Chimerism status is correlated to acute graft-versus-host disease after allogeneic stem cell transplantation.

To evaluate the correlation between chimerism status and acute graft-versus-host disease (aGVHD) following allogeneic hematopoietic stem cell transplantation. Chimerism of peripheral blood of 124 patients was monitored at regular intervals post-transplant. The chimerism of 124 post-transplant cases of CD3(+)T lymphocytes, 107 cases of CD3(-)CD56(+)CD16(+)NK lymphocytes, 49 cases of CD15(+) granulocytes, and 27 cases of CD19(+)B lymphocytes sorted by fluorescence-activated cell sorter were analyzed by polymerase chain reaction amplification of short tandem repeats. Differences were found in the time between establishment of full donor T-cell chimerism and the occurrence of aGVHD (P = 0.035, two related samples test). Patients with ≥69 % donor chimerism on day +7 in T-cells had higher rates of aGVHD. This study may provide a rational basis for treatment with adoptive immunotherapy at an earlier time, such as day 7 after SCT, than at present to prevent aGVHD.

JAK2 V617F positive essential thrombocythemia developing in a patient with CD5⁻ chronic lymphocytic leukemia.

Coexistence of chronic lymphocytic leukemia (CLL) and essential thrombocythemia (ET) in a patient is extremely rare, with only 10 cases reported thus far in literature. This paper describes a 94-year-old male having atypical B-CLL with CD5⁻ (CD5⁻) phenotype and ET. In this patient, we performed interphase fluorescence in situ hybridization (FISH) analysis which revealed 13q14.3 deletion in 31% of B-lymphocyte nuclei and RB1 deletion in 27% of B-lymphocyte nuclei, but not in neutrophils and T-lymphocytes. Furthermore, we identified JAK2 V617F mutation in the peripheral blood nucleated cells and neutrophils, but not in the B- and T-lymphocyte populations. Therefore, it was concluded that the occurrence of CD5− B-CLL and ET in this patient was pathogenically independent.

[The clinical evaluation of preemptive treatment of cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To retrospectively analyze the effect of preemptive treatment on cytomegalovirus (CMV) infection in patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: The data of one hundred and three patients who underwent allo-HSCT with preemptive treatment to prevent CMV associated diseases were retrospectively analyzed. Fluorescence quantitative PCR was used to detect CMV-DNA. The incidences of CMV viremia and CMV associated diseases were analyzed. RESULTS: CMV viremia was confirmed 63 times in 51 of the 103 patients. The incidence of CMV viremia was 49.5% and the median time of onset was 40 days after transplantation. All the patients with CMV viremia received preemptive antiviral therapy and 19 of them developed CMV associated diseases, including 14 hemorrhagic cystitis, 3 CMV associated pneumonia and 2 CMV associated enteritis. The total incidence of CMV associated diseases was 18.4%. After treatment with ganciclovir and/or foscarnet, 60 of the 63 times of CMV viremia disappeared. One patient was not included in the analysis because he died of intracranial hemorrhage and GVHD only 3 days after the treatment. The total response rate was 96.8% (60/62). The remaining two cases who did not respond to treatment died of CMV associated pneumonia in combination with acute GVHD. The direct mortality rate of CMV infection was 1.9% (2/103). CONCLUSION: The incidences of CMV viremia and CMV associated diseases do not increase in patients receiving preemptive therapy as compared with those receiving prophylaxis therapy. Preemptive treatment can not only prevent the progression of CMV viremia to CMV associated diseases in majority of the cases but also control CMV associated diseases effectively.

[The role of FDG-PET in staging of lymphoma and evaluation of therapeutic efficiency].

