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PubMed1997

Mobilization of hematopoietic progenitors in patients with chronic myeloid leukemia.

Although the mobilization and harvesting of Philadelphia chromosome-negative progenitors has been proven feasible by a number of groups, it is not clear which techniques should be used with regard to the monitoring of purging and evaluation of stem cell yield. Therefore, we isolated CD34-positive cells from leukapheresis samples of seven CML patients after induction therapy. These cells were analyzed using the colony-forming unit granulocyte-macrophage (CFU-GM) and long-term culture-initiating cell (LTCIC) assays. In addition, we analyzed frequency and clonogenicity of single stem cells using the LTCIC assay at limiting dilution. Individual colonies derived from these assays were subsequently analyzed for the presence of the bcr-abl gene with interphase fluorescence in situ hybridization (FISH) and for the presence of bcr-abl mRNA with RT-PCR. We compared these results with the cytogenetic analysis of the leukapheresis material. Molecular analysis of individual colonies correlated well with the results of cytogenetic analysis. However, in nine out of 18 samples, cytogenetic analysis exclusively demonstrated the presence of either Philadelphia chromosome-positive or -negative cells whereas with the molecular analysis of individual colonies tumor contamination or the presence of residual normal cells could be substantiated. We concluded that molecular analysis of individual colonies should be employed to further optimize induction protocols. With regard to stem cell mobilization we demonstrated that about 67 CFU-GM colonies and clusters were generated by one single LTCIC both for the CML samples and the samples obtained from patients with non-hematologic malignancy. This number is important since until now most centres use a number of four colonies and clusters generated by one LTCIC. Dividing the CFU-GM output in the LTCIC assay by four to determine LTCIC frequency could thus lead to an almost 20-fold overestimation of the LTCIC frequency. It is concluded that stem cell frequency analysis is an important tool with regard to the interpretation of mobilization protocols.

Adult↗
PubMed2005

[Significance of quantification of WT1 mRNA for monitoring minimal residual disease in acute myeloid leukemia patients].

OBJECTIVE: To evaluate the significance of quantification of WT1 mRNA for monitoring minimal residual disease (MRD) in patients with acute myeloid leukemia (AML). METHODS: WT1 mRNA level was detected with real-time quantitative RT-PCR (RQ-PCR) technique in bone marrow samples from 15 normal subjects (NBM) and 123 AML patients. Sixty-two AML samples were also detected AML1-ETO mRNA expression by RQ-PCR. Simultaneously follow-up of WT1 and AML1-ETO levels were carried out in 50 samples from 8 AML patients. WT1 and AML1-ETO levels were normalized by internal control ABL gene. RESULTS: All correlation co-efficiencies were over 0.99 for WT1, AML1-ETO and ABL standard curves. Co-efficiencies of both interassay and intraassay variation were below 4%. The WT1 expression levels in NBM were 0.001 to 0.019 with a median level of 0.008. Higher levels of WT1 expression were found in 61 of 67 (91%) newly diagnosed AML patients compared with NBMs and 37 of the 67 (55.2%) showed 100-fold higher WT1 levels than that in NBMs. WT1 mRNA levels were highest in M(4EO) and M(3) and lowest in M(1) and M(5) patients. There was an excellent correlation between WT1 and AML1-ETO gene expression levels (r = 0.88, P < 0.001). WT1 expression levels in three patients who were in continuous complete hematological remission (CHR) were within normal range. In three of four relapsing patients, WT1 expression levels increased 31.4, 11.4 and 4.0 fold respectively one month before hematological relapse. CONCLUSIONS: Quantification of WT1 expression level by RQ-PCR may be used to monitor MRD for most AML patients, but it is less sensitive than fusion gene. Continuous or significant increase of WT1 expression in CHR patients predicts an impending relapse.

Adolescent↗
PubMed1990

The (6;9) chromosome translocation, associated with a specific subtype of acute nonlymphocytic leukemia, leads to aberrant transcription of a target gene on 9q34.

