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Imatinib (ST1571) provides only limited selectivity for CML cells and treatment might be complicated by silent BCR-ABL genes.

Very promising results have been obtained in clinical trials on chronic-phase chronic myeloid leukemia (CP-CML) patients treated with imatinib mesylate (IM; Gleevecr, STI571), a BCR-ABL tyrosine kinase inhibitor. However, we found that IM caused considerable inhibition of normal hematopoietic progenitor cells upon treating control bone marrow (BM) cultures. In vitro IM treatment gave a decrease in the yield and size of colonies from BM of untreated CP-CML patients that was only two to three times that from the normal samples. Moreover, about 30% of myeloid progenitors (CFU-GM) from CML BM still formed colonies in the presence of IM, most of which had BCR-ABL RNA. About half of these treated colonies also displayed methylation of the internal ABL Pa promoter, a CML-specific epigenetic alteration, which was used in this study as a marker for BCR-ABL translocation-containing cells. However, ~5-8% of the treated or the untreated CML BM-derived colonies had no detectable BCR-ABL RNA by two or three rounds of RT-PCR despite being positive for the internal standard RNA and displaying hallmarks of CML, either t(9;22)(q34;ql 1) or ABL Pa methylation. Our results indicate that IM is only partially specific for CML progenitor cells compared to normal hematopoietic progenitor cells and suggest that some CML cells may have a silent BCR-ABL oncogene that could interfere with therapy.

Benzamides↗

PubMed1985

Activity of cyclic nucleotide phosphodiesterases in granulocytes of chronic myelogenous leukemia (CML). A preliminary report.

Cyclic 3':5'-adenosinmonophosphate phosphodiesterase (cAMP-PDE), high KM value type isoenzyme, and cyclic 3':5'-guanosino-monophosphate phosphodiesterase (cGMP-PDE), high KM value type isoenzyme, were determined in granulocytes of patients with chronic myelogenous leukemia (CML) in the chronic phase of the disease. Granulocyte cAMP-PDE activity was similar in the general group of CML patients to that in normal granulocytes; the cGMP-PDE, however, was somewhat higher. By dividing the CML general group into the "low leukocyte count" subgroup including granulocytes of patients with WBC number ranging from 10,300 to 40,000 per microliter (mean 22,000 per microliter), and the "high leukocyte count" subgroup including patients with leukocyte count above 40,000 per microliter (mean 67,000) remarkable differences in the activities of cyclic nucleotide phosphodiesterases between these subgroups could be found: cAMP-PDE activity for the high leukocyte count subgroups was significantly higher than for that in normals, and in CML-low leukocyte count subgroup. On the other hand, cGMP-PDE activity in granulocytes of the high leukocyte count subgroup was found to be remarkably lower than that in normals, in the low leukocyte count subgroup and general CML group. In CML patients the ratio of cAMP-PDE activity to cGMP-PDE activity was always considerably higher than that in controls. The obtained results suggest CML granulocytes to differ from normal ones in respect to their control of intracellular cyclic nucleotide levels. This difference is related to the accumulation of CML granulocytes.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1989

No correlation between locations of bcr breakpoints and clinical states in Ph1-positive CML patients.

The majority of patients with chronic myelogenous leukemia (CML) have a characteristic reciprocal translocation between chromosome 9 and 22, resulting in the Philadelphia (Ph1) chromosome. During this translocation, the c-abl oncogene on chromosome 9 is transferred to the Ph1 chromosome and linked to a breakpoint cluster region (bcr), which is part of a large bcr gene. This phenomenon results in the formation of a bcr-c-abl fusion gene, which is transcribed into an 8.5 kb chimeric mRNA encoding a 210 kd bcr-c-abl fusion protein. The fusion protein has tyrosine kinase activity implicated in the pathogenesis of CML. The breakpoint near the c-abl locus on chromosome 9 can occur within a large area. In contrast, the breakpoints on chromosome 22 cluster within the bcr region of 5.8 kb. A chronic phase lasts for an average of 2 to 3 years; and, subsequently, most patients enter blast crisis. In the present study, we examined 15 Ph1-positive CML patients (eight in chronic phase, one in accelerated phase, and six in blast crises) as to whether the identifiable difference in the locations of the bcr breakpoints exist between CML patients in chronic phase and those in blast crisis. In seven of eight CML patients in chronic phase, in one in accelerated phase, and in four out of six CML patients in blast crisis, the bcr breakpoints clustered in the 3' portion of the bcr. Thus, we could not find out the correlation between the locations of the bcr breakpoints and the clinical stage of the CML patients. This might imply that blastic transformation in Ph1-positive CML was caused by other mechanisms than the transition of the bcr breakpoints.