OBJECTIVE: To evaluate the application of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) to the staging and detecting residual masses of lymphoma. METHODS: The clinical data of 179 patients with lymphoma were analyzed retrospectively. The results of FDG-PET, computed tomography (CT) and bone marrow biopsy (BMB) were compared for detection of lymph node/extranodal lymphoid tissue and bone marrow infiltration. Therapeutic efficiency was assessed by International Workshop Criteria (IWC) and Revised Integrated International Workshop Criteria (IWC + PET). RESULTS: In the detection of 286 disease focuses in 98 patients before chemotherapy, the sensitivities of FDG-PET and CT were 73% and 70% (P < 0.01) in detecting nodal focus,and 87% and 45% (P < 0.01) in detecting extranodal lymphoma respectively. In detection of 104 lesions in 81 patients after chemotherapy,the sensitivities of FDG-PET and CT were 81% and 55% respectively (P < 0.01), and the specificities were 68% and 33%, respectively (P < 0.01) in detecting residual masses. According to IWC criteria, 33 patients achieved complete response/unconfirmed complete response (CR/CRu) , and 8 (24%) relapsed. Patients with PET-positive residual masses had a relapse rate of 40%, whereas only 21% of those with no such masses relapsed. Based on IWC + PET criteria, 25 patients achieved CR, with a relapse rate of 20%. Both FDG-PET and BMB produced positive results in 133/179 (74%) patients. Twenty-two patients with positive FDG-PET results were not detected by BMB. The sensitivities and specificities of FDG-PET for BM infiltration were 52% and 83%, respectively. CONCLUSIONS: FDG-PET is a high sensitive and specific technique in staging and detecting residual masses of lymphoma.

[Comparison of STR-PCR and FISH value for monitoring chimerism after sex-mismatched allogeneic hematopoietic stem cell transplantation].

This study was purposed to compare the significance of multiplex short tandem repeat polymerase chain reaction (STR-PCR) and fluorescent in situ hybridization (FISH) for monitoring chimerism after sex-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT). The chimerism of bone marrow or peripheral blood cells from 38 patients was analyzed by STR-PCR and FISH on days 14, 28 and at 3 months after allo-HSCT. The results indicated that on day 14, the complete chimerism (CC) was detected in 14 of 30 cases by STR-PCR and in 8 of 30 cases by FISH (p > 0.05). On day 28, the CC was detected in 26 of 31 cases by STR-PCR and in 15 of 31 cases by FISH (p < 0.01). At 3 months, the CC was observed in 22 of 24 cases by STR-PCR and 17 of 24 cases by FISH (p > 0.05). 14 cases were found to have a graft rejection or relapse among 28 cases which were continuously monitored more than 3 months post the transplants. Donor cell decrease in 9 of 14 cases was proved by FISH alone. It is concluded that FISH is more sensitive than STR-PCR in early monitoring chimerism status of post-transplant and in predicting graft rejection or disease relapse.

[Allogeneic hematopoietic stem cell transplantation following reduced intensity conditioning regimen for treatment of refractory leukemia].

OBJECTIVE: To evaluate the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) following reduced intensity conditioning (RIC) regimen for treatment of refractory leukemia. METHODS: Twenty patients with refractory leukemia received allo-HSCT following RIC regimen consisting of fludarabine plus small or moderate dose total body irradiation (TBI). Graft versus host disease (GVHD) prophylaxis was CsA plus mycophenolate mofetil (MMF) or short-term MTX, or these three drugs combination; CD25 monoclone antibody(McAb) and ATG were also used in some of the patients. RESULTS: Seventeen patients engrafted successfully, the median time for ANC > 0.5 x 10(9)/L was 13 (11 - 17) days, and for BPC > 50 x 10(9)/L 19 (12 -42) days. Detected by short tandem repeat (STR)-PCR, complete donor chimerism was confirmed in 16 patients with a median of 14 (7 -35) days. The incidence of acute and chronic GVHD was 47.1% (8/17) and 38.5% (5/13) respectively. The transplant related mortality (TRM) was 25.0% (5/20), mainly from graft failure, intracranial hemorrhage and severe infection. Up to now, 7 patients relapsed and 9 were alive with leukemia free. The overall survival (OS) at 2 year was (35. 3 +/- 14.2)% for all patients and (52.5 +/- 18.6)% for acute non-lymphocytic leukemia (ANLL) patients. CONCLUSION: Allo-HSCT following fludarabine and TBI based RIC regimen can be used for treatment of refractory leukemia with well tolerance and low TRM and there is a better prognosis for ANLL patients than that for acute lymphocytic leukemia patients.

