[Effects of Down-regulating PPP2R5C Expression on Expression Profile of TAL1-related Regulating Genes in Jurkat Cells].

OBJECTIVE: Based on our previous study showing the inhibition of lenkemia T cell proliferation by down-regulating PPP2R5C expression, this study was aimed to analyze the influence of down-regulating PPP2R5 expression via RNA interference on genes relatied with TAL1 signaling pathway by using gene chip technique. METHODS: The PPP2R5C-siRNA799 was transduced into Jurkat cells by nucleofection, the total RNA was isolated from treated Jurkat cells after culture for 48 hours; the target sequences were prepared by revevse transcription after mRNA purification, and were hybridized with affymetrix gene expression profile chip 3' IVT. The original image data were collected using affymetrix gene chip scanner 3 000, and the gene expression profile was analyzed using gene spring GX 11.0 soflware. RESULTS: The expression of all 26 genes related with TAL1 signaling pathway was changed, out of which the expression of 15 genes were up-regulated and the expression of 11 genes was down-regulated in PPP2R5C-siRNA 799-transfected Jurkat cells. The genes with significantly up-regulated expression were GATA1, TCF4, XRCC6 and TCF3, while the genes with significantly down-regulated expression were SIN3A and RUNX1. CONCLUSION: The down-regulation of PPP2R5C gene expression in Jurkat cells via RNA interference to a certain degree can inhibit TAL1 signaling pathway genes, thereby suppresses the proliferation of Jurkat cells.

[Overexpression of eIF4E gene in acute myeloid leukemia and its relation with disease progression].

The aim of this study was to detect the expression level of eIF4E gene in patients with non-treated, remission and non-remission/relapse acute myeloid leukemia (AML), and other non-malignant haematologic diseases so as to analyze and reveal the relationship of eIF4E gene expression with AML progression. SYBR Green I RT-PCR was used to assay the expression level of eIF4E mRNA extracted from bone marrow mononuclear cells in 30 patients with AML (6 in M2, 5 in M3, 8 in M4, 10 in M5, 1 in M6) and 20 patients with non-malignant hematologic diseases. The ß2-microglubin(ß2M) was used as internal reference and the formula 2(-ΔCt)×100% was applied to calculate the expression level of eIF4E gene. The results showed that the eIF4E expression level (7.098 ± 5.544)% in patients with non-treated and non-remitted/relapsed AML was significantly higher than that in patients with remission (0.964 ± 0.312)% (P < 0.01) and non-malignant hematologic diseases (0.248 ± 0.163)% (P < 0.01). There was no difference between latter two group patients, even though the expression level of eIF4E gene in patients with M4 and M5 was higher. As compared with non-malignant hematologic diseases, the expression level of eIF4E gene of patients with remission patients showed no significant difference. It is concluded that the over-expression of eIF4E gene has been found in patients with AML, and its level obviously decreases along with remission of disease, thus the eIF4E gene may be a surveillance parameter for disease progression.

[Analysis of CDR3 sequence of TCR Valpha6, Valpha10 and Vbeta23 oligoclonal T cells in one APL case].

AIM: To investigate the clonality and the pattern of CDR3 sequence of TCR Valpha and Vbeta repertoire in patients with acute promyelocytic leukemia (APL). METHODS: The CDR3 of TCR Valpha 29 and Vbeta 24 subfamily genes were amplified in mononuclear cells from peripheral blood of one case with APL using RT-PCR. The positive PCR products were further labeled with fluorescent and analyzed by genescan technique for the CDR3 size, to evaluate clonality of the detectable TCR Valpha and Vbeta T cells. The products of the oligoclonal T cells were analyzed by sequencing to define the sequence of CDR3. RESULTS: Oligoclonal T cells expressed TCR Valpha6, Valpha10 or Vbeta23 genes were identified from the APL patient. The molecular characteristics of the CDR3 of Valpha and Vbeta genes rearrangement were Valpha6NJalpha17, Valpha10NJalpha35 and Vbeta23NDbeta1NJbeta2.7. The amino acid sequence of CDR3 region in TCR Valpha 6, Valpha10 and Vbeta23 subfamilies were CAMRENSAGNK, YLCAGDELLWECA or CASSSKWAGGTYEQY, respectively. These sequences were accepted by GenBank (Accession Number EU544946, EU544947 and EU647219). CONCLUSION: Three idiotype Valpha and Vbeta sequences are identified in a case with APL, which is thought that may mediate the host specific immunity response for leukemia cells in APL. It might provide data for farther research on the specific immunotherapy of APL.

T cell receptor Vbeta repertoire usage and clonal expansion of T cells in chronic myelogenous leukemia.

