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To elucidate the possible mechanism of differentiation and/or apoptosis in NB4, K562 cells induced by tributyrin (TB), a histone deacetylase inhibitor (HDACi), the level of acetylated histone H3 was detected by Western blot, p21(WAF1) expression was detected by semi-quantitative RT-PCR. The results showed that histone H3 hyperacetylation was detected in NB4 (or K562) cells after treatment with TB 0.1 mmol/L (or TB 0.5 mmol/L) for 16 hours in a dose-dependent manner. p21(WAF1) dose-dependently increased at RNA level in these two cell lines treated by TB 0.1 mmol/L. The level of p21(WAF1) mRNA increased at 2 hours after TB treatment, reaching peak at 16 hours, and maintaining for 48 hours. In conclusion, the mechanism of differentiation and apoptosis in NB4, K562 cells induced by tributyrin may associate with its up-regulation of acetylated histone and p21(WAF1) mRNA level.

Objective: To detect the expression of the fusion genes resulting from chromosome abnormalities in childhood acute lymphoblastic leukemia(ALL) and its conformity to WHO classification., Methods: Sixty-two children with ALL were investigated. The expression of fusion genes was determined by multiplex reverse transcription-polymerase chain reaction (RT-PCR), karyotyping (R band) and immunophenotyping (by flow cytometry) were also performed., Results: Of the 62 patients, 23(37.1%) were found to carry 13 different fusion genes. The patients with immunophenotype of Pre-B-ALL were found to carry: TEL/AML1(3 cases); E2A/PBX1, E2A/HLF, TLS/ERG, MLL/AF4, MLL/AF9, MLL/AF10, MLL/AFX-MLL/AF6-MLL/ELL, MLL/AF6-MLL/ELL, dupMLL (one case for each); and HOX11 (6 cases). The patients with immunophenotype of Pre-T-ALL were found to carry: TAL1D (4 cases, one is also found to have HOX11 expression); and HOX11 (2 cases). The multiplex RT-PCR in combination with chromosome analysis revealed genetic abnormalities in 69.4%(43/62) of childhood ALL., Conclusion: Multiplex RT-PCR combined with chromosome analysis and immunophenotyping can provide reliable and helpful information for the diagnosis, therapy evaluation and prognosis prediction in childhood ALL, which may also serve as a basis on which to implement the criteria of WHO classification.

Objective: To study the DNA methylation status of WT1 gene promoter region in hematologic malignancy cell lines and its correlation with WT1 gene expression., Methods: 1. RT-PCR and methylation-specific PCR were performed for detecting WT1 gene expression and DNA methylation status in its promoter region in 8226, HL-60, Jurkat, K562, KG-1, NB4, Raji, SHI-1, U266 and U937 cell lines. 2. Treatment of U937 cells with 5-aza-CdR, a demethylation inducing agent and the changes in WT1 gene expression level and its promoter region methylation status were determined., Results: 1. HL-60, K562, KG-1, NB4 and SHI-1 cells showed higher levels while 8226, Jurkat, Raji, U266 and U937 cells showed extremely low levels of WT1 expression. DNA hypermethylation in WT1 gene promoter region was identified in 8226, Jurkat, Raji, U266 and U937 cells. 2. The WT1 gene expression in U937 was enhanced after treatment with 5-aza-CdR accompanied with the decrease of methylated and the increase of unmethylated levels in its promoter region., Conclusion: Modulation of the DNA methylation status in WT1 promoter region is one of the epigenetic mechanisms for regulating its expression.