Blast Crisis↗

PubMed1997

Apoptosis and proliferation (PCNA labelling) in CML--a comparative immunohistological study on bone marrow biopsies following interferon and busulfan therapy.

A comparative morphometric analysis was performed on smears and trephine biopsies of normal bone marrow and in chronic myelogenous leukaemia (CML) to assess the effects of therapy on apoptosis and cell proliferation. The in situ end-labelling (ISEL) technique was used for the demonstration of programmed cell death, in combination with the monoclonal antibody PG-M1 to identify macrophages. Cell proliferation was evaluated by employing the monoclonal antibody PC10 directed against proliferating cell nuclear antigen (PCNA). In CML (48 patients), significantly higher rates of apoptosis were observed than in normal bone marrow (smears, frozen sections, and paraffin-embedded samples) of 15 patients. In contrast, the PCNA labelling index of CML was not different from controls. In bone marrow tissue derived from CML patients, about 36 per cent of apoptotic bodies were ingested with CD68-positive macrophages. Study of the histotopographical distribution of labelled cells revealed that in CML, in contrast to the normal bone marrow, programmed cell death and PCNA activity were concentrated along the paratrabecular generation zone. In 28 patients with CML treated with interferon (IFN), sequential trephine biopsies displayed a significant enhancement of apoptosis which was associated with a decrease in PCNA reactivity. In contrast to this finding, no such alterations could be observed in 24 patients who received busulfan (BU) monotherapy. This study furthers the understanding of cell kinetics in CML. IFN therapy induces apoptosis and suppresses cell proliferation. The rate of programmed cell death prior to therapy and the extent of IFN-triggered apoptosis exert a significant predictive impact on survival. In this study, ISEL-positive (apoptotic) cells and bodies do not correspond to unscheduled cell repair as detected by PCNA immunoreactivity.

Adult↗

PubMed1988

Undetectable bcr-abl rearrangements in some CML patients are due to a deletion mutation in the bcr gene.

Most patients with chronic myelogenous leukemia (CML) have Philadelphia (Ph) chromosome. Breakpoints on chromosome 22 in CML occur in a small region designated as the breakpoint cluster region (bcr). More than 90 percent of CML patients have breakpoints in the bcr; the remaining patients had no detectable rearrangement. In our study, a commercially available 1.2 kb HindIII-BglII (1.2 HBg) bcr probe was used to locate breakpoints in the bcr, which were found in 22 of 24 patients. Furthermore, using a probe upstream from the 1.2 HBg probe, rearranged bands were clearly detected in the two patients in whom no extra bands had been found with the 1.2 HBg probe. These results strongly suggest that these two patients carry a deletion at the acr-abl recombination point encompassing the area of the 1.2 HBg probe. Therefore, in our series, all CML patients eventually had breakpoints in the bcr, and the involvement of rearrangement was demonstrated to be highly specific for CML. Our data indicate that hybridization of CML cellular DNA with several bcr probes is important in examining accurately the frequency of bcr-abl rearrangements in CML, as some cases contain a deletion within the region.

Chromosome Aberrations↗

PubMed2006

Gene therapy for BCR-ABL+ human CML with dual phosphorylation resistant p27Kip1 and stable RNA interference using an EBV vector.