[Distribution of abnormal cell clone with deletion of chromosome 20q in marrow cell lineages and apoptosis cells in myelodysplastic syndrome].

This study was aimed to investigate the distribution of abnormal clone in marrow cell lineages and apoptosis cells in myelodysplastic syndrome (MDS) with deletion of chromosome 20q. Monoclonal antibodies recognizing myeloid precursors (CD15), erythroid precursors (GPA), T cells (CD3(+)CD56(-)CD16(-)), B cells (CD19), NK cells (CD3(-)CD56(+)CD16(+)) were used to sort bone marrow cells in a MDS patient with del (20q) by fluorescence activated cell sorting (FACS). Annexin V-FITC and PI were used to sort bone marrow Annexin V(+)PI(-) and Annexin V(-)PI(-) cells by FACS. The sorted positive cells were detected by interphase dual-color fluorescence in situ hybridization (D-FISH) using a LSI D20S108 probe (Spectrum Orange) and a Telvysion TM 20p probe (Spectrum Green). FACS and FISH analysis were also performed on the samples from 4 cases with normal karyotype. The results showed that the proportions of MDS clone in the myeloid and erythroid precursors were 70.50% and 93.33% respectively, in the RAEB-1 patient with del (20q) and were obviously higher than that in control group (5.39% and 6.17%). The proportions of abnormal clone in T, B and NK cells were 3.23%, 4.32% and 5.77% respectively and were less than that in control group (5.76%, 4.85%, 6.36%). The percentage of apoptotic cells in the bone marrow nucleated cells was 16.09%. The proportions of MDS clone in Annexin V(+)PI(-) and Annexin V(-)PI(-) cells were 32.48% and 70.11%, respectively. It is concluded that most myeloid and erythroid precursors are originated from the abnormal clone in MDS with del (20q). A little part of apoptotic cells are derived from the abnormal clone.

[Inhibitory effect of Rnai on AML1 -ETO fusion gene expression in leukemia cells].

OBJECTIVE: By inhibiting AML1 -ETO fusion gene expression in Kasumi-1 cells with RNAi, to investigate the changes in cell proliferation and cell cycle. METHODS: The small interference RNAs (siRNAs) specifically targeting the AML1 -ETO fusion gene were synthesized in vitro and transfected into Kasumi-1 cells by electroporation, the non-specific siRNAs transfected cells were taken as control. EGFP plasmid was transfected into Kasumi-1 cell and the transfection efficiency was detected by FCM. Inhibitory effect of siRNAs were detected by real-time RT-PCR and Western blots. Cell proliferation was measured by CCK-8 assay. DNA content was detected by PI assay. RESULTS: The transfection efficiency was 44.5%. The AML1 -ETO specific siRNAs inhibited AML1 -ETO expression at both mRNA and protein levels. The cell proliferation rate in siRNAs treated group was lower than that in control group 72 h after transfection [(47.90 +/- 0.02)% vs (66.90 +/- 0.08)% , P < 0.05]. The cell cycle was blocked at G1 phase 72 h after siRNAs treatment, the cell proportion in G1 phase being 38.3% and 31.6% in control group, while in G2/M phase being 1.8% and 2.4% respectively. CONCLUSIONS: The synthesized siRNAs can inhibit AML1 -ETO fusion gene expression. AML1 -ETO specific siRNA induced the decline of AML1 -ETO fusion protein in Kasumi-1 cell, and then caused the cell cycle blocked in G1 stage and eventually inhibited the cell proliferation.