Objective: To study the potential effects of exogenous WT1 gene isoform on the differentiation of leukemia cell line NB4 and its possible molecular mechanisms., Methods: The recombinant eukaryotic expression vector (pCB6 + /WTA) containing full-length human WT1 isoform (WTA: -17AA/ -KTS) cDNA and the blank pCB6 + vector were transfected into leukemia cell line NB4 by electroporation. The WTA mRNA and protein in cells were detected by RT-PCR and Western blot. Cell morphology, NBT reduction and CD11b antigen expression in NB4 cells were assayed to evaluate cell differentiation. Expression of PML/RARalpha, p21 and c-myc genes was determined by semi-quantitative RT-PCR after transfection., Results: Compared with NB4/WTA cells, NB4 and NB4/CMV (NB4 cells transfected with pCB6 + vector) cells had higher morphological differentiation rates and higher CD11b expression levels after exposure to ATRA for 48 hours. The percentage of NBT reduction in NB4/WTA cells was lower than that in control groups. The difference in NBT reduction rate between NB4/WTA and control cells was gradually increased after treated with ATRA for three days. The expression levels of PML/RARalpha, p21 and c-myc genes in NB4/WTA cells were notably increased., Conclusion: Overexpression of exogenous WTA gene could partially inhibit the differentiation of NB4 cells by up-regulating the expression of PML/RARalpha, p21 and c-myc genes.

Objective: To study the clinical and laboratory features of childhood acute leukemia (AL) with MLL gene rearrangements., Methods: Sixteen of 298 cases of childhood AL with MLL rearrangements were studied by using MLL dual-color FISH, multiplex RT-PCR with 13 pairs of primers in combination with R banding karyotype analysis and cell immunophenotyping by flow cytometry., Results: Sixteen cases of childhood AL with MLL rearrangements accounted for 5.4% of 298 AL patients, and 56.3% of infant ALs. Among 106 cases analyzed by multiplex RT-PCR, MLL gene rearrangements were found in 11 cases, including MLL/AF4 fusion gene in 2, MLL/AF6 fusion gene in 1, MLL/AF6 and MLL/ELL combined with MLL/ AFX or HOX11 in one case each, MLL/AF9 in 2, MLL/AF10 in 1, MLL/ELL in 2. MLL partial tandem duplication in 1 and activated HOX11 in 1. In 27 cases assayed by FISH, 9 cases (36.0%) were demonstrated MLL gene rearrangements. In 16 patients with MLL gene rearrangements, 14 (87.5%) exhibited clonal chromosome abnormalities involved chromosome 11 in 11 cases: being t(4;11) in 2, t(6;11), t(8;11), t(7;8;11), t(9;11) in each trisomy 11 in 2 and 11q--in 3 cases. Among these 16 patients, 11 were B-ALL, and 5 AML-M5, 3 of the latter were CD7+ and CD2+. Of these 16 patients, 8 received chemotherapy and 7 of them achieved complete remission, while the other 8 patients gave up treatment., Conclusion: Multiplex RT-PCR combined with FISH provided a more accurate and sensitive method for detection of MLL gene rearrangements. Finding out MLL gene rearrangement is of most importance in guiding therapy and predicting prognosis in childhood AL.

Objective: To investigate retinoblastoma (Rb) associated protein 46 (RbAp46) gene expression levels in bone marrow (BM) cells of leukemia patients., Methods: Real-time quantitative reverse polymerase chain reaction (QRT-PCR) method was used for detecting RbAp46 expression levels in BM cells of 140 patients with acute leukemia (AL), 13 with chronic myelogenous leukemia in chronic phase (CML-CP), 7 with CML in blast crisis (CML-BC) and 32 with non-leukemic disorders., Results: The M-Estimators of RbAp46 were higher in 98 newly diagnosed ALs and 5 relapsed ALs than in 28 ALs in complete remission (CR) and 32 non-leukemic controls (178.23 and 213.65 vs 85.89 and 88.08, respectively). No statistic difference was found between the CR group and control group, or between the newly diagnosed group and relapsed group. The M-Estimators of RbAp46 in patients with CML-CP was 58.27, similar to that in control, but much lower than that in CML-BC (173.24). Among 98 newly diagnosed ALs, the M-Estimators of RbAp46 in M(3) and M(4) were the lowest in all of the subtypes. Furthermore, the RbAp46 expression levels were not correlated with the expression of the fusion genes of bcr/abl, PML-RARalpha, and multidrug resistant gene (mdr1), but were positively correlated with Wilms' tumor gene (WT1) expression levels and negatively with AML1/ETO fusion gene expression., Conclusion: RbAp46 expression levels in ALs and CML-BC were strikingly higher than that in non-leukemias and CML-CP, and might participate in leukemogenesis.

