| BackgroundLymphoma is a group of complex malignant tumours originating in lymphoid tissue that threaten seriously the health of the habitants in China. Recently, the incidence of lymphoma has rised year by year in our country, and the ascendant speed of which has taken the first place increasing over other malignant tumors. Precursor T-ALL/LBL (T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma) is an aggressive biologic unit originating from T lymphocyte, which make up a sizable proportion among lymphoma. In general, the therapeutic outcomes of precursor T-cell malignancies remain poor especially of those progressive or relapse types. So it is necessary to find a better therapeutic method for patients with precursor T-cell malignancy. As an important development in medical science, gene therapy has become a hopeful method in curing tumors. Fas (FS7-associated cell surface antigen) play a critical role in T-cell apoptosis by functioning as a major cell-surface DR (death receptor), which is important to maintain the T-cell homeostasis. Various defections in Fas have been found in patients with T-cell malignancy. Based on these facts, and in order to explore a potential effective therapeutic method for precursor T-cell malignancy, we studied the affect of over-expressed Fas to the apoptosis sensitivity of malignant precursor T-cell in vitro and to the tumorigenesis of malignant precursor T-cell in vivo.MethodsWild type Fas gene amplification. A peripheral blood sample of a healthy volunteer was obtained with institutional review board approval. The mononuclear cells were separated using Ficoll Reagent and total RNA was isolated using Trizol Reagent. Total cDNA was synthesized from the total RNA with SuperScript Reverse Transcriptase and Random Primers. Full-length Fas cDNA (1167bp) was amplified from the total cDNA by polymerase chain reaction (PCR) using high-fidelity Taq polymerase with primers. Eukaryotic vector construction. Fas cDNA was retrieved from agar gel and cloned into vector pMD-18T. The pMD-18T-Fas plasmid was transferred into competent cell DH5αto grow. The target gene of Fas cDNA was amplified using primers with EcoRI and XhoI cutting sites. After cut by EcoRI and XhoI, the target gene was cloned into eukaryotic expression vector pCDNA3.1(+) by ligase. The recombinant plasmid pCDNA3.1(+)-Fas were directly sequenced. Stable cell transfection. Jurkat cells were transfected with pCDNA3.1(+)-Fas and then grown in complete medium containing 500 mg/ml G418 for 7 days. The survive colonies were expanded continuously in 250 mg/ml G418 contained medium and were named as Jurkat-Fas cells. Analysis of Fas mRNA level in Jurkat cell and Jurkat-Fas cell. Total RNA of each cell line was isolated and reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the full-length Fas cDNA with GAPDH as an internal control. The PCR products and DNA ladder molecular weight marker were loaded onto agarose gel to proceed electrophoresis. Images were acquired under ultraviolet rays. 40 cycles of real-time PCR were performed to amplify 110 bp of FAS cDNA with GAPDH as an internal control. For each detection, the procedure was repeated 3 times. Analysis of Fas protein level in Jurkat cell and Jurkat-Fas cell. 4×105 cells of each cell line were collected and stained by PE anti-human Fas. After washed with cold PBS, the cells were scanned under confocal microscopy at a wavelength of 488 nm. 4×105 cells of each cell line were prepared and treated with PE anti-human Fas. The fluorescence intensity was detected by flow cytometry with PE mouse IgG1κisotype control as an internal control. For each detection, the procedure was repeated 3 times. Apoptosis sensitivity of Jurkat cell and Jurkat-Fas cell. After treated with human soluble Fas Ligandfor 0h, 12h, and 24h, respectively, 4×105 cells of each cell line were collected and stained with Alexa Fluor○R488 annexin V and propidium iodide (PI). The apoptotic ratios were analyzed by flow cytometry. For each detection, the procedure was repeated 3 times. Animal tumorigenesis of Jurkat cell and Jurkat-Fas cell. Eight SPF male BALB/c nude mice (4 weeks of age) were separated into two groups averagely and randomly. After exposed to medial lethal dose of ray (4Gy), the two groups of mice were subcutaneously inoculated with 5×107 Jurkat and Jurkat-Fas cells suspended in 100μl PBS at the upper right armpit, respectively. All mice were injected with 0.5ml 100ng/ml human soluble Fas Ligand through caudal vein every 7 days from the injection of tumor cells. Tumor growth was monitored through calculating size every 4 days for 40 days.ResultsSuccessful construction of pCDNA3.1(+)-Fas. The sequencing results of the recombinants plasmids pCDNA3.1(+)-Fas were consistent with GenBank accession no. M67454. Expected over-expression of Fas mRNA.The gel electrophoresis image showed apparent straps of GAPDH cDNA in both Jurkat cell line and Jurkat-Fas cell line, and apparently stronger strap of Fas cDNA in Jurkat-Fas cell line than that in Jurkat cell line. The mean relative expression level of Fas mRNA in Jurkat-Fas cells was significantly greater than that in Jurkat cells. (P=0.0007) Expected over-expression of Fas protein. The images of the Jurkat and Jurkat-Fas cells scanned by confocal microscopy displayed the Fas protein expressed on cell membrane surface. The protein level of Fas in Jurkat-Fas cells were significantly greater than that in Jurkat cells. (P<0.0001) Increased sensitivity to Fas-mediated apoptosis via over-expression of Fas. After treated with 25ng/ml FasL for 12h and 24h, the apoptosis rates of Jurkat-Fas cells were about 3-fold and 4-fold to Jurkat cell, respectively. The sensitivity to Fas-mediated apoptosis in Jurkat-Fas cells significantly increased compared to Jurkat cell. (12h, P=0.0012; 24h, P<0.0001) Inhibition to tumorigenesis via over-expression of Fas in vivo. After 24 days from the injection of tumor cells, while all of the four mice injected with Jurkat cells began to erupt tumors and the tumors’sizes increased stably with time proceed, none of the other four mice injected with Jurkat-Fas cells developed any tumors during the 40 days of observation. Over-expression of Fas significantly inhibited the tumorigenesis in vivo. (d24, P=0.0048; d28, P=0.0022; d32, P<0.0001; d36, P=0.0002; d40, P=0.0004)ConclusionThe comparison between Jurkat and Jurkat-Fas cell line demonstrated that the Fas protein level paralleled it’s mRNA level. Treated with FasL, the sensitivity to Fas-mediated apoptosis of Jurkat and Jurkat-Fas cells paralleled their Fas level, and the tumorigenesis of Jurkat and Jurkat-Fas cells negatively correlated with their Fas level. In a conclusion, stable over-expression of extrinsic Fas gene can significantly ameliorate the sensitivity to Fas-mediated apoptosis in human malignant precursor T cell, which indicate a novel strategy to improve therapeutic effects on T-cell malignancy. Other considerable problems such as target therapy should be studied in further work. As a specific feature of a malignant T lymphatic cell clone that makes it different from others, the idiotype (Id), immunogenic epitopes within the variable region of T cell receptor (TCR) may be a favorable candidate. |
PDF Full Text Request