[Sequential and quantitative analysis of chimerism after non-myeloablative stem cell transplantation].

OBJECTIVE: To investigate the exact kinetics of donor chimerism (DC), outcome of mixed chimerism (MC) and prognostic role of chimerism in the evaluation of engraftment, disease relapse, GVHD and long term survival after nonmyeloablative stem cell transplantation (NST). METHODS: 18 patients who received HLA compatible NST were evaluated. Peripheral blood and bone marrow were collected before and after transplantation at different time. DNA was extracted using QIAmp blood mini kit. Nine different STR markers were co-amplified in a single reaction by commercial AmpF/STR profiler plus PCR amplification kit. Separation of the PCR products and fluorescence detection were performed with ABI prism 310 genetic analyzer with capillary electrophoresis. Genescan and genotype software were used for size calling and quantification of peak areas. The formula to calculate donor chimerism values was based on different allelic distribution types between the donor and recipient. RESULTS: (1) Serial STR-PCR analysis revealed that donor chimerism became dominant (DC > 60%) by day 8; it preceded the detection of hematologic engraftment by an average of 4 days. It was also shown that chronic myeloid leukemia (CML) patients frequently had more delayed donor engraftment as compared with patients of acute leukemia or nonmalignant hematological diseases because the pretransplantation immune status of these two kinds of patients was different. (2) After NST, chimeric status had a process of conversion from the mixed chimerism (MC) to full donor chimerism (FDC). (3) The incidence of graft versus host disease (GVHD) of FDC group was higher than that of MC group (90.0% vs 62.5%). The average time between establishment of FDC and appearance of GVHD was 9 days. (4) Full donor chimerism and stable mixed chimerism with a high level of donor cells were compatible with disease free survival. On the contrary, progressive decrease of donor chimerism value was always followed by hematological relapse or graft rejection. CONCLUSIONS: Sequencial and quantitative detection of donor chimerism may be of great value to study the kinetics of engraftment of NST, to evaluate the status of engraftment, to predict the outcome and prognosis of patients posttransplant and to guide implementation of therapy at an early stage.

[Seventeen cases of nonmyeloablative stem cell transplantation using a conditioning regimen containing fludarabine].

OBJECTIVE: To explore the efficiency and toxicity of non-myeloablative stem cell transplantation (NAST) for hematological disease. METHODS: Seventeen patients, including 3 acute myeloid leukemia, 6 chronic myelogenous leukemia, 4 severe aplastic anemia, 2 non-Hodgkin's lymphoma, 1 multiple myeloma and 1 myelodysplastic syndromes received NAST from HLA-identical sibling donors. Peripheral blood stem cells were mobilized by G-CSF 300 microg/12 hours x 5 d. (2.15 -10.01) x 10(6) CD(34)(+) cells/kg were transplanted. A non-myeloablative conditioning regimen included fludarabine 30 mg.m(-2).d(-1) x 6 d;busulfan 4 mg.kg(-1).d(-1) x 2 d or cyclophosphamide 50 mg.kg(-1).d(-1) x 2 d and antilymphocytic globulin 12 approximately 15 mg.kg(-1).d(-1) x 4 d. Cyclosporin A was used to prevent graft versus host disease (GVHD) alone and no G-CSF was administered after NAST. RESULT: Hematopoiesis reconstitution resumed on day 8 to day 19 (average of day 13). Severe mucositis was absent. Hepatic venoocclusive disease did not occur. Infectious complications were rare. Acute and chronic GVHD each occurred in 5 patients. Idiopathic pneumonia was developed in 5 patients. In the follow-up duration of 120 to 425 days, 16 of the 17 cases had a stable mixed or complete chimerical states. Fourteen of 17 patients are alive. CONCLUSION: NAST is an effective therapy in the treatment of hematological diseases with less complications, less blood transfusion and lower cost.

[Electroporation-mediated gene transduction and expression of class 1 aldehyde dehydrogenase (ALDH1) and multidrug resistance gene (MDR1)].

BACKGROUND & OBJECTIVE: This study was designed to seek an efficient and safe method for transfer of genes of large size. METHODS: The retroviral vector containing different kinds drug resistance genes-class 1 aldehyde dehydrogenase (ALDH1) and multidrug resistance gene (MDR1) was linearized by the restriction enzyme NdeI digestion, and introduced into the packaging PA317 cells by electroporation. Selection was performed by vincristine (VCR) and 4-hydroperoxycyclophosphamide(4-HC) to obtain ALDH1 and MDR1 stably expressing cells. The integration of provirus, transcription and translation of foreign genes were confirmed by Southern blot, reverse transcription(RT)-PCR and flow cytometry, respectively. The safety of this delivery system was verified by testing helper virus(envelop gene) using nested PCR. RESULTS: Both ALDH1 and MDR1 were successfully transduced into PA317 cells by electroporation. Stable integration of foreign genes in host cells genome was determined by Southern hybridization blot. The transcription of ALDH1 and MDR1 was demonstrated by RT-PCR. The overexpression of P-glycoprotein encoded by the downstream gene MDR1 with approximately a 4-fold increase in 98% cells was analyzed by flow cytometry. No helper virus can be detected by nested PCR assay. CONCLUSION: These results implicate that the introduction and overexpression of both ALDH1 and MDR1 genes in vitro is attainable by a simple and convenient electroporation method, with the character of safety and high efficiency.