Objective: To investigate the significance of monitoring Wilms' tumor gene (WT1) expression level in bone marrow of leukemia patients following allogeneic bone marrow transplantation (allo-BMT)., Methods: Real-time quantitative reverse transcription polymerase chain reaction method was established for measuring WT1 and GAPDH expression levels in bone marrow cells of 15 patients with leukemia, including a total of 111 specimens during the follow-up, and in 23 non-leukemia patients by using LightCycler. Normalized WT1 expression level (WT1(N)) was determined as a ratio of WT1 to GAPDH times 10(4)., Results: The median expression levels of WT1(N) in 17 samples of newly diagnosed patients, 6 samples of relapsed patients, 88 samples from leukemia patient in complete remission and 23 samples of non-leukemic controls were 40.18 (5.48 to 510.27), 125.89 (34.50 to 273.95), 4.80 (0 to 56.96) and 1.47 (0 to 8.56) respectively. Nonparameter statistic analysis (Mann-Whitney U test) showed that the WT1(N) expression levels in the newly diagnosed group and relapsed group were statistically higher than in those in the complete remission group and non-leukemic controls (all P < 0.01), without significant differences between the complete remission group and control group (P = 0.692) and between the newly diagnosed group and relapsed group (P = 0.595). In general, the dynamic curves of WT1(N) levels following allo-BMT were consistent with the tendency of changes in expression levels of corresponding fusion genes for minimal residual disease (MRD) monitoring. Spearman Rho correlation analysis revealed that the correlation coefficient between the WT1(N) expression levels and BCR/ABL, AML/ETO, PML/RARalpha and MLL/AF17 fusion genes expression were 0.678 (P = 0.00), 0.677 (P = 0.00), 0.806 (P = 0.00) and 0.553 (P = 0.049) respectively. Three patients relapsed after allo-BMT and one patient relapsed before allo-BMT during the follow-up. A re-increment of WT1(N) expression level during follow-up could be detected 40 to 180 days earlier to hematological relapse., Conclusion: The WT1 expression level of leukemia patients following allo-BMT measured by real time RT-PCR can be a useful tool for monitoring MRD and warning the clinical relapse during follow-up.

Objective: To investigate Wilms' tumor gene (WT1) expression levels in bone marrow (BM) of acute leukemia patients (ALs)., Methods: A real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) method was established for detecting WT1 and internal reference GAPDH expression levels in BM of 108 ALs and 23 non-leukemia controls by Light Cycler., Results: The median expression levels of WT1 in 70 newly diagnosed ALs and 11 relapsed ALs were statistically higher than those in 23 ALs in complete remission (CR) and 23 non-leukemic controls (75.10 and 89.56 vs 2.07 and 1.51 respectively). No statistic differences was found between the CR group and control group, nor between the newly diagnosed group and relapsed group. Of the 70 newly diagnosed ALs, median WT1 expression level of acute granulocytic leukemias was significantly higher than that of acute monocytic leukemias (M(5)), but there was no statistic differences among the M(1), M(2), M(3) and ALL subtypes. Furthermore the WT1 levels were not correlated to peripheral WBC counts, BM blast percentage and multidrug resistant gene (mdr1) expression at presentation, but correlated to chromosome karyotypes. Dynamic analysis of WT1 levels of 2 patients on treatment showed that WT1 expression levels predicted relapse., Conclusion: WT1 expression levels in ALs were strikingly higher than that in non-leukemias. WT1 can be a marker for detecting MRD and evaluating therapy efficacy in leukemias.