[Clinical significance of dynamic monitoring of cell chimerism following allogeneic hematopoietic stem cell transplantation].

OBJECTIVE: To evaluate the relationship of chimerism status of cell subsets with engraftment, occurrence of acute graft versus host disease (aGVHD), graft rejection and disease relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: Chimerism status in peripheral blood (PB) and bone marrow (BM) of 65 patients received allo-HSCT were monitored at regular intervals post-transplant. Fluorescence-activated cell sorter (FACS) was used to sort CD3(+)T lymphocytes in 65 cases, CD3(-)CD56(+)CD16(+)NK cells in 52 cases, CD15(+) granulocytes in 32 cases and CD19(+)B lymphocytes in 20 cases post transplants. The chimerism status of different lineage cells was analyzed by polymerase chain reaction amplification of short tandem repeats (PCR-STR). RESULTS: On day +7, NK-cells donor chimerism (DC 55.5%) was higher than other cell subsets. T lymphocyte was the latest one to reach complete donor chimerism (CDC) with a median on day +21. Patients whose T lymphocytes donor chimerism was more than 70% on day +7 and more than 95% on day +14 had a high risk for acute aGVHD. In all cases except those with ALL, the decreased DC of T lymphocytes were observed before molecular or hematological relapse occurred. CONCLUSION: Serial and quantitative T cell chimerism analysis provides a reliable and rapid screening method for the early detection of engraftment, graft rejection, disease relapse and occurrence of aGVHD, therefore, is a prognostic tool to identify patients at high risk of aGVHD and disease relapse following allo-HSCT.

The association of up-regulation of X-linked inhibitor of apoptosis protein with cell adhesion-mediated drug resistance in U937 cells.

An increasing body of evidence indicates that environmental factors may contribute to the drug resistance of acute myeloid leukaemia (AML). CAM-DR (cell adhesion-mediated drug resistance) is a reversible, de novo drug resistance induced by adhesion of tumour cell lines to fibronectin (FN). Adhesion was demonstrated to directly regulate the apoptotic machinery. And it was observed in previous studies that high levels of X-linked inhibitor of apoptosis protein (XIAP) were related to resistance to chemotherapeutics in many cancer cell lines. However, whether XIAP is relevant to CAM-DR of AML cells is unknown. In this report, we demonstrated that the mRNA and protein levels of XIAP were increased by 96.15% and 120.92%, respectively in U937 cells cocultured with FN as compared with controls. Antisense oligonucleotides targeting XIAP down-regulated the expression of XIAP and sensitized U937 cells to daunorubicin. In addition, we investigated the signalling pathway involved in the upregulation of XIAP. The levels of phosphorylated Akt (Ser473) were elevated in U937/FN cells and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 suppressed XIAP expression and restored the chemosensitivity to daunorubicin. Our findings suggested that adhesion-dependent activation of the PI3K/Akt/XIAP pathway may be one of the factors involved in the CAM-DR of U937 cells. Targeting this pathway may be a useful approach to improve the therapeutic responsiveness of leukaemia cells.

[Detection of cytogenetic abnormalities involving chromosomes 5,7 and 8 in myelodysplastic syndromes with fluorescence in situ hybridization and its clinical significance].

OBJECTIVE: To identify the abnormal karyotypes by fluorescence in situ hybridization (FISH) and explore prognostic implications in patients with myelodysplastic syndromes (MDS). METHODS: FISH was used to detect the frequently occurring chromosome abnormalities (-5/5q, +8, -7/7q-) in 37 MDS cases. SPSS 11.5 software and correlation analysis were used to analyze the relativity among the abnormal chromosomes, the prognosis and the disease conversion in 37 MDS patients. RESULTS: Karyotype abnormalities were found in 21 (56.8%) of 37 cases, among which 6 (16.2%) were complex karyotypes, 9 (24.3%) +8, 2(5.4%) -5/5q-, 2(5.4%) -7/7q-. In the median time of follow-up of 12 months, 12 cases transformed into acute leukemia. Complex karyotypes were significantly associated with the poor prognosis and leukemia transformation. + 8 and -7/7q- abnormalities were correlated with the death. CONCLUSIONS: FISH was more sensitive than conventional cytogenetics for detecting mini-clonal abnormality. There are some differences in abnormal karyotypes between patients in China and the western countries. Multi-probes used in cytogenetic detections may predict the patient' s prognosis more accurately. The higher proportion of abnormal karyotypes the poorer prognosis.