[The supportive effect of primary bone marrow stromal cell layers on retroviral-mediated transduction of human hematopoietic stem/progenitor cells].

To elucidate the effect of established primary bone marrow stromal layers on the gene transduction of human hematopoietic stem/progenitor cells (HSC/HPC), mononuclear cells (MNC) from adult bone marrow were isolated by centrifugation on Ficoll-Hypaque gradients and plated in stromal culture medium. The cells were incubated until passage 4 to establish primary stromal layers. The HSC/HPC prestimulated by cytokines were transduced by retroviral supernatant containing mdr1 gene in presence of irradiated stroma-contact support. Transduced cells were plated in a colony-forming unit assay with and without vincristine (VCR) to assess the efficiency of transduction. Individual colonies were also analyzed by polymerase chain reaction (PCR) for the presence of provirus. The results showed that the mixed adherent cell layers were formed when adult bone marrow stromal cells were incubated for four to six weeks, mainly being composed of fibroblasts. In the presence of stroma-contact support, the average of gene transduction efficiency in marrow-derived progenitors increased 2.1 to 3.3 folds measured by colony-forming assay and/or PCR, significantly higher than those without support of stroma. It is concluded that the presence of bone marrow stroma support in combination with cytokine facilitates augmenting the extent of retroviral-mediated gene transduction.

Aldehyde-dehydrogenase gene-transduced hematopoietic cell line K562 overcomes the cytoxicity of cyclophosphamide in vitro.

The identification of genes inducing resistance to anticancer chemotherapeutic agents and their introduction into hematopoietic cells represents a promising approach to overcome bone marrow toxicity, the limiting factor for most high-dose chemotherapy regimens. Because resistance to cyclophosphamide has been correlated with increased levels of expression of the aldehyde-dehydrogenase (ALDH1) gene in tumor cells lines in vitro, this study tested whether ALDH1 overexpression could directly induce cyclophosphamide resistance. Results showed that a retroviral vector was used to transduce full-length human ALDH1 cDNA into human hematopoietic cell line K562 that was then tested for resistance to 4-hydroxycyclophosphamide (4-HC), an active analogue of cyclophosphamide. Overexpression of the ALDH1 gene resulted in a significant increases in cyclophosphamide resistance in transduced K562 cells (50% inhibition concentration, IC50 = 10 micro mol/L). These findings indicate that ALDH1 overexpression is sufficient to induce cyclophosphamide resistance in vitro and provide a basis for testing the efficacy of ALDH1 gene transduction to protect bone marrow cells from high-dose cyclophosphamide in vivo.

[Multidrug resistance mechanisms in cell line HL-60/VCR].

BACKGROUND & OBJECTIVE: Drug resistance is a major factor in chemotherapeutic failure of leukemia. Multidrug resistant cell lines are the good models for investigating the mechanisms and reversal of acquired drug resistance. This study was designed to explore the multidrug resistance (MDR) mechanisms in cell line HL-60/VCR. METHODS: Flow cytometry and a panel of antibodies were used to analyze the expression of MDR proteins (P-gp, MRP, LRP, BCRP, GST-pi) and apoptosis-modulating proteins (bcl-2, bcl-x, bax, bad) in MDR cell line HL-60/VCR and drug sensitive cell line HL-60. RESULTS: The expression levels of MDR proteins (P-gp, MRP, BCRP, GST-pi) were (18.62, 1.19, 1.50, 1.32-flod) higher in HL-60/VCR than in HL-60, while the expression of LRP level was similar. The levels of apoptosis-modulating proteins(bcl-2, bcl-x, bad) were (2.48, 1.25, 1.08-fold) higher in HL-60/VCR than in HL-60, while the pro-apoptosis protein bax contrarily decreased in HL-60/VCR. CONCLUSION: Various MDR mechanisms were involved in multi-drug resistance HL-60/VCR cell line, which including increasing expression of drug-resistance protein (P-gp, MRP, BCRP, and GST-pi); the apoptosis-modulating proteins (bcl-2, bcl-x, bax, and bad) might take part in the mechanism of drug resistance.