Objective: To investigate the effects of tributyrin (TB), a histone deacetylase inhibitor, on the growth, differentiation and apoptosis of SHI-1 leukemia cells and explore its possible mechanism., Method: Cell proliferation and viability were determined by cell counting, trypan blue dye exclusion. Cell morphological analysis, Annexin binding, DNA electrophoresis, expression of CD11b and CD14, NBT reduction were performed to evaluate differentiation and apoptosis of SHI-1 cells. The level of acetylated histone H3 was detected by Western blot and p21(WAF1/CIP1) expression by semi-quantitative RT-PCR., Results: TB inhibited the proliferation and viability of SHI-1 cells in a time-dose dependent manner. The morphology of SHI-1 cells cultured in the presence of 0.1 mmol/L TB for 72 hs was more mature with higher NBT positivity and up-regulated expressions of CD11b and CD14 than that of control group. Exposed to 0.5 - 1.0 mmol/L TB for 48 hs, SHI-1 cells exhibited the morphological hallmarks of apoptosis, the increasing of Annexin binding and the DNA ladder on gel electrophoresis. The level of acetylated histone H3 and p21(WAF1) mRNA extracted from SHI-1 cells were increased by the treatment of TB., Conclusion: TB can inhibit proliferation, induce differentiation and apoptosis of SHI-1 cells. The mechanism may associate with its up-regulation of acetylated histone and the expression of p21(WAF1).

cDNA microarray, recently applied to analyze gene expression profile of cancers, was difficult to be utilized in myelodysplastic syndrome (MDS) for a special need of excessive mRNA hardly provided by ordinary bone marrow aspiration in MDS patients. The aim of this study was to investigate the feasibility of exploring the molecular events underlying MDS by using cDNA microarray and mixing mRNA from multiple patients. A commercially purchased BioStar H141 cDNA microarray containing 14,110 clones of cDNA or EST was employed to analyze the gene expression profile of bone marrow mononuclear cells from two cases of MDS. Equal amount of total RNA from each patient was mixed, reversely transcribed to cDNA and labeled with Cy5. Mixture of Cy5-labeled cDNA and Cy3-labeled cDNA from normal bone marrow cells was concomitantly hybridized to H141 microarray in duplicate. In H141 chips, 1,064 cDNAs were spotted at least twice targeting different fragments of a single gene cDNA. The results showed that among these 1,064 cDNA clones, the expression level of 625 (58.7%) and 630 (59.2%) ones was consistent within these two chips, respectively, 297 (27.9%) and 191 (18.0%) inconsistent, 21 (2.0%) and 11 (1.0%) in opposite. Among 411 duplicately spotted cDNAs with complete data, expression levels of 400 (97.3%) was consistent between two chips. 488 genes with known function were identified as differentially expressed in MDS, among which 101 genes were involved in hematopoiesis regulation, including those encoding transcription factors, cell cycle-regulating proteins, metabolism-relating enzymes, and adhesive molecules. It is concluded that cDNA microarray can be used for profiling gene expression of mixed MDS samples and replication shall be necessary for reducing the data bias caused by experimental operation.

Objective: To study the gene expression profiles of patients with myelodysplastic syndrome (MDS) and try to identify some genes with pathogenetic and diagnostic relevance., Methods: The bone marrow mononuclear cells (BMMNCs) of 10 patients with MDS, including 4 cases of refractory anemia (RA), 1 case of refractory thrombocytopenia (RTC), 4 cases of RA with excess blasts (RAEB), and 1 case of RAEB in transformation (RAEBt), were isolated and the total RNA was extracted and underwent transcriptome analysis by using customized cDNA microarray with 500 gene clones. Seventeen specimens of normal bone marrow, as controls, were collected from the ribs of 17 patients with chest tumors. Cluster software was used to make analysis. Semiquantitative RT-PCR was carried out to confirm the results of microarray analysis., Results: 95 genes, such as the genes of thrombospondin 1 (THBS1), phosphatase and tensin homolog (PTEN), MAX dimerization protein (MAD), DNA-damage-inducible transcript 3 (DDIT3), ets variant gene 1 (ETV1), G1 to S phase transition 1 (GSPT1), were shown to be abnormally expressed in at least 5 MDS patients compared to the normal controls, involving cell growth and differentiation regulation, cell cycle control, signaling, and redox. The 10 MDS patients in different stages were clustered into two distinct groups, whereas a case with refractory thrombocytopenia and other RA patients were clustered into two subgroups. Semiquantitative RT-PCR revealed the identical results in 3 (60%) of the 5 genes determined by microarray analysis. Further analysis on samples from 50 MDS patients confirmed the different expression levels of RNA helicase-related protein (RNAHP), GSPT1, and DDIT3 between the MDS patients and the normal controls., Conclusion: The technology of microarray can reveal the intrinsic molecular features of MDS patients and the detection of DDIT3 and RNAHP expression may be useful for the diagnosis of MDS.