XIAP is upregulated in HL-60 cells cocultured with stromal cells by direct cell contact.

Apoptosis resistance is an important mechanisms of drug resistance mediated by bone marrow stromal cells (BMSCs). BMSCs influence tumor cells survival through several mechanisms including direct cell-cell contact and the effects of soluble factors. In this research we investigated the role of X-linked inhibitor of apoptosis protein (XIAP) in the apoptosis resistance mediated by stromal cells in HL-60 cells and the signaling pathway involved. We found that bone marrow stromal-derived soluble factors and direct cell contact both prevented apoptosis of HL-60 cells. XIAP is upregulated by direct cell contact but not by soluble factors. Phosphatidylinositol 3-kinase (PI3K) and Akt were activated and LY294002 downregulated XIAP and increased apoptosis in HL-60 cells co-cultured with BMSCs. The results indicated that PI3K/Akt/XIAP is an important pathway involved in the apoptosis resistance of HL-60 cells co-cultured with BMSCs by direct cell contact. Inhibition of this signaling pathway may provide a new therapeutic strategy for acute myeloid leukemia treatment.

Simultaneous suppression of multidrug resistance and antiapoptotic cellular defense induces apoptosis in chemoresistant human acute myeloid leukemia cells.

The emergence of acquired drug resistance is a major hurdle in the successful treatment of leukemia. Researches indicated that the main mechanisms of most cancers included so-called "pump" and "nonpump" resistance. We studied the mechanisms involved in the drug resistance of HL-60/ADR and found that its drug resistance was associated with the simultaneous overexpression of XIAP and MRP. We compared the reversal effects of XIAP and MRP ASO used in combination and separately. Reverse transcription-PCR and Western blot were applied to examine the changes of mRNA and protein levels, respectively. The results showed that XIAP and MRP ASO used separately could down-regulate the expression of XIAP and MRP in HL-60/ADR cells, respectively. XIAP and MRP ASO used in combination did not enhance the inhibition expression of XIAP and MRP of HL-60/ADR cells. The apoptosis of co-transfection group was significantly higher than XIAP ASO (P<0.05). The cytotoxicity was determined by MTT cell viability/proliferation assay. When used in combination the sensitivity of HL-60/ADR cells to DNR was increased significantly compared with XIAP or MRP ASO used separately (P<0.05). The results indicated that both XIAP and MRP were involved in the drug resistance mechanisms of HL-60/ADR cells. The sensitivity to DNR could be enhanced significantly when XIAP and MRP ASO used in combination.

[Prognostic factor analysis of 77 old patients with acute myelogenous leukemia].

