THE DISCOVERY, ISOLATION AND CHARACTERIZATION OF THE BCR-ABL ONCOGENE AND THE LATEST THERAPEUTIC STRATEGY FOR THE TREATMENT OF CHRONIC MYELOID LEUKEMIA
The bcr-abl oncogene is a product of a translocation between chromosomes 9 and 22. This fusion product is also called the Philadephia chromosome and is responsible for the cancer seen in myeloid cells, chronic myeloid leukemia (CML).
Chronic myelogenous leukemia (CML) is a clonal hematopoietic stem cell disorder with an annual incidence of one to two cases per 100,000 per year (Mauro Druker, 2001). The disease proceeds in four distinct phases the stable (chronic) phase, advanced phase, accelerated phase, and blast crisis. The first phase, which is the chronic phase, is characterized by massive expansion of myeloid cells. Leukemic cells lose their capacity to terminally differentiate in the other phases, resulting in an acute leukemia, which is highly noncompliant with therapy. The Philadelphia (Ph) chromosome has been found to be the cytogenetic hallmark of all phases of chronic myeloid leukemia and some subsets of acute lymphoblastic leukemia (ALL). The Ph chromosome is a shortened chromosome 22 that results from a reciprocal translocation between the long arms of chromosomes 9 and 22 (Mauro Druker, 2001).
CML is distinguished from other conditions by the presence of a distinctive molecular abnormality, namely a translocation involving the bcr gene on chromosome 22 and the abl gene on chromosome 9 (Kumar, Abbas, Fausto, 2004).
Researches conducted on oncogenic viruses gave some of the first clues to the mechanisms by which normal cells became malignant. These viruses have been found to fall into two major groups. These include chronic leukemia viruses and acute transforming viruses. Chronic leukemia viruses are replication competent and are not known to transform cells in vitro (Rosson Reddy, 1988). However, several of the chronic leukemia viruses have been implicated in tumors when injected into newborn animals after a lengthy latent period of several months to years.
Discovery
The abl oncogene was initially found in the Abelson murine leukemia virus (Ab-MLV). This virus was isolated about 6 years prior to the discovery that genes contained in some retroviruses were homologous to normal cellular genes and was initially of interest because, unlike most murine leukemia viruses Ab-MLV induced lymphomas after a very short latent period (Ramakrishnan Rosenberg, 1989). The ability of Ab-MLV to change both fibroblasts and lymphoid cells in vitro emphasized the biological differences that differentiate the Ab-MLV from other murine leukemia viruses. However, these early experiments only did little to explain the varied and complex responses of cells to abl expression. The identification of the Ab-MLV transforming protein and the finding that many normal cells encode a related protein pointed the way for experiments that culminated in the cloning of the viral genome and the normal cellular homologue of the viral transforming gene (Ramakrishnan et al, 1989).These seminal experiments helped define the properties of the Ab-MLV and was a starting point for understanding the relationship of the virus to the normal cellular abl gene.
The ABL-BCR was first discovered and described by Peter Nowell from the University of Pennsylvania School of Medicine and David Hungerford from Fox Chase Cancer Centres Institute for Cancer Research in 1960. The Chromosome was therefore named after the City in which both institutions were located- Philadephia (Philadelphia chromosome).
The discovery of the Philadelphia chromosome as a hallmark of chronic myelogenous leukemia in 1960 by Peter Nowell provided evidence for a genetic link to cancer (Koretzky, 2007)
Isolation of the bcr-abl oncogene
Different methods have been successfully used to isolate the bcr-abl oncogene. Some of them include
Isolation of BCR-ABL Transfected Ml Clones An expression vector coding for BCR-ABL p210 (pLSV-p210) together with a plasmid conferring resistance to G418 neomycin (pSV2neo) is introduced by electroporation into the S-6 subclone of Ml cells. Stable neo-resistant clones are analyzed for expression and incorporation of the BCR-ABL gene. Northern blot analysis showed that two isolated clones expressed high levels of the BCR-ABL large mRNA transcript. There was no signal either in a control neo clone or in the non transfected parental cells. As a result of the low abundance of the endogenous ABL mRNA transcripts (of 5.3 and 6.5 kilobases) it was not possible to detect them in total RNA samples. The p210 protein, the functional expression of the BCR-ABL gene product, was established by in vitro autokinase assay in immune complexes produced by anti-BCR antibodies. It should be noted that the two BCR-ABL-transfected clones displayed normal growth kinetics and cell cycle distribution, characteristic of parental Ml cells (Zafriri, Argaman, Canaan Kimchi, 1992).