The specific (6;9)(p23;q34) chromosomal translocation is associated with a defined subtype of acute nonlymphocytic leukemia (ANLL). The 9q34 breakpoint is located at the telomeric side of the c-abl gene. Through a combination of chromosome jumping, long-range mapping, and chromosome walking, the chromosome 9 breakpoints of several t(6;9) ANLL patients were localized within a defined region of 8 kilobases (kb), 360 kb telomeric of c-abl. Subsequent cDNA cloning revealed that this region represented an intron in the middle of a gene, called Cain (can), encoding a 7.5-kb transcript. Disruption of the can gene by the translocation resulted in the expression of a new 5.5-kb can mRNA from the 6p- chromosome. Isolation of chromosome 6 sequences showed that breakpoints on 6p23 also clustered within a limited stretch of DNA. These data strongly suggest a direct involvement of the translocation in the leukemic process of t(6;9) ANLL.

Blotting, Southern↗
PubMed2002

Modulation of the F-actin cytoskeleton by c-Abl tyrosine kinase in cell spreading and neurite extension.

The nonreceptor tyrosine kinase encoded by the c-Abl gene has the unique feature of an F-actin binding domain (FABD). Purified c-Abl tyrosine kinase is inhibited by F-actin, and this inhibition can be relieved through mutation of its FABD. The c-Abl kinase is activated by physiological signals that also regulate the actin cytoskeleton. We show here that c-Abl stimulated the formation of actin microspikes in fibroblasts spreading on fibronectin. This function of c-Abl is dependent on kinase activity and is not shared by c-Src tyrosine kinase. The Abl-dependent F-actin microspikes occurred under conditions where the Rho-family GTPases were inhibited. The FABD-mutated c-Abl, which is active in detached fibroblasts, stimulated F-actin microspikes independent of cell attachment. Moreover, FABD-mutated c-Abl stimulated the formation of F-actin branches in neurites of rat embryonic cortical neurons. The reciprocal regulation between F-actin and the c-Abl tyrosine kinase may provide a self-limiting mechanism in the control of actin cytoskeleton dynamics.

Actins↗
PubMed2006

Heterologous expression implicates a GATA factor in regulation of nitrogen metabolic genes and ion homeostasis in the halotolerant yeast Debaryomyces hansenii.

The yeast Debaryomyces hansenii has a remarkable capacity to proliferate in salty and alkaline environments such as seawater. A screen for D. hansenii genes able to confer increased tolerance to high pH when overexpressed in Saccharomyces cerevisiae yielded a single gene, named here DhGZF3, encoding a putative negative GATA transcription factor related to S. cerevisiae Dal80 and Gzf3. Overexpression of this gene in wild-type S. cerevisiae increased caffeine and rapamycin tolerance, blocked growth in low glucose concentrations and nonfermentable carbon sources, and resulted in lithium- and sodium-sensitive cells. Sensitivity to salt could be attributed to a reduced cation efflux, most likely because of a decrease in expression of the ENA1 Na(+)-ATPase gene. Overexpression of DhGZF3 did not affect cell growth in a gat1 mutant but was lethal in the absence of Gln3. These are positive factors that oppose both Gzf3 and Dal80. Genome-wide transcriptional profiling of wild-type cells overexpressing DhGZF3 shows decreased expression of a number of genes that are usually induced in poor nitrogen sources. In addition, the entire pathway leading to Lys biosynthesis was repressed, probably as a result of a decrease in the expression of the specific Lys14 transcription factor. In conclusion, our results demonstrate that DhGzf3 can play a role as a negative GATA transcription factor when expressed in S. cerevisiae and that it most probably represents the only member of this family in D. hansenii. These findings also point to the GATA transcription factors as relevant elements for alkaline-pH tolerance.

Adenosine Triphosphatases↗
PubMed1988

Selective transformation of primitive lymphoid cells by the BCR/ABL oncogene expressed in long-term lymphoid or myeloid cultures.