BACKGROUND & OBJECTIVE: The manifestations of old acute myelogenous (AML) patients have their special biological and clinical characteristics, with lower response rate to therapy and shorter survival time. This study was to investigate the prognostic factors of elderly patients with AML retrospectively. METHODS: 77 patients aged> or =60 years with AML from 1994 to 2005 were admitted to our study and all the possible prognostic factors were analyzed with Kaplan-Meier survival analysis. The significant factors were further analyzed by Cox proportional hazard model analysis. RESULTS: Seventy-two patients were evaluated. The patients aged 60-70 (median survival time was 350 days) had significantly longer survival time than those aged more than 70 (median survival time is 60 days)(P<0.001), which their CR ratios were 71.4% and 29.4% (P=0.001). The patients with performance status 0 or 1 (median survival time was 402 days) had significantly longer survival time than those with performance status 2, 3 or 4 (median survival time was 31 days)(P<0.001), which their CR ratios were 75% and 15% (P<0.001). The patients with primary AML (median survival time was 98 days) had significantly longer survival time than those with secondary AML (median survival time was 32 days)(P=0.007), which their CR ratios were 50% and 0% (P=0.023). The patients treated with sub-standard dosage of anthracycline (median survival time was 293 days) had significantly longer survival time than those treated with reduced dosage of anthracycline (median survival time was 35 days)(P=0.006), which their CR ratios were 63.6% and 33.3% (P=0.02). The patients with bone marrow blast cell ratio< or =50% (median survival time was 98 days ) had significantly longer survival time than those with bone marrow blast cell ratio >50% (median survival time was 55 days)(P=0.006). The patients with favorable karyotype (median survival time was 293 days) had significantly longer survival time than those with unfavorable or normal karyotype (median survival time was 31 days)(P=0.005). The patients without CD34 expression (median survival time was 201 days) had significantly longer survival time than those with CD34 expression(median survival time was 36 days)(P<0.001). The patients with the peripheral blood white blood cell count (PBWBC)>10x10(9)/L (50%) had significantly higher CR ratio than those with PBWBC< or =10x10(9)/L (25%)(P=0.043). The patients received chemotherapy (50%) had significantly higher CR ratio than those received supportive therapy (0%)(P=0.001). In the stepwise COX proportional hazard regression model, all the seven factors related to OS remained independent and significant. CONCLUSIONS: Factors, including age >70, PS 2 to 4, percentage of blasts in bone marrow >50%, secondary AML, unfavorable karyotype, expression of CD34, lower dosage.

[Evaluation of the subsets of lymphocytes and their activated status in patients with myelodysplastic syndrome].

This study was purposed to investigate the clinical significance of the amount and activated status of T cell subsets, B cells, NK cells in peripheral blood from patients with myelodysplastic syndrome (MDS). The proportion of T cells, B cells, NK cells in peripheral blood from 30 patients with MDS and their surface activation markers of CD28, CD45RA, CD45RO, CD69, HLA-DR were analyzed by flow cytometry. Twenty-two patients were in the low risk group (RA + RAS) while eight patients were in the high risk group (RAEB + RAEBT). The result showed that the amounts of T cells (CD3+ cells) in peripheral blood from patients with MDS were lower than those in control group. The amounts of naive CD4+ cells (CD4+ CD45RA+ cells) in MDS patients were lower than those in control. The expression rates of early activation marker (CD69) and late activation marker (HLA-DR) on CD3+ cells in MDS patients were significantly higher than those in control. The abnormalities of the immunologically competent cells were mainly observed in the low risk group (RA + RAS), and were characterized by the high expression rates of CD69+ and HLA-DR+ on CD3+ cells, the decrease of B cell amounts. The amount abnormalities of T cell subsets were mainly observed in high risk group (RAEB + RAEBT), and were characterized by the decrease of CD3+ cells and CD3+ CD4+ CD8- cells (Th cells) amounts without high expression of the CD69 and HLA-DR, the decrease of NK cells amounts. It is concluded that there are the abnormalities of T cell subsets and function in the patients with MDS and may change with disease progression, so the measurement of amount and activated status of T cell subsets in peripheral blood from MDS patients can have predictive role for diagnosis of disease progression and guide of therapy.

[An analysis of leukocyte subsets chimerism following allogeneic peripheral blood stem cell transplantation].

