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Objective: To observe the effects of matrix metalloproteinases (MMP)-2 and MMP-9 secreted by leukemic cells on tight junction proteins ZO-1, claudin-5 and occluding and the permeability of the blood-brain barrier (BBB) and explore the mechanisms of MMP-2 and MMP-9 in leukemic cell infiltration of the central nervous system (CNS). Methods: The mRNA expressions of MMP-2 and MMP-9 in leukemic cell lines SHI-1, HL-60 and U937 were detected by quantitative RT-PCR. The MMP inhibitor GM6001 was used to inhibit the secretion of MMP-2 and MMP-9. RNA interference (RNAi) was used to knock down the expression of MMP-2 and MMP-9. Zymography was used to analyze the secretion of MMP-2 and MMP-9 in the supernatant of different leukemia cell lines treated or untreated with drugs, as well as the RNAi-treated cells. An in vitro BBB model composed of human brain microvascular endothelial cells (BMVECs) was developed on a Matrigel-based insert. Cell invasion through a barrier of Matrigel-based human basement membrane and the BMVECs-based human BBB barrier was assayed to measure the invasive capacity and the capacity to breakdown the BBB of different leukemia cell lines treated or untreated with drugs, as well as the RNAi-treated cells. The morphologic changes of BMVECs after co-culture with different leukemia cell lines treated or untreated with drugs, as well as the RNAi-treated cells in vitro BBB models were observed by invert microscopy and tight junction proteins in these BMVECs were analyzed with a laser-scanning confocal microscope. Results: ①The mRNA expression in different leukemic cell lines shown a pronounced transcription of MMP-2 and - 9, and the transcriptional level in SHI-1 cells was the highest among all leukemic cell lines tested ( P <0.01). The data of activities of MMP-2 and -9 were consistent with the results of mRNA expression and SHI-1 displayed higher capacity of invasion ( P <0.01). ②After incubation 24h with different leukemic cells, the BMVECs disrupted to loss cell-cell contacts and grew in single cell. Confocal imaging showed down-regulations of ZO-1, claudin-5 and occluding accompanied by the disruption of BBB in vitro models. SHI-1 cells had stronger alterations to BMVECs, tight junction proteins and the permeability of the BBB than HL-60 and U937 cells. However, GM6001 and the knock-down of MMP-2 and MMP-9 altered the responses of BBB. They reduced the degradation of three tight junction proteins with a decreased permeability of BBB. Conclusion: MMP-2 and MMP-9 secreted by leukemic cells could disrupt the BBB by degrading the tight junction proteins ZO-1, claudin-5 and occluding, which contributed the infiltration of leukemic cell into CNS.

Objective: To construct a pcDNA3.1-AML1-ETO expression vector and investigate its effects on proliferation and differentiation of U937 leukemic cells., Methods: AML1-ETO gene was amplified by PCR from pCMV5-AML1-ETO and inserted into eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO. The recombinant plasmid was transfected into U937 cells by Lipofectamin 2000. Individual clones selected with G418 were isolated. The integration and the expression levels of AML1-ETO in transfectants were determined by PCR, RT-PCR and Western blot analysis respectively. Trypan blue refusal staining method was used to detect the proliferation of U937 cells. Light microscope was applied to observe the morphologic changes of the cell. The expression of myeloid cell differentiation antigen was detected using flow cytometry., Results: The recombinant pcDNA3.1-AML1-ETO was confirmed by enzyme digestion and sequencing. The highly expressing AML1-ETO subclone was established. AML1-ETO was expressed in U937 cells transfected with pcDNA3.1-AML1-ETO. The growth of the monoclonal cells was inhibited evidently (P < 0.05). The expression of CD11b in transfected group \[(4.17 ± 0.31)%\] was lower than that in empty plasmid transfected group and non-transfected group \[(11.40 ± 0.17)% and (11.03 ± 0.15)%\] respectively (P < 0.001). Transfected cells displayed morphology of less differentiation. The expression level of CDl1b was unchanged in transfected cells treated with TPA (P > 0.05)., Conclusion: The eukaryotic expression vector for AML1-ETO gene was successfully constructed and expressed in U937. AML1-ETO inhibits the proliferation and differentiation of transfected cells. It provides the basis for further study of mechanisms of AML1-ETO in leukemogenesis.