The BCR/ABL gene, formed by the Philadelphia chromosome translocation (Ph1) of human chronic myelogenous leukemia, encodes an altered ABL gene product, P210. P210 is strongly implicated in the malignant process of chronic myelogenous leukemia, but it precise role is unknown. Infection of long-term bone marrow cultures enriched for B-lymphoid cell types with a Moloney murine leukemia virus retroviral vector containing the BCR/ABL cDNA resulted in clonal outgrowths of immature B-lymphoid cells which expressed abundant P210 kinase activity. Surprisingly, infection of long-term myeloid lineage-enriched cultures also resulted in clonal outgrowths of immature B-lymphoid cells. The P210-expressing lymphoid cell lines resulting from either type of culture were resistant to the lethal effects of corticosteroids. These findings indicate that high levels of P210 expressed from a Moloney murine leukemia virus long terminal repeat preferentially stimulate the growth of immature B-lineage cells, and this effect is apparent even in myeloid lineage-enriched cultures, in which few if any lymphoid cells can be detected prior to infection.

B-Lymphocytes↗
PubMed1989

Juvenile chronic myeloid leukemia: oncogene characterization.

A 31/2-year-old female presented with thrombocytopenia and anemia. The bone marrow showed marked hemophagocytosis with an increase in macrophages. Over the next 2 months, there was a progressive de-differentiation of this monocytic population with the accumulation of blasts in the blood and bone marrow. The blasts had a normal 46XX karyotype and showed no fusion of the bcr and abl genes associated with Philadelphia chromosome positive leukemia. Intensive chemotherapy produced a transient hypoplastic state during which a bone marrow transplant was performed. The bone marrow after transplant again demonstrated a large population of macrophages. These cells continued to de-differentiate over the ensuing year up until the time of the patient's death. The mononuclear blast cell population was inducible toward monocytic maturation in tissue culture by low doses of ARA-c or daunorubicin. These mononuclear blasts expressed c-myc and c-fos mRNA at high levels, a further marker of their proliferative state and monocytic origin.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1991

[Advances in molecular genetic diagnosis of leukemia].

Recently, Molecular genetics has remarkably advanced and it is introduced in medicine. The use of recombinant DNA methods for the diagnosis of leukemias is reported with special reference to the contribution of cytogenetic findings, such as specific chromosome aberrations previously obtained. Therefore, cytogenetic studies on Ph1 chromosome and other specific aberrations found in leukemias are historically reviewed. Using Southern blotting, PFGE, PCR, and in situ chromosome mapping techniques we have analyzed many cases with CML and cases with ALL. We found M-bcr rearrangements not only in standard Ph1, but also in complex types and in Ph1 (-) ve CML. Chromosomal in situ hybridization was very informative identifying transposition of bcr and abl genes between chromosomes 22 and 9. In this connection, FISH (fluorescence in situ hybridization) technique was developed by us, which is expected to have an exceptional power of analysis. ALL had either M-bcr or m-bcr rearrangements, the latter being identified by PFGE. Next, application of PCR technique that enables to obtain more than 10(5) copies of target sequences could monitor minimal residual diseases in CML. Recently, the relevant gene were cloned respectively in FAB-M2 and APL (FAB-M3), so that detection of minimal residual diseases will be successfully performed in these types of leukemia. Finally, targeting chemotherapy using antisense sequences is prospectively described.

Blotting, Southern↗
PubMed1994

ABL and BCR genes are not imprinted in androgenetic and gynogenetic human tissues.

In the translocation leading to the formation of the Philadelphia chromosome, the hallmark of chronic myeloid leukemia (CML), the translocated chromosome 9 (ABL), is of paternal descent whereas chromosome 22 (BCR) is of maternal origin (1). To study possible imprinting of the human ABL and BCR genes, we used human tissues exclusively endowed with their maternally (benign teratoma) or paternally (complete hydatidiform mole) inherited chromosomes. Using the sensitive PCR technique followed by northern blotting, we demonstrate here that ABL and BCR are expressed to a similar extent in androgenetic and gynogenetic human tissues, thus suggesting that ABL and BCR genes are not imprinted in these human tissues.

Base Sequence↗
PubMed2006

[Expression of Bcl-2 protein and the amplification of c-myc gene in patients with chronic myeloid leukemia].