BACKGROUND: In general, it is very important to understand the state of T cell immune response against tumor cells in leukemia patients and it is especially critical to assess the T cell repertoire of untreated patients. As we know, few studies have dealt with the distribution of oligoclonal T cells in leukemia, so we investigated the distribution and clonality of TCR Vbeta repertoire of T cells in patients with chronic myelogenous leukemia (CML) in chronic phase. METHODS: The complementarity determining region 3 (CDR3) of TCR Vbeta24 subfamily genes were amplified in peripheral blood mononuclear cells from 27 cases with CML using reverse transcription-polymerase chain reaction (RT-PCR). In order to observe the distribution of TCR Vbeta repertoire, the PCR products were further analyzed by genescan technique to evaluate clonality of the detectable TCR Vbeta T cells. The PCR products of the oligoclonal T cells from three cases were analyzed by direct sequencing to define the sequence of CDR3. RESULTS: The expression pattern of TCR Vbeta repertoire in different individuals are different. Vbeta2-21 subfamilies could be detected in CML cases. The frequent usage Vbeta repertoire in CML was Vbeta1, Vbeta2 or Vbeta13. Most of the PCR products from 27 patients displayed polyclonality, while a part of the PCR products from 21 out of 27 samples displayed clonal expansion pattern. The clonal expanded T cells in CML could be found in Vbeta16 subfamilies. The frequent usage of Vbeta genes in clonal expansion was Vbeta3, Vbeta13 or Vbeta21. Multiple Vbeta clonal expansion was a general phenomenon in the same patient. The CDR3 sequence of Vbeta21 oligoclonal T cells from 3 cases showed some difference in splice regions and in the usage of J segments. CONCLUSIONS: These results indicated that clonal expanded T cells could be found in patients with CML and were tendentious in Vbeta3, Vbeta13 and Vbeta21 subfamilies that may be related to the specific immune response for leukemia cell associated antigen.

[Clonal expansion of TCR Vbeta subfamily T cells induced by bcr3-abl2 peptide].

OBJECTIVE: To investigate the clonal expansion of T cell receptor (TCR) Vbeta subfamily T cells from cord blood induced by bcr3-abl2 peptide in vitro. METHODS: T cells from 3 units of cord blood were amplified by anti-CD(3) monoclonal antibody (McAb) and IL-2 with or without synthetic b3a2 peptide. T cell specific cytotoxicity was analyzed by lactate dehydrogenase (LDH) assay, TCR Vbeta subfamilies by using reverse transcriptase-polymerase chain reaction (RT-PCR) and genescan technique. RESULTS: bcr3-abl2 peptide specific cytotoxicity T cells were successfully induced from the 3 units of cord blood by synthetic b3a2 peptide. Compared with that in CD(3) McAb induced cells, distribution pattern of TCR Vbeta repertoire was different in T cells induced with b3a2 peptide. Oligoclonal and oligoclonal tendency TCR Vbeta subfamily T cells could be identified in cord blood T cells induced by b3a2 peptide in 1 or 2 weeks, whereas those induced by anti-CD(3) McAb and IL-2 were mostly polyclonal. CONCLUSION: The cytotoxicity T cells with anti-CML specificity could be induced by b3a2 peptide. The specific anti-CML cytotoxicity may be derived from the clonal expansion TCR Vbeta subfamily T cells.

[Analysis of selected usage and clonal expansion of TCR Vbeta repertoire of peripheral blood T cells in patients with non-Hodgkin's lymphoma].

To investigate the distribution and clonal expansion of TCR Vbeta subfamily T cells in patients with B-NHL and T-NHL, the CDR3 of TCR Vbeta 24 subfamily genes was amplified in peripheral blood mononuclear cells from 4 cases with B-NHL and 2 cases with T-NHL using RT-PCR, and to observe the usage of TCR Vbeta repertoire, the PCR products were further labeled with fluorescence and analyzed by genescan technique for the CDR3 size, to evaluating clonality of the detectable TCR Vbeta T cells. The results indicated that only selected expression of 6-12 Vbeta subfamily T cells could be identified in the 6 cases with NHL, and Vbeta1, Vbeta8, Vbeta13 and Vbeta19 were expressed in all samples, Vbeta2 and Vbeta16 could be found in 5 samples, whereas Vbeta4-6, Vbeta10-12, Vbeta15, Vbeta17-18, Vbeta20 and Vbeta22-23 were absent in all samples. Genescan analysis showed that clonal expansion of T cells could be found in 1-3 Vbeta subfamilies from 2 cases with B-NHL and 1 case with T-NHL. In conclusions, the similar selected usage of TCR Vbeta subfamily T cells could be found in peripheral blood from patients with B and T NHL, clonal expansion of T cells which were considered to be related to lymphoma cell antigen could be detected in a part of patients with B or T NHL.

[The feature of TCR V alpha40 with Jdelta1, Ddelta3 or psiJalpha gene rearrrangements in T cells].

The rearrangement segments of TCR Valpha40 gene with Jdelta1, Ddelta3 or psi Jalpha were amplified in genomic DNA from peripheral blood mononuclear cells of 10 normal subjects, sorted CD3(+) cells from peripheral blood of 4 cases and thymocytes from 7 cases, by using nested PCR. Different amounts of DNA from all samples were amplified to estimate the frequency of Valpha40 gene rearrangements. The results indicated that the rearrangements of TCR Valpha40 gene with Jdelta1, Ddelta3 or psi Jalpha could be found respectively in the most samples of peripheral blood T cells or thymocytes. The frequencies of Valpha40 rearrangements were different in peripheral blood T cells and thymocytes by analysis of PCR with different amounts DNA. It is concluded that the TCR V alpha40-psi Jalpha was the most frequent rearrangement in mature and immature T cells, whereas TCR Valpha40-Ddelta3 was more frequently rearranged in immature T cells