Objective: To evaluate preliminarily the significance of detecting the Wilms' tumor (WT1) gene level on monitoring minimal residual disease (MRD) and predicting the clinical outcome in patients of acute leukemia following hematopoietic stem cell transplantation (HSCT)., Methods: The mRNA expression levels of WT1 and house-keeping gene ABL were dynamically measured with Real-time quantitative reverse transcription polymerase chain reaction (RQ-RT-PCR) on 326 bone marrow samples from 63 post-HSCT patients in our hospital from December 2001 to September 2009. After comparing the WT1 levels of patients with different post-transplantation outcomes, the investigators used the receiver operating characteristic (ROC) curve to determine the WT1 threshold so as to predict their clinical relapses. Then different prognoses of WT1 positive and negative patients were analyzed., Results: The levels of WT1 expression showed significant difference between the 19 relapsing and 44 non-relapsing patients with the median expression levels of 1270 (55 - 47 596) and 132 (0 - 2959) respectively (P < 0.01). In 19 relapsing patients, except for 1 patient discontinuing the detection of WT1, 10 mortality cases due to recurrence had higher levels of WT1 expression than other 8 patients (P > 0.05). According to the ROC curve, the cut-off value of WT1 at 585 could separate 63 patients into the WT1-positive group (> 585) and the WT1-negative group (≤ 585). The WT1-negative group was found to have a longer relapse-free survival (RFS) and overall survival (OS) than the positive group (all P < 0.01). Twenty-one WT1-positive patients were followed up for 3, 4 - 6, 7 - 9 and 9 months respectively. The cumulative post-HSCT recurrence rates in those WT1-positive cases were 8/8, 2/4, 2/4 and 3/5 (P = 0.063) respectively. And the intervention was ineffective., Conclusion: WTl gene may be an independent factor of monitoring MRD. And WT1 > 585 is a poor post-HSCT prognostic factor for the patients of acute leukemia.

Objective: To observe the effect of leukemic cells on blood-brain barrier (BBB) in mice with central nervous system leukemia (CNSL) by establishing mice CNSL model and an in vitro BBB model and explore the mechanism of leukemic cell infiltrating central nervous system (CNS)., Methods: After splenectomy, cytoxan intraperitoneal injection, and sublethal irradiation, 10 BALB/c nu/nu mice were transplanted intravenously with 1.2 × 10(7) of SHI-1 human monocytic leukemic cells. Mice were monitored for survival and clinical manifestation of nerve palsy. The leukemic cells engrafted were examined by RT-PCR, histopathology and bone marrow (BM) smears. Immunofluorescence analysis with laser scanning fluorescence confocal microscopy was used to determine the expression of fibrinogen and tight-junction protein ZO-1. An in vitro BBB model composed of human brain microvascular endothelial cells (BMVECs) was developed on a Matrigel-based insert. Different leukemic cell lines were seeded onto the upper compartment of transwell insert. After incubated for 24 h with BMVECs, cells that had migrated into the lower compartment were counted and analyzed., Results: (1) Paralysis with or without sight loss was developed in half the mice 30-35 d after innoculated with SHI-1 cells. Leukemic cells infiltrates were observed in BM and in different part of brain tissues including brain parenchyma. The transcriptions of human MLL/AF6 fusion gene were also detected in BM and brain tissues in paralysis mice. The fibrinogen expression and ZO-1 disruption were detected in the infiltrated tissue. (2) After 24 h incubation with leukemic cells, the BMVECs sheets were disrupted and grew singly and ZO-1 expression was down-regulated markedly. SHI-1 cells showed more injurious to BMVECs and higher invasive rate \[(40.33 ± 1.53)% vs (11.83 ± 1.44)%, P < 0.05\] than HL-60 cells did., Conclusion: One of the mechanisms of leukemic cells infiltrates CNS in CNSL is injure to the BBB.

This study was purposed to investigate the expression of the growth-factor independence 1 (GFI1) in patients with leukemia and its clinical significance. Bone marrow mononuclear cells were obtained from 65 newly diagnosed leukemia patients including 24 acute myeloid leukemia (AML), 18 chronic myelogenous leukemia (CML), 6 acute lymphoblastic leukemia (ALL), 17 blast crisis of chronic myelogenous leukemia and 13 patients with iron deficiency anemia (IDA) were used as controls. The relative expression of gene gfi1 was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and taqman quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR). The results showed that gene expression of gene gfi1 in leukemia patients was obviously higher than that in controls and the difference was statistically significant (p < 0.01), in which the expression of gene gfi1 in newly diagnosed CML patients was higher than that in newly diagnosed AML, newly diagnosed ALL, CML-BCP patients and the difference was significant (p < 0.01). Expression of gene gfi1 in lymphocytic blast crisis of CML was higher than that in nonlymphocytic blast crisis of CML, and the difference was significant. It is concluded that gene gfi1 may play an important role in leukemia, especially in CML incidence and progression. The high level expression of gene gfi1 may be participate in the development of lymphoma.