Expression of v-abl-derived p60 in E. coli A plasmid expression vector, pAS1-Abl-60, is constructed which permits controlled expression of a truncated product of the A-MuLV v-ab1 transforming gene in E. coli (Fergusson, Pritchard, Field, Rieman, Greig, Poste Rosenberg, 1985).
In an bid to get high level buildup of v-abl-derived tyrosine kinase in E. coli, fusion of the v-ab1 coding sequence in-frame with the coding sequence of the first 7 amino acid residues of a product (influenza virus NS1) known to be expressed at high levels in E. coli are carried out. This approach is designed to ensure that translation of the fusion product will initiate efficiently (Fergusson, Pritchard, Field, Rieman, Greig, Poste Rosenberg, 1985).
Characterization
The Philadelphia chromosome is found in more than 95 of patients with chronic myeloid leukemia. This chromosome contains 5 BCR gene sequences fused to the ABLgene at its 5 end. The gene product formed is a Bcr-Abl protein that has a greatly elevated protein tyrosine kinase activity. Two forms of the Bcr-Abl protein have been described earlier. Depending on the location of the breakpoint within BCR, either a 210- (P210BCR-ABL) or 185-kDa (P185BCR-ABL) protein is produced (Liu, Wu, Ma, Dailu, Haataja, Heisterkamp, Groffen, Arlinghaus, 1995). The two forms of the Bcr-Abl protein are very important factors in the induction and maintenance of leukemias, as confirmed by mouse studies.
The defect in the BCR-ABL gene is a translocation. Segments of chromosome 9 and 22 swap places which results in a gene (a fusion gene) between the BCR ( breakpoint cluster region) gene from chromosome 22 (region q11) and the Abl1 gene (Abelson, the name of a leukemia virus that carries a similar protein) located on chromosome 9.
Because of its discovery and the early recognition of its role in human cancer, BCR-ABL is one of the most highly studied oncogenes (Etten, 2004).
The translocation results in the oncogene, BCR-ABL gene which is located on the short derivative chromosome 22 and it encodes the BCR-ABL fusion protein with its molecular weight ranging from 185kDa to 210kDa. The Abl gene expresses a membrane-associated protein, tyrosine kinase and the bcr region expresses serinethreonine kinases but the tyrosine kinase function is of great clinical importance as it is the target for drug therapy.
Treatment of Chronic Myeloid Leukemia
Chronic myeloid leukemia (CML) is a pluripotent hematopoietic stem cell disorder characterized by accumulation of mature and immature granulocytes in peripheral blood and bone marrow due to uncontrolled growth and resistance to apoptosis (Rongzhen, Qinhua, Yingzi, Xiaoying, Xiaoxian, Dong, Qinghua, Xiaohua, Xiao-Fang, 2005).
A breakpoint cluster region (bcr) was identified on chromosome 22 the DNAs of all (over 30) Ph-positive CML patients examined to date have breakpoints in this region of up to 5.8 kb (Grosveld, Verwoerd, Agthoven, Klein, Ramachandran, Heisterkamp, Stam, Groffen, 1986).
Imatinib
In the late 1990s, Imatinib was first identified by Norvatis pharmaceuticals. Imatinib is a potent inhibitor of tyrosine kinase activity. Gleevec (also called Imatinib or STI571) is a small-molecule inhibitor that binds to the kinase domain of bcrabl oncoprotein and stabilizes the protein in its closed, inactive conformation, thereby inhibiting its activity, and is now a rst-line therapy for the majority of CML cases because of its high efficacy and relatively mild side-effects. (Rongzhen et al, 2005).
The Bcr-Abl fusion protein kinase causes chronic myeloid leukemia and is targeted by the signal transduction inhibitor STI-571Gleevecimatinib (STI-571). Sequencing of the BCR-ABL gene in patients who have relapsed after STI-571 chemotherapy has revealed a limited set of kinase domain mutations that mediate drug resistance (Azam, Latek, Daley, 2003).
In 80 of newly diagnosed cases of CML, imatinib induces the bone marrow to be totally free of the Philadephia chromosome(Levinson, Reid, Burt, Harrison, Fleming, 2008).
Chronic myelogenous leukemia (CML) is a clonal hematopoietic stem cell disorder with an annual incidence of one to two cases per 100,000 per year (Mauro Druker, 2001). The disease proceeds in four distinct phases the stable (chronic) phase, advanced phase, accelerated phase, and blast crisis. The first phase, which is the chronic phase, is characterized by massive expansion of myeloid cells. Leukemic cells lose their capacity to terminally differentiate in the other phases, resulting in an acute leukemia, which is highly noncompliant with therapy. The Philadelphia (Ph) chromosome has been found to be the cytogenetic hallmark of all phases of chronic myeloid leukemia and some subsets of acute lymphoblastic leukemia (ALL). The Ph chromosome is a shortened chromosome 22 that results from a reciprocal translocation between the long arms of chromosomes 9 and 22 (Mauro Druker, 2001).