To establish leukemic cell lines stably transfected by RbAp46 gene, electroporation was performed after optimizing the transfection condition for suspended cells. Under conditions of low voltage and high capacitance, RbAp46 was transfected into U937 by electroporation. Individual clones selected with G418 for 3 weeks were isolated. The integration and the protein levels of the exogenous RbAp46 in transfectants were determined by PCR and Western blot analysis, respectively. The subclone expressing high level of RbAp46 was then established. Viability of transfected cells was assayed by trypan blue exclusion. Cell number was counted daily to determine the growth rate. The results showed that growth rate of U937 cell lines expressing exogenous RbAp46 was about 50% lower than that in control. It is concluded that leukemic cell lines stably expressing exogenous RbAp46 were established and overexpression of RbAp46 inhibits the growth of U937 leukemic cells.

Objective: To study the expression of CXCR4 in acute leukemic cells and its clinical significance., Method: Bone marrow samples from 73 acute leukemia patients and leukemic cell lines were investigated by flow cytometry (FCM), the expression of SDF-1 in human marrow stromal cells and meninges were studied by using reverse transcription polymerase chain reaction (RT-PCR). Adhesion, migration and invasion of U937, NB4 and K562 cells were studied in vitro., Results: The expression rates of CXCR4 in ALL and AML patients was 65.6% and 17.1%, respectively. And it was 0.2%, 41.0% and 52.0% in K562, U937 and NB4 cells, respectively. The extramedullary infiltration rates were 61.9% and 18.2% for CXCR4 positive and negative groups of ALL, respectively (P < 0.05); while in AML, the number of peripheral white blood cells in CXCR4 positive group was lower than that in CXCR4 negative group (P < 0.05). SDF-1alpha could enhance the adhesion, migration and invasion capacity of leukemic cells in vitro., Conclusion: Overexpression of CXCR4 in AL cells might be the molecular mechanism of extramedullary infiltration in leukemia.

Background: This study was designed to quantitatively measure WT-1 expression levels in patients with chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) during follow-up and to clarify the value of WT-1 as a molecular marker in minimal residual disease monitoring., Methods: The TaqMan quantitative real-time RT-PCR method was established by using cloned WT-1 cDNA or synthesized oligonucleotides resembling WT-1 cDNA fragments in limit dilution as template until a stable and reliable standard curve was obtained. In a 25-month follow-up, the transcriptional levels of WT-1, Bcr-Abl, and Abl gene, were quantitatively measured in bone marrow cells from 25 CML or acute lymphoblastic leukemia (ALL) patients with the Ph chromosome. In addition, the expression of these genes in 40 samples of normal peripheral blood was also examined using the same method. The ratios of WT-1/Abl and Bcr-Abl/Abl were both plotted, and the two expression patterns were compared as well as their clinical significance., Results: The levels of WT-1 expression in normal peripheral blood were detectable. In CML and Ph positive ALL patients, WT-1 expression levels changed in parallel with the Bcr-Abl expression pattern as the disease progressed or responded to effective treatment., Conclusion: WT-1 expression provides a novel molecular marker in addition to Bcr-Abl for monitoring minimal residual disease (MRD) and targeting therapy in Ph chromosome-positive leukemia patients.