BACKGROUND/AIM: Chronic myeloid leukemia (CML) represents a malignant myeloproliferative disease developed out of pluripotent hematopoietic stem cell that contains the fusion bcr-abl gene. Disorders that occur in the process of apoptosis represent one of the possible molecular mechanisms that bring about the disease progress. The aim of our study was to carry out the analysis of the presence of the amplification of the c-myc oncogene, as well as the analysis of the changes in the expression of Bcl-2 in the patients with CML. METHODS: Our study included 25 patients with CML (18 in chronic phase, 7 in blast transformation). Using an immunohistochemical alkaline phosphatase-anti-alkaline phosphatase (APAAP) method, we analyzed the expression of cell death protein in the mononuclear bone marrow cells of 25 CML patients. By a differential PCR (polymerase chain reaction) method, we followed the presence of amplified c-myc gene in mononuclear peripheral blood cells. RESULTS: The level of the expression of Bcl-2 protein was considerably higher in the bone marrow samples of the patients undergoing blast transformation of the disease. The amplification of c-myc gene was detected in 30% of the patients in blast transformation of the disease. CONCLUSION: The expression of Bcl-2 protein and the amplification of c-myc gene are in correlation with the disease progression.

Bone Marrow Cells↗
PubMed2002

Purging of chronic myelogenous leukemia cells by retrovirally expressed anti-bcr-abl ribozymes with specific cellular compartmentalization.

In patients with chronic myelogenous leukemia (CML), abnormal expansion of myeloid cells is maintained by expression of the p210(bcr-abl) fusion protein. Thus, this protein and its mRNA represent primary targets to inhibit proliferation of these cells. Here we describe the properties of a ribozyme against the bcr-abl mRNA, expressed as a fusion transcript with the human U1 small nuclear RNA or the adenovirus VA1 RNA and delivered to the cells through retroviral vectors. These fusion ribozymes are specifically localized in the nucleus or in the cytoplasm, respectively. Transduction of 32D-LG7 myeloid cells, whose growth is IL-3 independent thanks to deregulated bcr-abl expression, imposed strong negative selective pressure on cell growth and induced restoration of an IL-3-dependent phenotype. Although expressed at a level similar to that of the U1-fusion ribozyme, the cytoplasmic VA1 ribozyme was a more powerful inhibitor of p210(bcr-abl) gene expression. In cells transduced with the vector expressing this ribozyme, the levels of the bcr-abl transcript were reduced up to 10(4)-fold, the p210(bcr-abl) protein became undetectable, and the cells underwent massive apoptosis when cultured in the absence of IL-3. Transduction of primary hematopoietic cells obtained from bone marrow of patients with CML resulted in remarkable reduction of bcr-abl mRNA levels, starting a few days after transduction. These results show the feasibility and efficacy of vector-expressed anti-bcr-abl ribozymes for purging of CML cells.

Animals↗
PubMed2002

Novel targeted therapies for Bcr-Abl positive acute leukemias: beyond STI571.

In the pathophysiology of CML, the constitutive activity of the Bcr-Abl tyrosine kinase (TK) is, most likely, the sole molecular abnormality of the chronic phase. It also remains a critical molecular determinant of malignant behavior of the leukemic progenitors in the accelerated and blastic phase of CML. Therefore, downregulation of the levels and activity of Bcr-Abl is clearly the lynchpin of a rational therapeutic strategy against all phases of CML. Support for this has only been strengthened by the observations that resistance to imatinib mesylate (imatinib) commonly involves a breakthrough and the persistent activity of Bcr-Abl TK. This is due to either mutations that inhibit imatinib action on Bcr-Abl TK or amplification of the bcr-abl gene. Recent studies have demonstrated that other small molecule tyrosine kinase inhibitors that also inhibit Bcr-Abl TK may be highly active in inducing differentiation and apoptosis of CML progenitors, regardless of their sensitivity to imatinib. Small molecule inhibitors that downregulate the levels of Bcr-Abl by inhibiting its translation, e.g., arsenic trioxide, or promoting its proteasomal degradation, e.g., geldanamycin analogues, have also been identified. Finally the identification of other potent survival and antiapoptotic signaling pathways in imatinib-resistant CML progenitors indicates that inhibitors of these pathways will eventually be treatment strategies for advanced phases of CML.