BACKGROUND: BCR-ABL-mediated chronic myelogenous leukemia (CML) CD34(+) cell proliferation mostly depends on the nucleo-cytoplasmic ratio of the cyclin-dependent kinase inhibitor p27. The ubiquitin-ligase SCF(Skp2) promotes degradation of phosphorylated p27 at T187 in the nucleus, resulting in G1/S progression of the cells. On the other hand, phosphatidylinositol-3-kinase (PI3K)-directed T157 nuclear localization signal (NLS) phosphorylation results in cytoplasmic sequestration of p27, leading to abnormal integrin-mediated proliferation of CD34(+) CML cells. METHODS: We demonstrate the generation of an engineered Epstein-Barr virus (EBV) vector with a BAC backbone that has the unique capacity to carry doubly modified (DM) p27 (i.e. T187A, T157A p27) along with the BCR-ABL siRNA expression construct. The HSV-tk suicide gene has also been incorporated in the same vector, which promotes apoptosis in a BCR-ABL-independent pathway. RESULTS: Expression of DM p27 markedly inhibits proliferation of BCR-ABL(+) primary human CML cells. Moreover, DM p27 strongly inhibits the growth of imatinib-resistant CML cells, compared to the T157A p27 (SM p27). The CML growth inhibition is found to be the result of significant G1/S arrest with concomitant increase in hypophosphorylated retinoblastoma (Rb). Moreover, the EBV vector mediated stable RNA interference induces apoptosis in K562 cells and reduces myeloid colony forming units. CONCLUSIONS: We therefore propose a multi-gene delivery strategy for BCR-ABL(+) CML cells by targeting not only the fusion transcript, but also the downstream signaling, to overcome drug resistance in the acute phase of CML.

Active Transport, Cell Nucleus↗

PubMed1982

CML characterization of a product of a second class I locus in the rat MHC.

In the rat, genes that control the expression of target antigens detected by cell-mediated lympholysis (CML) are present in the major histocompatibility complex (MHC). The relationship of these loci, CT and Ag-L, to each other and to other loci within the MHC is unknown. In this report, we demonstrate the existence of a CML target antigen in the (DA X BN)F1 anti-DA.1I(BI) strain combination. The gene coding for this antigen is linked to the RT1 complex as indicated by the CML reactivity of targets from backcross and congenic animals. Inhibition studies demonstrated that this antigen has the widespread tissue distribution characteristic of class I antigens, and the gene coding for this CML antigen maps coincident with the RT1.E class I locus as indicated by the lysis of targets from the recombinant strains r10 and r11. The CML can be blocked by antisera directed against a product of the RT1.E locus. The locus controlling this CML reactivity, like CT and Ag-L, has been separated from RT1.A by recombination; unlike CT and Ag-L, the produce of this CML locus appears to be identical with an RT1.E allelic product that has been serologically identified and biochemically characterized.

Animals↗

PubMed2001

Biologically based risk estimation for radiation-induced CML. Inferences from BCR and ABL geometric distributions.

Chronic myeloid leukemia (CML) invites biologically based radiation risk modeling because CML is simultaneously well-understood, homogeneous and prevalent. CML is known to be caused by a translocation involving the ABL and BCR genes, almost all CML patients have the BCR-ABL translocation, and CML is prevalent enough that its induction is unequivocally detected among Hiroshima A-bomb survivors. In a previous paper, a linear-quadratic-exponential (LQE) dose-response model was used to estimate the lifetime excess risk of CML in the limit of low doses of gamma-rays, R gamma. This estimate assumed that BCR-ABL translocation dose-response curves in stem cells for both neutrons and gamma-rays, differ only by a common proportionality constant from dicentric aberration dose-response curves in lymphocytes. In the present paper we challenge this assumption by predicting the BCR-ABL dose response. The predictions are based on the biophysical theory of dual radiation action (TDRA) as it applies to recent BCR-to-ABL distance data in G0 human lymphocytes; this data shows BCR and ABL geometric distributions that are not uniform and not independent, with close association of the two genes in some cells. The analysis speaks against the previous proportionality assumption. We compute 11 plausible LQE estimates of R gamma, 2 based on the proportionality assumption and 9 based on TDRA predictions. For each estimate of R gamma we also compute an associated estimate of the number of CML target cells, N; the biological basis of the LQE model allows us to form such estimates. Consistency between N and hematological considerations provides a plausibility check of the risk estimates. Within the group of estimates investigated, the most plausible lifetime excess risk estimates tend to lie near R gamma = 0.01 Gy-1, substantially higher than risk estimates based on the proportionality assumption.