OBJECTIVE: To analyse the relationship of T lymphocyte and granulocyte chimerism following allogeneic peripheral blood cell transplantation and the occurrence of relapse, graft failure and graft versus host disease. METHODS: 21 patients underwent allogeneic peripheral blood stem cell transplantation (allo-PBSCT). Fluorescence-activated cell sorter (FACS) sorted CD3+ T lymphocytes and CD15+ granulocyte from peripheral blood of all the patients were analyzed for short tandem repeats in 7 days interval for 1 month starting from the day of PBSCT, then 1 month interval for 6 months, and then 3 months interval to the end of one year. RESULTS: Chimerism of granulocyte was higher than T lymphocyte on day 7 posttransplant in 4 patients given myeloablative conditioning. The median donor chimerism of granulocyte and T lymphocyte was 95% and 55% respectively. The other 17 patients had higher chimerism of T lymphocyte than granulocyte on day 7, which was 60% (15%-76%) and 0% (0%-40%) respectively. 20 patients reached complete donor chimerism (CDC) on day 21 (14-102 days) for T lymphocyte and on day 14 for granulocyte, except one relapsed on day 28. Seven patients had decreasing mixed chimerism when disease relapsed or graft failure occurred. T cell donor chimerism decreased earlier than myeloid cells, however, bone marrow sample and granulocyte still remained in complete donor chimerism or stable mixed chimerism, bone marrow smear showed normal at the same time. CONCLUSION: Blood leukocyte subset chimerism analysis, especially T cell chimerism analysis may provide earlier information of engraftment, relapse and graft failure than blood and bone marrow samples, therefore immunomodulatory therapies may be given to recipients earlier and overall survival may be improved.

[Role of XIAP in the drug resistance of HL-60 cells].

OBJECTIVE: To explore the role of X-linked inhibitor of apoptosis protein (XIAP) in the fibronectin (Fn)-adhesion mediated drug resistance of HL-60 cells. METHODS: Culture plates were coated with Fn and bovine serum albumin (BSA) (as control), respectively. Colorimetric CCK-8 assay was used to determine the effects of Fn on the cytotoxicity of DNR to HL-60 cells. Intracellular DNR accumulation was assayed with flow cytometry. Reverse transcription-PCR and Western blot were used to examine the mRNA expression and XIAP, bcl-2, MRP and mdr1 proteins, respectively. HL-60 cells were added to Fn coated Culture plates. The fully phosphorothioate antisense oligonucleotide (AS-ODNs) and the control ODNs of XIAP were delivered into HL-60 cells in the form of liposome-ODN complexes. IC(50) was calculated by linear regression of survival percent versus drug concentration. RESULTS: HL-60 cells adhered to Fn-coated plates had a significant survival advantage over those grown on BSA coated plates and in suspension when exposed to DNR, the IC(50) of Fn group being significantly higher than that of BSA group and suspension group (0.526 micromol/L vs 0.132 micromol/L, 0.123 micromol/L, respectively, P < 0.05). XIAP was up-regulated significantly in Fn group compared with BSA group and suspension group (P < 0.05), whereas there was no difference in the expressions of bcl-2, MRP and mdr1 among the three groups (P > 0.05). The intracellular concentration of DNR in Fn-adhered HL-60 cells was similar to that in BSA group and suspension group (P < 0.05). AS-ODNs of XIAP down-regulated the XIAP expression in Fn-adhered HL-60 cells. In addition, AS-ODNs sensitized HL-60 cells to the cytotoxic effects of DNR. CONCLUSION: The increased XIAP protein level contributes to the drug resistance induced by adhesion to Fn. AS-ODNs of XIAP might reverse the drug resistance.

[Partial deletion of chromosome 13 long arm in multiple myeloma and its clinical significance].

OBJECTIVE: To explore the specific locus deletion of the long arm of chromosome 13 and its relationship with the clinical behavior and prognosis of multiple myeloma (MM). METHODS: FISH analysis was performed on bone marrow smears from 68 patients with MM to study the deletion of Rb-1 gene and locus 13q14.3 on chromosome 13. The statistic value of its effect on clinical features were determined. RESULTS: 35 out of the 68 (51%) cases were found with deletion of chromosome 13; deletion of Rb-1 gene were found in 29 (43%) cases; deletion of locus 13q14.3 were found in 23 out of 44 (52%) cases; the analysis results were same in 66% of the cases (29/44) with the above two probes. Chi-square test showed that partial deletion of chromosome 13 was associated with clinical behavior, early chemotherapy response and 1 year survival. CONCLUSION: Deletion of Rb-1 gene and locus 13q14.3 were both common cytogenetic changes in MM patients with effect on the biological behavior of the disease.