Objective: The cytochrome P450 subfamily IIIA5 (CYP3A5) gene is responsible for the metabolism of many clinically used anticancer agents. So far the studies on CYP3A5 gene has only been focused on the leukemia cell lines. This study examined the polymorphism of CYP3A5 and tried to find the possible relationship between CYP3A5 gene expression and treatment outcome or prognosis in children with acute leukemia., Methods: The genotype distribution of CYP3A5-6986A/G gene polymorphism was detected with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in 66 children with newly diagnosed acute leukemia (AL) and 22 control individuals. Quantitative real-time RT-PCR was used to examine wt-CYP3A5 and SV1-CYP3A5 mRNA levels in the bone marrow., Results: Three genotypes of CYP3A5-6986A/G polymorphisms were found: CYP3A5*1/*1, CYP3A5*1/*3 and CYP3A5*3/*3. There were significant differences in the wt-CYP3A5 mRNA expression among the AL patients with different genotypes (p<0.05). In patients with acute lymphocytic leukaemia (ALL), the complete remission (CR) rate in the group with a low expression of wt-CYP3A5 mRNA was significantly higher than that in the group with a high expression (p<0.05). A dynamic monitoring for wt-CYP3A5 mRNA expression was performed in two cases of ALL. The expression increased before ALL relapse compared with that in CR in a patient, while in the other patient, the expression was kept in a low level and the patient remained in CR CONCLUSIONS: wt-CYP3A5 mRNA expression was associated with the treatment outcome and prognosis in children with AL. Dynamic monitoring for wt-CYP3A5 mRNA expression in the bone marrow may be useful in the evaluation of the disease severity in childhood acute leukemia.

Objective: To investigate the effects of targeting elimination of the leukemic CD34+ progenitor cells by using cytotoxic T lymphocytes (CTLs) specifically against Wilms tumor gene (WT1)-derived peptide., Methods: A 9-mer WT1 peptide (CMTWNQMNL) containing HLA-A * 0201-binding anchor motifs was synthesized. The suspended cells were collected from the culture of the peripheral blood mononuclear cells and divided into 2 groups: Group D (pure T cells) and Group C (IL2 + T cells). The dendritic cells (DCs) generated from the peripheral blood mononuclear cells of an HLA-A* 0201-positive healthy donor were repeatedly loaded with WT1 peptide so as to elicit cytotoxic T cells (CTLs) specifically for WT1 peptide, and restricted by HLA-A * 0201 (Group A). The specific lysis effects of WT1 peptide specific CTLs upon leukemic bone marrow CD34+ progenitor cells positively expressing WT1 (3 being HLA-A * 0201(+) and 3 being HLA-A*0201(-)), peripheral blood CD34+ cells from healthy persons (2 being HLA-A * 0201(+) and 1 being HLA-A * 0201(-)), and leukemia cells of the lines of NB4 (HLA-A * 0201(+)/WT1(+)), U937 (HLA-A * 0201(+)/WT1(-)), and K562 (HLA-A * 0201(-)/WT1(+)) were measured by methyl thiazolyl tetrazolium (MTT) chromatometry assay. The colony-forming unit-granulocyte macrophage (CFU-GM) forming capability of the leukemic bone marrow CD34+ progenitor cells and peripheral blood CD34+ cells from healthy persons treated with WT1 peptide specific CTLs were also determined by methylcellulose medium colony-forming unit assay. The CTLs elicited by DCs without WT1 peptide loading(Group B)and T cells cultured with IL-2 (Group C) were taken as controls., Results: The CTLs specific for WT1 peptide and restricted by HLA-A * 0201 (Group A) exerted a specific lysis upon the 3 cases with HLA-A * 0201(+)/WT1(+) leukemic bone marrow CD34+ progenitor cells and NB4 leukemia cells, the specific lysis levels were 55.3% +/- 2.8%, 67.1% +/- 3.2%, 49.4% +/- 3.8% and 55.0% +/- 3.7% respectively, all significantly higher than those upon the 3 cases with HLA-A * 0201(-)/WT1(+) leukemic bone marrow CD34+ progenitor cells, normal healthy peripheral blood CD34+ cells of the healthy persons, and leukemia cell of the lines K562 and U937 (the specific lysis levels were all < 20%). The specific lysis level of Group A CTLs was significantly higher than those of Group B and Group C CTLs (both P < 0.01). The relative colony formation rates of WT1-CTLs in Group A upon the 2 cases with HLA-A * 0201(+)/WT1(+) leukemic CD34+ progenitor cells were 17.8% +/- 4.0% and 20.8% +/- 3.41% respectively, both significantly lower than those of the none WT1-CTLs in Group B (88.9% +/- 3.4% and 91.8% +/- 5.7% respectively, both P < 0.01). There was no significant difference in the relative colony formation rate upon HLA-A * 0201(+)/WT1(-) leukemic bone marrow CD34+ progenitor cells and the HLA-A * 0201(-)/WT1(-) or HLA-A * 0201(+)/WT1(-) normal peripheral blood CD34+ progenitor cells between the CTLs of Groups A and B., Conclusions: The CTLs specific for WT1 and restricted by HLA-A * 0201 can exert specific lysis upon leukemic CD34+ progenitor cells highly expressing WT1 gene, and can inhibit the CFU-GM colony formation of such leukemic CD34+ progenitor cells. The product of expression of WT1 gene may be used as a target in the leukemic CD34+ cells.