Fusion Proteins, bcr-abl↗

PubMed2005

Additional clonal abnormalities in Philadelphia-positive ALL and CML demonstrate a different cytogenetic pattern at diagnosis and follow different pathways at progression.

The cytogenetic patterns in addition to the Philadelphia translocation in Philadelphia-positive (Ph+) ALL and chronic myeloid leukemia (CML) is heterogenous. We investigated 154 patients with Ph+ ALL at diagnosis or at relapse and 174 patients with different phases of CML. Ph+ ALL at diagnosis demonstrated a heterogenous pattern with a high frequency of numerical and structural chromosomal aberrations. CML at diagnosis presented with rare additional chromosomal changes. In Ph+ ALL, the pathway from diagnosis to relapse was characterized by the acquisition of a higher number of chromosomal aberrations, but the pattern of frequent aberrations at relapse did not differ from that observed at diagnosis. In contrast, in CML the pathway from chronic to the advanced phases was characterized by the acquisition of new chromosomal changes and by the development of karyotype complexity. In addition, the investigation of 10 cases of lymphoid blast crisis of CML showed significant differences in the karyotype in comparison to Ph+ ALL at diagnosis. Therefore, the karyotype can be helpful in discriminate de novo lymphoid blast crisis of CML from de novo Ph+ ALL. In conclusion, we were able to demonstrate that the cytogenetic patterns of Ph+ ALL and of CML are different at diagnosis and furthermore follow different pathways during progression or relapse.

Blast Crisis↗

PubMed1987

Chromosome abnormalities in CML.

The Ph chromosome is the hallmark of CML, where it is found in more than 90% of the cases. Cytogenetically, it usually results from a t(9;22)(q34;q11). The Ph arises in a stem cell and in chronic phase is found in all haematopoietic cell lineages, although it causes only increased granulopoiesis, and sometimes increased thrombopoiesis; furthermore blast crisis may occur in all differentiative patterns of the pluripotent stem cell. Recently, molecular investigations of Ph positive CML cases have revealed a consistent genomic recombination between two genes, BCR on chromosome 22 and the ABL oncogene. The latter is translocated from 9q34, its normal site, to the 22q- or Ph chromosome. This molecular rearrangement expresses a unique 8.5 kb BCR-ABL hybrid mRNA transcript, that encodes an altered BCR-ABL protein of approximately 210 kD with enhanced in vitro tyrosine kinase activity. The breakpoints on chromosome 22q- are clustered in a 5 kb DNA fragment, allowing their study using Southern blot analysis. Cytogenetic variant forms of the Ph translocation involving three or more chromosomes are found in about 5% of the cases. Southern blot and in situ hybridization studies have demonstrated that these variants are cytogenetically more complex than the standard t(9;22) but molecularly they show the same essential genomic recombination. This is also true for a small number of cases of Ph negative CML. Clonal progression, indicated by the presence of clonal, non-random chromosome abnormalities, in addition to the Ph is rare during chronic phase but is found in 80% of blast crisis. These additional aberrations may precede BC by weeks or months and have therefore a clear prognostic value. Ph is not restricted to CML, since it is also found in ALL (20% of adult cases) and rarely in AML. Ph in acute leukaemia is cytogenetically indistinguishable from Ph in CML, but molecular studies have shown that in 50% of the cases the breakpoint on chromosome 22 is different from the very consistent and characteristic breakpoint in CML. Nevertheless genomic recombination takes place that results in a novel ABL protein at least in some of the cases. Despite extensive cytogenetic and molecular investigations, the mechanisms underlying the formation of the Ph as well as the pathogenesis of Ph positive CML are still unknown but are now the object of intensive research.

Blast Crisis↗

PubMed2002

Inhibition of Bcr-Abl kinase activity by PD180970 blocks constitutive activation of Stat5 and growth of CML cells.