Antineoplastic Agents↗
PubMedDate not supplied

BCR-ABL fusion peptides and cytotoxic T cells in chronic myeloid leukaemia.

The BCR-ABL gene that arises in chronic myeloid leukaemia (CML) is a neoantigen. Peptides derived from the BCR-ABL fusion junction may therefore be immunogenic, if appropriately presented to the immune system. This article reviews data demonstrating that certain junctional peptides will bind to HLA molecules, and that these peptides will elicit specific T-lymphocyte responses in vitro, in both normal subjects and in CML patients. The clinical relevance of these observations is discussed.

Antigen Presentation↗
PubMed1996

ts BCR-ABL kinase activation confers increased resistance to genotoxic damage via cell cycle block.

Using a temperature-sensitive mutant of the p210 BCR-ABL gene, transfected into a growth factor-dependent cell line (BaF3), we show that transient BCR-ABL kinase expression increases single cell and clonogenic resistance to apoptosis arising from genotoxic damage induced by ionizing radiation and VP-16/etoposide. This effect is achieved in the absence of any detectable changes in the levels of BCL-2, BAX or BCL-x proteins and is independent of proliferative, MAP kinase-dependent effects of BCR-ABL kinase. In contrast to parental cells that transiently arrest in G2 and then apoptose, p210 BaF3 cells show a pronounced and sustained G2 arrest following radiation coupled with enhanced phosphorylation of cdc2. A cell cycle block in early M phase induced by the mitotic spindle poison, nocodazole, does not provide protection from apoptosis. Reversal of G2 arrest by caffeine abolishes the protective effect of BCR-ABL kinase. These data provide further insight into the transforming properties of BCR-ABL and are relevant to the clinical intransigence of Ph-positive leukaemias.

Antineoplastic Agents, Phytogenic↗
PubMed1988

Negative regulation of c-abl tyrosine kinase by its variable N-terminal amino acids.

Activation of the oncogenic potential of c-abl proto-oncogene has been correlated with the activation of its tyrosine kinase activity. The oncogenes derived from c-abl, e.g., gag/v-abl in Abelson murine leukemia virus or bcr/abl in chronic myelogenous leukemia, lack N-terminal coding sequences of the normal c-abl gene. In mouse and human cells, two sets of N-terminal amino acids encoded by 5'-variable exons are found in c-abl proteins. To assess the importance of N-terminal deletion in the activation of c-abl tyrosine kinase, a full length or an N-terminal deleted c-abl protein was expressed in bacteria and in monkey COS cells. Measurements of the autokinase activity of these two c-abl proteins showed that deletion of the N-terminal amino acids led to a three to five fold increase of the c-abl tyrosine kinase activity. Thus, the N-terminal deletion is important in the activation of c-abl proto-oncogene.

Amino Acid Sequence↗
PubMed1991

Arg encodes a widely expressed 145 kDa protein-tyrosine kinase.

Arg encodes a protein highly related to the c-abl gene product with regard to overall structural architecture as well as the amino acid sequences of their tyrosine kinase, and src-homologous 2 and 3 domains. The two genes form a distinct subfamily of non-receptor tyrosine kinases and share a common homolog in Drosophila. In this study we characterized the arg protein product by expression of its coding sequence in bacteria. The recombinant arg protein was detected in bacterial lysates by immunoblotting and exhibited a molecular mass of 145 kDa. Phosphoamino acid analysis of the arg gene product following an immune complex autokinase reaction revealed tyrosine phosphorylation and established that it possesses tyrosine kinase activity. High-titer antibody capable of detecting the cellular arg gene product was generated by expressing a carboxy-terminal segment of arg in bacteria and using the recombinant protein as an immunogen. The arg gene product was identified in cultured human cells as a 145 kDa protein that exhibited autokinase activity. Analysis of arg expression in murine tissues revealed that arg, like c-abl, is widely expressed, further extending the similarities between the two genes, and suggesting that arg probably functions in signaling pathways fundamental to many cell types.