Background & Objective: Regulation of expression of human telomerase reverse transcriptase(hTERT) is regarded as the major determinant of telomerase activity. Several studies suggested that c-myc could regulate hTERT expression. This study was designed to examine the telomerase activity, the expression of hTERT and c-myc, and to analyze their correlations in acute leukemia., Methods: The telomerase activity was semi-quantitatively assayed in mononuclear cells (MNCs) of bone marrow (BM) from 35 acute leukemia patients and 15 control cases by polymerase chain reaction enzyme- linked immunosorbent assay (PCR-ELISA). The mRNA expression of hTERT and c-myc were determined using reverse transcription polymerase chain reaction (RT-PCR) at the same time., Results: The mean telomerase activity, the expression levels of the hTERT mRNA and c-myc mRNA in acute leukemia were 0.71+/-0.32, 0.47+/-0.36, and 0.85+/-0.31, respectively, which was significantly higher than those of control cases (P< 0.01). The telomerase activity and the expression level of c-myc mRNA were associated with the expression level of hTERT mRNA (P< 0.01). In the 25 follow-up patients, the mean telomerase activity and the expression level of the hTERT were 0.61+/-0.39 and 0.29+/-0.31 in complete remission cases, while 0.83+/-0.23 and 0.53+/-0.31 in none complete remission cases, respectively. There was no significant difference between the two groups., Conclusion: The telomerase activity and the expression of hTERT and c-myc mRNA were up-regulated in acute leukemia, and the elevated expression of hTERT may be an important adjuster of telomerase activity. It is likely that c-myc protein can stimulate the expression of hTERT and thereby enhance telomerase activity, which may involve in carcinogenesis of leukemia.

Objective: To study the expression of gelatinase A (MMP2) in acute leukemic cells and its clinical implication., Methods: The bone marrow samples from 46 acute leukemia patients were investigated by reverse transcription polymerase chain reaction (RT-PCR) and Gelatin Zymography method. Matrigel invasion assay was studied on U937, NB4, K562 and SHI1 cells in vitro., Results: The expression of MMP2 was positive in 24 of 46 patients with AL (52.2%). In MMP2 positive and negative groups of AL, the extramedullary infiltration rates were 50.0% and 18.2% (P < 0.05), whilst the percentage of blast cell in peripheral white blood cells were (77.21 +/- 13.9)% and (62.95 +/- 17.2)% (P < 0.01), respectively. The positive expression of TIMP2 and MT1-MMP in 46 AL patients were 27 (58.7%) and 33 (71.7)%. The matrigel invasion assay suggested that MMP2 is involved in migration of leukemic cell through extracellular matrix (Matrigel)., Conclusion: The active form of MMP2 might be essential to the egress of leukemic cells from bone marrow into peripheral blood, followed by an extramedullary infiltration.

Background & Objective: Vascular endothelial growth factor (VEGF) play an important role in tumor angiogenesis through a paracrine mechanism, but the importance of its autocrine has not been fully elucidated. This study was designed to explore the gene coexpression pattern of VEGF and its receptors (Flt-1 and KDR) in variety of human malignant cell lines., Methods: After isolation of RNA from thirty tumor cell lines and four endothelial cells, gene expressions of VEGF and its receptors were measured by semi-quantitative reverse transcriptase polymerase chain reaction using the transcriptional level of the house-keeping gene for internal calibration., Results: VEGF transcript was detected in the majority of tumor cell lines (29/30) and in all endothelial cell lines at moderate or high level, while the expression of VEGF was low in human umbilical vein endothelial cell (HUVEC). Moreover, Flt-1 gene expression was found in 50% of hematopoietic malignancies (6/12), 28% of solid tumors (5/18), and endothelial cells (EA. hy926 and HUVEC). In contrast, the expression of KDR gene was found in 2 (16.7%) hematopoietic cell lines and 6 (33.3%) solid tumor lines. In endothelial cells, KDR gene expression was detectable in HMEC-1 and HUVEC only. However, ECV304 cells express neither Flt-1 nor KDR gene., Conclusion: Overexpression of VEGF gene in all tumor cell lines provides a potential biological marker for malignancies and the coexpression of VEGF with its receptors suggesting an autocrine pathway exists in tumor cells.

