPI-PLD eDNA with the size of approximately 2.6 kb (GenBank accession number AY007546) from human bone marrow stromal cells. It is known that several GPI anchored CD on human cell membranes, such as CD55, CD59, are important on the human immunity function. Many GPI-anchored proteins have been shown to associate with src-family kinases and heterotrimeric G proteins on membrane raft in activity lymphocytes and growing lymphocytes.Chronic myeloid leukemia (CML) is characterized by abnormal tyrosine kinase activity of the fused bcr/abl gene. It is as yet unknown whether this is the only and sufficient cause of the disease, or whether other supporting and co-active abnormalities exist. Many experimental evidences have elucidated that GPI-anchored proteins was associated with cancer and tumor malignancy, for example, the leukemia. GPI-PLD mRNA has very low expression in tumor cells of different malignancy including K562, HL-60 and U937 cells. Because the GPI-PLD is able to release GPI-anchored proteins and regulate their expression, the abnormalities of GPI-anchored proteins and GPI-PLD activities may be one cause of CML. We suppose that the change of GPI-PLD expression in CML patients maybe contribute to the tumor cells' immune escape mechanism. The aim of this research is to explore the disparity in GPI-PLD expression levels between leukemic cells of CML and healthy controls and elucidate any relationship of GPI-PLD expression levels to complement and T lymphocyte mediated killing of leukemic cells.Methods: 1. The competitive RT-PCR was used to detect quantitatively the GPI-PLD mRNA in mononuclear cells from healthy control and patients with CML. The GPI-PLD activities in mononuclear cells and sera were estimated by partitioning in triton-X-114 with GPI anchored placental alkaline phosphatase (PLAP) as the substrates. GPI-anchored CD55 and CD59 on mononuclear cells were analyzed by flow cytometer. Combination of triton-X-114 partitioning with Western bloting afftrmed CD55 and CD59 released from K562 cell membrane by GPI-PLD.2. The plasmid, pcDNA3.1(+)/GPI-PLD, was built by gene cloning. The K562 cells were transfected with pcDNA3.1(+)/GPI-PLD by liposome transfection. The positive clones were screened with G418 and their functions were also identified.3. The over-expression of GPI-PLD in K562 cells was induced by treatment with insulin. The changes of GPI-PLD mRNAs and activities before and after treating with insulin were detected.4. Complement-mediated lysis was assessed by trypan blue dye exclusion, T lymphocyte mediated killing of K562 cells by MTT colofimetric assay.Results: 1. The mean value of GPI-PLD activities in both mononuclear cells and plasma from CML patient group (n = 80) were 21.2 + 2.8% and 28.9 + 3.6%, which were significantly lower than those in control group (n = 110) [41.7±3.8% and 41.9±4.5%, respectively]. The enzyme activities of mononuclear cells and plasma in patient group were decreased by 100% and 70%, respectively, as compared with those in the control group. Significant differences in the percentages of reduction existed between both groups (P＜0.001). Furthermore, the GPI-PLD activities of mononuclear cells in patient group reached only 75% as those of its plasma (P＜0.001). However, obvious differences in GPI-PLD activities between mononuclear cells and plasma of control group were not found (P＞0.05).2. The expression levels of GPI-PLD mRNAs in mononuclear cells from 20 patients with CML and 20 normal controls were 0.438×103±30 and 1.237×103±42 copies/ng total RNA, respectively. These results demonstrated that copies of GPI-PLD mRNA in CML patients were very significantly lower than those in normal controls (P＜0.001). The percentages of decline almost reached 3-fold. However, at the tenth day after therapy with bone marrow transplantation (BMT), the copies of GPI-PLD mRNA and GPI-PLD activities in 6 younger patients almost recovered to the levels of healthy subjects3. The expressions of both CD55 and CD59 were all positive in 40 patients with CML and 20 healthy subjects. However, the fluorescence intensities of CDS5 and CD59 in the former (ranging from 330.7 to 673.8 and 545.6 to 782.4) distributed with a style more scattered than the latter (ranging from 438.8 to 478.6 and 641.2 to 704.6). The mean value of CD55 and CD59 expression in CML patients (537.2±25 and 747.9±35) displayed significant difference to that in healthy subjects (458.0±13 and 677.4±19).4. The cloned K562 cells over-expressing GPI-PLD steadily were obtained after the eukaryotic expression plasmid pcDNA3.1 (+)/GPI-PLD was successfully constructed and transfected to K562 cells by liposome transfection followed by the screening with G418.5. After treatment with insulin(10-7 mol/L) for 24 h and 48 h, the cellular GPI-PLD activity and mRNA levels in wild K562 cells were all increased about 4 and 7-fold, respectively. In simultaneity, those items in the GPI-PLD transfected K562 cells were all increased by another 10% and 20% over their originally high expression level.6. Combination of triton-X-114 partitioning with Western bloting atTtrmed that the GPI-anchored CD55 and CD59 on wild and transfected K562 cell surfaces have been released into culturing media by GPI-PLD after induction with insulin.7. The killing rate of complement-mediated cell lysis was increased almost 2 and 3 times in wild K562 cell after the insulin induction for 24 h and 48 h. The killing rates for the GPI-PLD transfected K562 cell were all increased by another 112% and 137% over their originally high killing rate, respectively.8. The killing rate of T lymphocyte mediated cell lysis was increased almost 3 and 5 times in wild K562 cell after the insulin induction for 24 h and 48 h. The killing rates for the GPI-PLD transfected K562 cell were all increased by another 119% and 133% over their originally high killing rate, respectively. Conclusion: 1. The GPI-PLD expression levels in CML patients are lower than those in healthy adults. After therapy with BMT, the copies of GPI-PLD mRNA and GPI-PLD activities in patients almost recovered to the levels of healthy subjects.2. We successfully screened the GPI-PLD gene transfected K562 cells, which over-expressed GPI-PLD steadily.3. Enhanced GPI-PLD expression of K562 cells by insulin is able to induce the release of GPI-anchored CD55 and CD59 and it can increase the killing of these cells by complement and T lymphocyte.4. The molecular mechanism of GPI-PLD acting in CML disease development and in tumor immunological escapology has been elucidated for the first time by us. |
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