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr-Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr-Abl kinase. In this study, we show that blocking Bcr-Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC(50) of 5 nM. Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr-Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr-Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr-Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr-Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML.

Apoptosis↗

PubMed1996

Primary proliferating immature myeloid cells from CML patients are not resistant to induction of apoptosis by DNA damage and growth factor withdrawal.

Induction of apoptosis by growth factor deprivation or gamma-irradiation-induced DNA damage was directly studied in proliferating primary haemopoietic cells derived from CD34-positive cells of 13 CML patients and 12 normal controls. CD34-positive cells were cultured in the presence of appropriate concentrations of SCF and G-CSF for 5-7 d. After gamma irradiation with 500 rad or growth factor deprivation, the fraction of apoptotic cells was assessed by two independent methods applying either measurement of cells incorporating FITC-labelled dUTP by terminal transferase or assessment of the fraction of cells with a less than 2N DNA content in flow cytometry. Proliferating CML cells were not resistant to the induction of apoptosis either after gamma irradiation or subsequent to growth factor deprivation. A similar fraction of normal and CML cells underwent apoptosis 48 h after withdrawal of growth factors. CML cells displayed an increased susceptibility to induction of apoptosis after DNA damage. A significantly higher proportion of apoptotic cells were detected in samples derived from CML patients after irradiation with 0.5 Gy. These results, in conjunction with conflicting observations by other investigators, on the induction of apoptosis by gamma irradiation in various bcr-abl positive cells, suggest that bcr-abl-dependent effects on apoptosis strongly depend on the cells used. Our observations in CML cells derived exclusively from newly diagnosed CML patients demonstrate that bcr-abl expression per se is not sufficient to induce resistance to apoptosis.

Apoptosis↗

PubMed1999

Interferon-alpha and bcr-abl antisense oligodeoxynucleotides in combination enhance the antileukemic effect and the adherence of CML progenitors to preformed stroma.

We have studied the in vitro effect of IFN-alpha and bcr-abl antisense oligodeoxynucleotides (As ODN) alone and in combination with the aim of enhancing the antileukemic activity of the two single agents and evaluating whether the two agents in combination might restore the adherence capacity of chronic myeloid leukemia (CML) progenitors to preformed stroma. We have also correlated the increased adhesion found after in vitro treatment with the expression of adhesion molecules on leukemic progenitors. Incubation of the BV173 cell line with escalating doses of IFN-alpha (100-10000 U/ml) showed a colony growth inhibition between 10 and 30%. IFN-alpha and junction-specific As ODN in combination showed a greater antiproliferative effect compared to that observed with the two agents used alone. In particular, As ODN at a concentration of 40 microg/ml in combination with IFN-alpha at 100 and 1000 U/ml showed a greater inhibitory effect compared to that obtained with IFN-alpha only. Addition of As ODN to IFN-alpha at 10000 U/ml did not result in a greater BV173 inhibition. In a further set of experiments, primary cells from 16 CML patients at diagnosis were incubated with 40 microg/ml of J-spec As ODN, several control ODNs and IFN-alpha at 1000 U/ml alone and in combination. A significantly greater elimination of CML progenitors was found after treatment with the combination of IFN-alpha and J-spec As ODN, compared to any other treatment group, confirmed also by a more marked effect on p210 expression. The deficient adhesion of CML progenitors on human preformed stroma was restored at levels similar to that of normal bone marrow cells after treatment with IFN-alpha and/or J-spec As ODN, while the phenotypic analysis showed that the combined treatment increased significantly the expression of CD49b and CD62L on CML CD34+ cells. However, when the expression of adhesion molecules was blocked with specific monoclonal antibodies, only CD49d (expressed on more than 90% of CML CD34+ cells) appeared to influence the functional activity of adhesion molecules. In conclusion, IFN-alpha and bcr-abl As ODN in combination exert a marked in vitro antileukemic activity and could be a useful approach for in vitro purging of CML cells prior to autologous transplantation.

Antigens, CD34↗

PubMed2004

Identification of mcl-1 as a BCR/ABL-dependent target in chronic myeloid leukemia (CML): evidence for cooperative antileukemic effects of imatinib and mcl-1 antisense oligonucleotides.