DNA-Directed RNA Polymerases↗
PubMed1998

Reverse transcription polymerase chain reaction is a reliable assay for detecting leukemic colonies generated by chronic myelogenous leukemia cells.

Single-colony karyotyping (SCK) and reverse-transcription polymerase chain reaction (RT-PCR) are two increasingly used techniques for the quantification of leukemic colonies generated by chronic myelogenous leukemia (CML) cell fractions purged or selected in vitro. Recently, the existence of Philadelphia (Ph) chromosome positive progenitors with a silent BCR-ABL gene has been reported, thus raising concerns on the use of RT-PCR for detecting BCR-ABL positive progenitors. In order to investigate this issue further, colonies (n = 204) generated by mononuclear (MNC) or CD34+ CML cells were individually harvested, divided into two aliquots and analyzed both at the cytogenetic level to detect the Ph chromosome, and the molecular level to detect BCR-ABL transcripts. The mean (+/- s.d.) percentages of colonies analyzable by either SCK or RT-PCR were 74 +/- 16% and 86 +/- 16%, respectively. A significant percentage of colonies (67 +/- 19%) could be successfully analyzed by both SCK and RT-PCR. Although the majority of these colonies (97 +/- 5%) were Ph-positive and BCR-ABL-positive, a negligible percentage (4%) of progenitors were Ph-positive but BCR-ABL-negative. In order to test the influence of colony size on the outcome of molecular analysis, the efficiency of our RT-PCR assay in detecting BCR-ABL transcripts was investigated by means of experiments in which the number of cells used to start RNA extraction was serially reduced. These experiments showed that at least 150 cells were necessary to achieve a reproducible amplification of BCR-ABL transcripts. By correlating the size of harvested colonies with the outcome of molecular analysis, it was evident that BCR-ABL-negative but Ph-positive colonies represented false negative results occurring when a number of leukemic cells below the detection limit of our RT-PCR assay was analyzed. In conclusion, our data demonstrate that individual CML colonies grown in semisolid culture assays can be indifferently analyzed by SCK or RT-PCR, and support an extensive use of a carefully standardized RT-PCR assay to estimate the leukemic burden within samples which have been purged and selected in vitro.

Adult↗
PubMed2001

Targeting c-kit mutations in solid tumors: scientific rationale and novel therapeutic options.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. These tumors span a wide clinical spectrum from benign to malignant and have long been recognized for their nearly absolute resistance to chemotherapy and radiation treatment. Surgery is the primary treatment modality for GISTs, but GISTs represent an incurable malignancy for patients with metastatic or unresectable disease. Thus, novel approaches to the treatment of GISTs were desperately needed. Gastrointestinal stromal tumors are characterized by expression of the transmembrane receptor tyrosine kinase KIT, which is defined by the CD117 antigen and is the product of the c-kit proto-oncogene. Activating or gain-of-function mutations in the c-kit gene have been identified in the majority of GIST cases. The resulting constitutive KIT tyrosine kinase activity was hypothesized to provide growth and survival signals to GIST cells and to be crucial to the pathogenesis of the disease. This hypothesis became testable with the identification of the signal transduction inhibitor imatinib mesylate (formerly STI571, [Gleevec]; Novartis Pharmaceuticals Corp, East Hanover, NJ), which blocks the tyrosine kinase activity of KIT as well as the kinase activity of the normal c-abl gene product, the oncogenic Bcr-Abl chimeric fusion protein of chronic myeloid leukemia, and the platelet-derived growth factor receptor. Preclinical experiments showed rapid inhibition of ligand-independent KIT phosphorylation, decreased cellular proliferation, and induction of apoptosis after exposure of GIST cells to imatinib mesylate in vitro. These results provided the rationale to move forward with clinical testing of imatinib mesylate as an anticancer therapy for GIST. In early 2000, a dramatic clinical and radiographic response to imatinib mesylate was shown in a single patient with advanced, chemotherapy-resistant GIST. The powerful scientific rationale for this proof-of-concept study, together with the durable and significant response observed in this first GIST patient treated with imatinib mesylate, have provided the driving force for rapid clinical development of this targeted therapy in this solid tumor indication.

Antineoplastic Agents↗