Objective: To establish a real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) for quantitative detection of the common molecular markers that have affirmative clinical significance in the acute and chronic leukemia patients, and evaluate its significance in diagnosing leukemias and monitoring minimal residual disease (MRD)., Methods: Primers and TaqMan probes were designed for detecting various fusion transcripts and normal abl gene was used as the internal control. The expression level of fusion transcripts in 202 newly diagnosed leukemias were determined., Results: In absolute quantity, expression level of the fusion transcripts in various leukemias was b3a2 (b2a2) 47614.63, e1a2 98847.53, AML1-ETO 300029.51, PML-RAR alpha 25506.28, respectively, while in relative quantity to abl, the levels were 1.05, 0.91, 5.33 and 0.55, respectively., Conclusion: The relative quantification of gene expression level by using RQ-RT-PCR to abl control gene is more accurate and direct viewing. Different levels of transcription of corresponding fusion genes are found in various subtypes of leukemias at diagnosis, among which the level of AML1-ETO was higher and PML-RAR alpha lower.

Objective: To study T-cell reconstitution by the assessment of T-cell receptor excision circle (TRECs) and T-cell receptor clonal repertoire after HLA-matched sibling bone marrow transplantation (MSD-BMT) in leukemia patients and try to reveal the rule of T-cells repopulation in MSD-BMT., Methods: Real-time quantitative PCR detection of TRECs was carried out in the DNA of peripheral blood mononuclear cells from 23 leukemia patients who underwent MSD-BMT. The content of TRECs in 70 normal donor individuals was also detected to determine the normal range of TRECs in healthy subjects. Among them, 10 patients received RT-PCR to amplify 24 subfamily genes of T-cell receptor B variable (TCRBV) and five normal donors served as control. The PCR products were further analyzed with genescan to evaluate the clonality of BV subfamily and calculate the usage rate of BV families., Results: The mean value of TRECs in normal donors was (3351.1 +/- 3711.1) copies/10(5) cells. There was an inverse correlation between the value of TRECs and the age of healthy subjects. Before transplantation, all the patients were detected for the number of TRECs, the mean TRECs number was (307.9 +/- 433.3) copies/10(5) cells and it was far lower than that of healthy subjects. From one month to five months after bone marrow transplantative (BMT), the TRECs levels were low and in some patients they even could not be detected. Six months later, TRECs levels increased obviously and maintained that high nearly one year. 24 months after BMT, the number of TRECs increased and reached the pretransplantation status. One patient was detected 2 m and 3 m after transplantation and found that there was a tendency of additional use of BV families and an increase of the expression of CDR3 polymorphism. 2-15 months after transplantation, in ten of the patients, there were 7-16 BV subfamilies expressing and in more than 48% the expression was polyclinic. Monoclones and oligoclones existed in 24 BV subfamilies., Conclusions: At an early stage after BMT, the number of TRECs was low and remained so for a long time. TCRBV CDR3 repertoire showed certain BV families expressing. 6 months after BMT, thymus produced certain number of naive T cells and TCRBV CDR3 repertoire showed additional use of BV families and increased expression of polymorphism.