Antiapoptotic members of the bcl-2 family have recently been implicated in the pathogenesis of chronic myeloid leukemia (CML), a hematopoietic neoplasm associated with the BCR/ABL oncogene. We have examined expression of MCL-1 in primary CML cells and BCR/ABL-transformed cell lines. Independent of the phase of disease, isolated primary CML cells expressed myeloid cell leukemia-1 (mcl-1) mRNA and the MCL-1 protein in a constitutive manner. The BCR/ABL inhibitor imatinib (=STI571) decreased the expression of MCL-1 in these cells. Correspondingly, BCR/ABL enhanced mcl-1 promoter activity, mcl-1 mRNA expression, and the MCL-1 protein in Ba/F3 cells. BCR/ABL-dependent expression of MCL-1 in Ba/F3 cells was counteracted by the mitogen-activated protein-kinase/extracellular signal-regulated kinase (MEK) inhibitor, PD98059, but not by the phosphoinositide 3-kinase inhibitor, LY294002. Identical results were obtained for constitutive expression of MCL-1 in primary CML cells and the CML-derived cell lines K562 and KU812. To investigate the role of MCL-1 as a survival-related target in CML cells, mcl-1 siRNA and mcl-1 antisense oligonucleotides (ASOs) were applied. The resulting down-regulation of MCL-1 was found to be associated with a substantial decrease in viability of K562 cells. Moreover, the mcl-1 ASO was found to synergize with imatinib in producing growth inhibition in these cells. Together, our data identify MCL-1 as a BCR/ABL-dependent survival factor and interesting target in CML.

Animals↗

PubMed2002

Hypoxia modifies proliferation and differentiation of CD34(+) CML cells.

We previously showed that hypoxia (1% O(2)) favors the self-renewal of murine and human normal hematopoietic stem cells. This study represents the first attempt to characterize the effects of hypoxia on the maintenance of chronic myeloid leukemia (CML) progenitors. CD34(+) cells isolated from apheresis products of CML patients were incubated in hypoxia (1% O(2)) and normoxia (20% O(2)). After 8 days of culture, their proliferation, capacity for colony-forming-cell (CFC) generation in secondary cultures (pre-CFC), and phenotype (CD34 and platelet-activating factor receptor [PAF-R]) were compared with those of normal cells, and tyrosine phosphorylation in CML cells was measured. Hypoxia inhibits the proliferation of CD34(+) cells and preserves the pre-CFC capacity and cell-surface CD34 expression of CML cells better than normoxia. The PAF-R expression, which was absent on freshly isolated cells, was detected at the cell surface in both populations after 8 days of culture, but with a lower percentage of positive cells in CML cell cultures. Incubation in hypoxia suppressed the PAF-R expression of normal cells and increased it in CML cells, resulting in a similar expression in the two populations. These effects could be linked to inhibition by hypoxia of the tyrosine hyperphosphorylation of cellular proteins, a major hallmark of CML cells.

Antigens, CD34↗

PubMed2000

Preliminary study of HLA-ABCDR antigens in CML, ANLL, thalassemia and severe aplastic anemia in Thais.

The objective of this study was to analyse human leukocyte antigen (HLA) and disease association in common blood diseases [chronic myelogenous leukemia (CML), acute nonlymphocytic leukemia (ANLL), thalassemia and severe aplastic anemia] in Thais. The subjects were patients from the Hematological Clinic, Departments of Medicine and Pediatrics, Ramathibodi Hospital who were referred for HLA typing for bone marrow transplantation (BMT) at the Histocompatibility Laboratory from March 1988 to September 1997. A total of 129 patients had complete HLA-ABC typing. The patients included 45 CML, 40 ANLL, 26 thalassemia (Thal) and 18 severe aplastic anemia (SAA). Of these, 88 patients were typed for HLA class II. The HLA class I (ABC) and II (DR, DQ) typings were performed by microlymphocytotoxicity test. It was found that HLA class I was associated with CML, ANLL and Thal, whereas, HLA class II was associated with SAA. HLA-B8 and HLA-B18 were increased in CML with R.R. values of 12.2 and 3.9, respectively, whereas, HLA-B18 was increased in ANLL with R.R. value of 4.5. In addition, HLA-DR2 and DR3 were increased in SAA with R.R. values of 3.8 and 4.8, respectively. For Thal, HLA-A2 and B46 were increased in Thal in Central Thais with R.R. values of 3.3 and 6.1, respectively, whereas, HLA-B13 was increased in Thal in Northern Thais with R.R. value of 8.5. On the other hand, HLA-B7 was absent in CML. HLA-Cw7 was decreased in CML and SAA, whereas, HLA-DR6 was decreased in ANLL and SAA. Furthermore, HLA-Cw6 was also decreased in CML, whereas, HLA-A33 and Bw4 were decreased in SAA. Although the sample size of each disease was small, the increase of HLA-DR2 was observed in SAA in Thais which was similar to other studies in different ethnic groups. These preliminary data may be useful for further study in HLA and blood disease association.