TNF-related apoptosis-inducing ligand (TRAIL) is a newly identified member of the tumor necrosis factor (TNF) family. TRAIL induces apoptosis by activating caspase cascades, stimulating a loss of mitochondrial membrane potential (Delta Psim) and cytochrome C release in the FADD/caspase-8 dependent pathway. However, TRAIL can also trigger transcriptional activations of the pro-oncogene of c-fos, JNK, and NF-kappaB by other signaling pathways downstream of FADD/caspase-8. MAPK/ERK activation has a dominant protecting effect over apoptotic signaling from the death receptors. The functional expression of TRAIL by leukemic cells may be involved in tumor cells evasion of immunosurveillance. Somatic mutations of TRAIL-R1 and TRAIL-R2 genes may play a role in the pathogenesis of some tumors. TRAIL can induce apoptosis on various continuous transformed cell lines and primary tumor cells, including several of hematopoietic origin, displaying minimal toxic effects on normal tissues. Because of the abilities of induction of both cytotoxic (apoptosis) and cytostatic (cell cycle perturbation) effects on the leukemic cells, TRAIL is currently considered as a potential(co) therapeutic drug against tumors.

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.

WT1 gene encodes a zinc finger transcription factor that regulates transcription of its downstream genes. Some of target genes for WT1 are involved in regulating both cell cycle and cellular proliferation and differentiation. However, WT1 itself is regulated by its upstream genes such as NF-kappaB and GATA-1. Thus there exists a pathway of transcriptional regulation mediated by WT1, which controls development of hematopoietic system. Leukemia results from disrupting the homeostasis among hematopoietic proliferation, differentiation and apoptosis, which is often the consequence of an inappropriate expression of transcription factors and subsequent disruption of the normal gene expression pattern. This article reviews the relationship between the WT1-mediated pathway of transcriptional regulation and leukemia.

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.

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.

To study the telomerase activity in the bone marrow MNCs from patients with myelodysplastic syndromes in comparison with that in normal individuals and acute leukemia patients. The intracellular telomerase activity was semi-quantitatively examined by PCR-ELISA assay in the marrow cells of 20 normal individuals, 21 cases of myelodysplastic syndromes (MDS) and 32 cases of acute leukemia. Telomerase activity in normal marrow cells was 0 - 0.30 U, with a mean level of (0.11 +/- 0.08) U, in which 3 cases were considered positive according to the standard set by the Kit. In 32 acute leukemia patients, the mean level of telomerase activity was (0.42 +/- 0.26) U (ranged 0 - 0.96 U) with a positive rate of 78.1%, showing a significantly higher activity in acute leukemia (P < 0.01). Moderate telomerase activity was detected in 21 cases of MDS, with a mean level of (0.27 +/- 0.19) U (0 - 0.97 U) from which the positive rate was 66.7%. This value was significantly higher than that in the normal BM (P < 0.05). Moreover, a significantly higher telomerase activity was shown in the high-risk group of MDS (P < 0.05). Based on the international scoring system evaluating the prognosis of MDS (IPPS), telomerase activity in HIGH subgroup was significantly higher than that in INT-1 and INT-2 subgroup (P < 0.05). The level of telomerase activity was not correlated to the chromosome aberrations. These results show that a borderline telomerase activity could be found in normal bone marrow cells. Telomerase activity was markedly higher in acute leukemia. BM of MDS patients demonstrated a moderate telomerase activity. Higher telomerase activity could be found in high-risk group and correlated with poor prognosis.

In order to study the relation between WT1 gene expression and differentiation of NB4 leukemic cells, a competitive RT-PCR method was established by using recombinant DNA technique to detect the expression of WT1 gene quantitatively during differentiation of NB4 leukemic cells induced by retinoic acid. The expression of CD11b was simultaneously determined by an indirect immunofluorescence staining technique and flow cytometry. Our results showed that WT1 gene expression rapidly decreased during the differentiation of NB4 cells. The molecular number of WT1 gene in 1 micro g total RNA was 4 x 10(6), 1.56 x 10(6) and 0.4 x 10(6), respectively, prior to and at 12 or 24 hours after exposure to retinoic acid, and it was in accordance with the change of CD11b. These data suggest that the abnormally high expression of WT1 gene in leukemic cells was associated with the block of cell differentiation. The detection of WT1 expression with competitive RT-PCR combined with measure of CD antigens will contribute to study on the differentiation induction of leukemic cells.