Objective: To predict T-cell immune reconstitution by investigating T cell receptor excision circles (TREC) and T-cell receptor beta-chain variable region (TCRBV) clonal repertoire in leukemia patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT)., Methods: Real-time quantitative PCR was used to detect the TREC in 43 leukemia patients undergoing matched sibling donor bone marrow transplantation (MSD BMT), matched unrelated donor (MUD) BMT, or haploidentical-stem cell transplantation (HID-SCT), and in 70 normal individuals. RT-PCR was used to amplify 24 subfamily genes of T-cell receptor beta-chain variable region (TCRBV) in 24 of the 43 patients and 5 normal donors as control. The PCR products were further analyzed by genescane to evaluate the clonality of BV subfamily, characteristics of complementarity determining region 3 (CDR3), and the usage rate in BV subfamily., Results: There were (335.1 +/- 782.5) copies/10(5) cells in the 43 patients before transplantation, far lower than the normal value. The TREC values of the patients of the 3 groups all decreased obviously 3 months after transplantation. The TREC value of the MSD-BMT group recovered faster than the other two groups and reached the value before transplantation in 24 months. The recovery of TREC value in the HID-BMT group was delayed. 3 - 19 months after transplantation, the usage of TCRBV subfamilies was still restricted. There were 6 - 16 BV subfamilies expressed and 33% - 48% of them were polyclonals, the others were monoclones and oligoclones and existed in 24 BV subfamilies, no common monoclone BV subfamilies was expressed., Conclusion: Investigation of the TREC and TCRBV clonal repertoire showed that the number of naive T cell is lower and the usage of TCRBV subfamilies skewed 3 - 24 months after allo-HSCT. The immune deficiency of the patients undergoing HID-BMT is more prominent and consistent with the clinical process.

Objective: Successful allogeneic hematopoietic stem cell transplantation (HSCT) requires reconstitution normal T-cell immunity. Measurement of T-cell receptor excision circles (TRECs) and T-cell receptor beta (TCRBV) CDR3 repertoire is a means of quantifying recent thymic T-cell production and reflecting antigen-specific T-cell clones proliferation., Methods: We used real-time quantitative PCR to detect TRECs from 43 Chinese patients who underwent three kind of allo-HSCT without T-cell depletion. RT-PCR was performed to amplify 24 subfamily genes of TCRBV in 24 patients of them., Results: For haploidentical-D group, the TRECs numbers were lower up to 24 months. For matched-sibling donor (MSD) group, the recovery of TRECs was faster than those of other two groups. TRECs values in matched-unrelated donor (MUD) were in the middle. During 2-19 months after transplantation, there were 6-16 BV subfamilies expressed and 33-48% of them were polyclones. The usage rate of TCRBV and percentage of polyclones in haploidentical-D were less than those of other two groups. Twenty-three CDR3 molecules were obtained from nine patients who were potentially associated with GVHD or CMV infection., Conclusions: Analyzing the changes of TCRBV repertoire and measuring TRECs during immune reconstitution would be useful to determine the host's current immune status and ability of T-cell immune reconstitution and also to find antigen-specific T-cell clones in the three kinds of HSCT.

This study was aimed to explore the usefulness of multiplex reverse transcription-polymerase chain reaction (multiplex RT-PCR) in detection of fusion genes associated with specific translocations in acute leukemia (AL) patients with normal karyotypes. 37 AL patients with normal karyotypes were analyzed by multiplex RT-PCR. The results showed that 4 types of fusion genes such as PML/RARA, AML1/ETO, CBFbeta/MYH11, BCR/ABL were detected in 8 (21.6%) patients by multiplex RT-PCR. In conclusion, multiplex RT-PCR is useful in detection of fusion genes associated with specific translocations in acute leukemia (AL) with normal karyotypes and it would refine the karyotype analysis. When the normal karyotypes were detected in acute leukemia patients by conventional cytogenetic method, the multiplex RT-PCR should be performed for them.

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 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.

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.