[Effects of combined therapy of human leucocyte antigen haploidentical related bone marrow transplantation and Chinese medicine in treating leukaemia].

OBJECTIVE: To explore the feasibility of human leucocyte antigen (HLA) haploidentical related T-cell undepleted allogeneic bone marrow transplantation (Allo-BMT) combined with Chinese medicine for the treatment of leukaemia. METHODS: Four patients with chronic myeloblastic leukemia (CML) and 4 with acute myeloid leukemia (AML) received allo-BMT with graft from 1 - 3 HLA-mismatched related donors. All patients were pre-treated with standardized conditioning regimen consisting of high dose Ara-c, cyclophosphamide (CY) and total body irradiation (TBI) or busulfan. Donors were given G-CSF 250 microg/d for 7 days prior to marrow harvest. To prevent GVHD, besides application of CSA and MTX, ATG 2.5 mg/kg was given everyday for 4 days before transplantation, and MMF 1.0 g per day starting from the 7th day after transplantation. Chinese medicine for replenishing qi and yin and strengthening Pi and Wei was administrated orally after transplantation. RESULTS: Successful haematopoietic reconstruction was seen in all patients. The median days for reaching of granulocyte >0.5 X 10(9)/L and platelet > 20 x 10(9)/L were 12 (range 10-14) and 20 (range 18-25) days respectively. GVHD of skin in various grade was seen in 7 patients, among whom only one advanced to grade IV; besides, 2 accompanied with GVHD of gut, one with hemorrhagic cystitis, one died for concurrent infection on the 81st day, and one left hospital of his own accord on the 46th day. The median follow-up duration was 18 (range 2-32) months, during this period six patients were alive in a disease-free situation. CONCLUSION: Combined therapy of HLA haploidentical related T-cell undepleted Allo-BMT and Chinese medicine plus immunosuppressants with pre-harvest G-CSF application in doner could effectively reduce the incidence of acute severe GVHD and raise the disease-free survival rate in treating leukaemia.

[Apoptosis of HL-60 cells induced by artesunate in vitro].

The study was aimed to investigate the effect of artesunate (ART) on the apoptosis of HL-60 cells in vitro and the expression of Bcl-2 and ICAD in the process of apoptosis induced by ART. The inhibition of ART on HL-60 cells were evaluated by means of MTT assay; cell apoptosis was detected by light microscopy, agarose gel electro-phoresis, flow cytometry; Western blot was used to analyze the expression of Bcl-2 and ICAD in cells during apoptosis induced by ART. The results showed that ART could significantly inhibit the proliferation of HL-60 cells in time-and dose-dependent manner. After treating HL-60 cells with ART for 48 hours, the IC(50) values was 18.33 microg/ml and its inhibition effect contributed to the induced apoptosis. Bcl-2 and ICAD proteins both all expressed in HL-60 cells, the level of expression declined as concentration increased. It is concluded that artesunate may induce apoptosis of HL-60 cells in vitro, Bcl-2 and ICAD may be an important control factor in the signal transduction pathway of ART-induced apoptosis.

[Immune response of dendritic cells capturing antigens from apoptotic U937 cells induced by artesunate].

The objective of study was to investigate whether U937 cells-loaded dendritic cells (DCs) could induce anti-leukemic immune activity. The apoptosis of U937 cells was induced by artesunate (ART). DCs derived from peripheral blood mononuclear cells of health donors were loaded with apoptotic U937 cells, and induced to maturation in the presence of TNF-alpha. Matured DCs were cocultured with autologous T-lymphocytes, and combined with IL-2 in order to induce the leukemia-specific CTL. The phenotypes of DCs and T lymphocytes were tested by flow cytometry. The ability of DC capturing antigens was measured by Dextran-FITC endocytosis. The IL-12p70 level was assayed by ELISA kit. The proliferation of CTL and CTL activity were measured by MTT assay. The results showed that the apoptotic rate of the U937 cells was 51.2% when U937 cells were induced by 1 microg/ml ART for 48 hours in vitro. DCs had the most powerful ability of endocytosis in its immature phase. Apoptotic U937 cells could not induce the features of DC maturation, and apoptotic U937 cell-pulsed immature DCs could be matured with TNF-alpha. The IL-12p70 level secreded by apoptotic U937 cell-loaded mature DCs (mDC-(Apo)U937) was higher than that of non-loaded mDC. The proliferation of autologous T lymphocytes co-cultured with mDC-(Apo)U937 was significantly remarkable and the content of CD8(+) CTL was significantly higher in comparison with any other groups. CTL induced by mDC-(Apo)U937 had stronger killing effect on U937 cells than NB4 (p < 0.01). It is concluded that the mDC-(Apo)U937 can effectively generate T cell-mediated dendritic antileukemic responses in vitro.

[Overexpression of S3 ribosomal protein gene is involved in drug resistance in K562/DOX cells].

OBJECTIVE: To investigate the effect of overexpression of S3 ribosomal protein (S3rp) gene on the resistance of leukemia cell to antitumor drugs. METHODS: Both sense and antisense cDNA recombinants of S3rp gene were constructed with pcDNA3.1 expression vector. Subsequently, the sense S3rp cDNA recombinant was transfected into K562 cells while the antisense one into K562/DOX cells (a multidrug resistant cell line). In addition, empty pcDNA3.1 vector was transfected into the corresponding cells as negative controls. The chemosensitivity of cells was evaluated by MTT assay. RESULTS: Sense S3rp cDNA transfected K562 cells were 5.8 times more resistant to doxorubicin than control cells did, whereas antisense S3rp cDNA transfected K562/DOX cells were 3.2 times less resistant to doxorubicin than control cells did. CONCLUSION: Overexpression of S3rp gene plays an important role in the development of drug resistance in K562/DOX cells.

[Identification of multidrug resistance related genes in leukemia by suppression subtractive hybridization].

OBJECTIVE: To clone and screen genes related to multidrug resistance (MDR) in leukemia. METHODS: Suppression subtractive hybridization (SSH) was performed to profile differentially expressed genes between a MDR leukemia cell line (K562/DOX, as tester) and its parent cell line (K562, as driver). Reverse Northern dot blot was carried out to further screen the subtracted cDNA library. The overexpressed cDNA fragments in K562/DOX cells were sequenced and compared with known genes in Genbank. RT-PCR and Northern blot were employed to confirm the differential expression of some identified genes. RESULTS: Eleven genes were identified being overexpressed in K562/DOX, including S3 ribosomal protein (S3rp) gene, NADH dehydrogenase subunit 2 (ND2) gene and My023 gene, which have not been reported to be related to MDR in cancer. CONCLUSION: Several genes, which might be involved in MDR were identified, indicating novel mechanisms of MDR in leukemia.