Adult↗

PubMed2002

[The effect of multi-unit ribozymes on the growth inhibition and apoptosis induction of CML K562 cells].

OBJECTIVE: To investigate the possibility of multi-unit ribozymes to purge bone marrow of chronic myelocytic leukemia (CML), its in vitro cleavage ability and the reversal effect on CML cell's malignant phenotype. METHODS: As bcr-abl fusion gene plays an important role in CML pathology, three single-unit ribozymes were designed and synthesized in 44 base pairs near the fusion point, two enzyme cleavage sites on bcr gene and one on abl gene. Multi-unit ribozymes' in vitro transcription and retroviral vector through gene recombination were constructed. Then, its in vitro cleavage ability was tested and the retroviral vector was transfected into K562 cell. Through MTT assay, the incorporation rate of (3)H-TdR, RT-PCR, Southern and Northern blot hybridization, flow cytometry, transmission and scanning electron microscopy were used to study the effect of multi-unit ribozymes on CML cell proliferation, cell structure, cell cycle and the induction of apoptosis. RESULTS: Multi-unit ribozymes had in vitro cleavage efficiency of 70.8%. After the transfection of multi-unit ribozymes retroviral vector into K562 cell, cell proliferation and DNA synthesis were greatly reduced with an inhibition rate of about 50% after 96 hours of transfection. Multi-unit ribozymes could cleave K562 cell's RNA with a reduction rate about one 1 000 th of the original. By flow cytometry (FCM), 18.4% cells underwent apoptosis after 72 hours transfection with most of the cells blocked in the G phase. Here, the ratio in S phase was lowered by 41.9%. Under transmission and scanning electron microscope, compaction of nuclear chromation and apoptosis bodies were observed in the transfected cells. CONCLUSION: Multi-unit ribozymes possess high cleavage ability in vitro. The ribozymes, whose retroviral vector being transfected into CML cell, are able to express a lasting ability to cleave the fusion gene, induce apoptosis, reduce cell proliferation, revert the malignant phenotype. It is possible to make use of multi-unit ribozymes to purge CML bone marrow. Therefore, multi-unit ribozymes may very well be valuable in the gene therapy of CML.

Apoptosis↗

PubMed1977

Regulatory mechanisms in cell-mediated immune responses. II. Comparison of culture-induced and alloantigen-induced suppressor cells in MLR and CML.

Two antigen-nonspecific T cell-dependent suppressor systems were compared for their effects upon CML and MLR. Suppressor cells generated by an in vitro culture of spleen cells were compared with suppressor cells generated by in vivo priming with alloantigen. Culture-induced suppressor cells were themselves unable to respond in CML or MLR; were able to suppress actively the CML and MLR responses of untreated responding cells; were mitomycin-sensitive; and, produced no easily demonstrable suppressive supernatant. Alloantigen-primed cells were able to respond in CML and LR; could suppress proliferation in MLR, but were able to suppress CML only after mitomycin treatment; and, produced suppressive supernatants active in suppressing both CML and MLR. In addition to cataloging the differences and similarities between these suppressor populations, the data have been employed to analyze the mechanisms by which suppression occurs in CML and MLR.

Animals↗