[A modified cytogenetic study for multiple myeloma].

OBJECTIVE: To evaluate the effect of modified culture method used to cytogenetic analysis and the clinically significance of chromosomal abnormalities to multiple myeloma (MM). METHODS: Mononuclear cells were isolated from bone marrow aspirate of 20 MM patients; and then cultured for 3 days without any cytokines, and 6 days in the presence of IL-6 (10 ng/mL) and GM-CSF (30 ng/mL) before RHG banding analysis; the remained part of aspirates were treated directly. Eight cases of iron deficiency anemia were taken as control. RESULTS: The experiment was failure in 2 cases because of blood clot, and another 2 cases could be analyzed only by direct method due to inadequate cells. The karyotype abnormalities were found from 4 cases of 16 available patients. Of them, three cases had complex karyotypes. The abnormalities were detected after 6 days culture with addition of cytokines. No abnormalities were detected from those groups of directly analysis and 3 day culture. Meantime, the clinical data showed that the patients with cytogenetic abnormalities were in stage III, and had a high percentage of MM cells (25%-56%) in their bone marrow, and also poor responses to prior chemotherapy. No cytogenetic abnormalities were found from control individuals in all groups. CONCLUSION: Extended culture in the presence of cytokines could improve the efficiency of cytogenetic analysis to MM. Complex karyotype was common cytogenetic abnormalities in MM patients with poor response to chemotherapy.

[Drug resistance reversal of HL-60/ADR cells by simultaneous suppression of XIAP and MRP].

This study was purposed to explore the mechanisms of drug resistance of HL-60/ADR cells and to compare the reversal drug-resistance effects of antisense oligonucleotides (AS ODN) of XIAP (X-linked inhibitor of apoptosis protein) and AS ODNs of MRP (multidrug resistance-associated protein) by use alone or in combination. Reverse transcription-PCR and Western blot were applied to detect the expression of XIAP, BCL-2, MRP and MDR1 in mRNA and protein levels of HL-60 cells and HL-60/ADR cells, respectively. Fully phosphorothioated AS ODN of XIAP and MRP was delivered into HL-60/ADR cells with Lipofectamine 2000 in the form of liposome-ODN complexes alone or in combination. CCK-8 cell viability assay was used to determine the effect of AS ODN of XIAP and MRP used alone or in combination on the chemotherapy sensitivity of HL-60/ADR cells to daunorubicin (DNR). Reverse transcription-PCR and Western blot were applied to examine the changes of XIAP, MRP in mRNA and protein levels respectively. The results showed that MRP and XIAP were both significantly higher in HL-60/ADR cells than those in HL-60 cells. AS ODN of XIAP and MRP down-regulated the expression of XIAP and MRP in HL-60/ADR cells and increased the sensitivity of HL-60/ADR cells to DNR, respectively. AS ODN of XIAP + MRP did not enhance the inhibition expression of XIAP in HL-60/ADR cells but increased the sensitivity of HL-60/ADR cells to DNR significantly as compared with AS ODN of XIAP (P < 0.05). AS ODN of XIAP + MRP did not increase the concentration of DNR nor enhanced the inhibition expression of MRP in HL-60/ADR cells but increased the sensitivity of HL-60/ADR cells to DNR significantly (P < 0.05), as compared with AS ODN of MRP. It is concluded that both XIAP and MRP may be involved in the drug resistance mechanisms of HL-60/ADR cells. Drug-resistance of HL-60/ADR cells can be reversed significantly when antisense oligonucleotides of XIAP and MRP were used in combination.