CML is distinguished from other conditions by the presence of a distinctive molecular abnormality, namely a translocation involving the bcr gene on chromosome 22 and the abl gene on chromosome 9 (Kumar, Abbas, Fausto, 2004).
Researches conducted on oncogenic viruses gave some of the first clues to the mechanisms by which normal cells became malignant. These viruses have been found to fall into two major groups. These include chronic leukemia viruses and acute transforming viruses. Chronic leukemia viruses are replication competent and are not known to transform cells in vitro (Rosson Reddy, 1988). However, several of the chronic leukemia viruses have been implicated in tumors when injected into newborn animals after a lengthy latent period of several months to years.
Discovery
The abl oncogene was initially found in the Abelson murine leukemia virus (Ab-MLV). This virus was isolated about 6 years prior to the discovery that genes contained in some retroviruses were homologous to normal cellular genes and was initially of interest because, unlike most murine leukemia viruses Ab-MLV induced lymphomas after a very short latent period (Ramakrishnan Rosenberg, 1989). The ability of Ab-MLV to change both fibroblasts and lymphoid cells in vitro emphasized the biological differences that differentiate the Ab-MLV from other murine leukemia viruses. However, these early experiments only did little to explain the varied and complex responses of cells to abl expression. The identification of the Ab-MLV transforming protein and the finding that many normal cells encode a related protein pointed the way for experiments that culminated in the cloning of the viral genome and the normal cellular homologue of the viral transforming gene (Ramakrishnan et al, 1989).These seminal experiments helped define the properties of the Ab-MLV and was a starting point for understanding the relationship of the virus to the normal cellular abl gene.
The ABL-BCR was first discovered and described by Peter Nowell from the University of Pennsylvania School of Medicine and David Hungerford from Fox Chase Cancer Centres Institute for Cancer Research in 1960. The Chromosome was therefore named after the City in which both institutions were located- Philadephia (Philadelphia chromosome).
The discovery of the Philadelphia chromosome as a hallmark of chronic myelogenous leukemia in 1960 by Peter Nowell provided evidence for a genetic link to cancer (Koretzky, 2007)
Isolation of the bcr-abl oncogene
Different methods have been successfully used to isolate the bcr-abl oncogene. Some of them include
Isolation of BCR-ABL Transfected Ml Clones An expression vector coding for BCR-ABL p210 (pLSV-p210) together with a plasmid conferring resistance to G418 neomycin (pSV2neo) is introduced by electroporation into the S-6 subclone of Ml cells. Stable neo-resistant clones are analyzed for expression and incorporation of the BCR-ABL gene. Northern blot analysis showed that two isolated clones expressed high levels of the BCR-ABL large mRNA transcript. There was no signal either in a control neo clone or in the non transfected parental cells. As a result of the low abundance of the endogenous ABL mRNA transcripts (of 5.3 and 6.5 kilobases) it was not possible to detect them in total RNA samples. The p210 protein, the functional expression of the BCR-ABL gene product, was established by in vitro autokinase assay in immune complexes produced by anti-BCR antibodies. It should be noted that the two BCR-ABL-transfected clones displayed normal growth kinetics and cell cycle distribution, characteristic of parental Ml cells (Zafriri, Argaman, Canaan Kimchi, 1992).
Expression of v-abl-derived p60 in E. coli A plasmid expression vector, pAS1-Abl-60, is constructed which permits controlled expression of a truncated product of the A-MuLV v-ab1 transforming gene in E. coli (Fergusson, Pritchard, Field, Rieman, Greig, Poste Rosenberg, 1985).
In an bid to get high level buildup of v-abl-derived tyrosine kinase in E. coli, fusion of the v-ab1 coding sequence in-frame with the coding sequence of the first 7 amino acid residues of a product (influenza virus NS1) known to be expressed at high levels in E. coli are carried out. This approach is designed to ensure that translation of the fusion product will initiate efficiently (Fergusson, Pritchard, Field, Rieman, Greig, Poste Rosenberg, 1985).
Characterization
The Philadelphia chromosome is found in more than 95 of patients with chronic myeloid leukemia. This chromosome contains 5 BCR gene sequences fused to the ABLgene at its 5 end. The gene product formed is a Bcr-Abl protein that has a greatly elevated protein tyrosine kinase activity. Two forms of the Bcr-Abl protein have been described earlier. Depending on the location of the breakpoint within BCR, either a 210- (P210BCR-ABL) or 185-kDa (P185BCR-ABL) protein is produced (Liu, Wu, Ma, Dailu, Haataja, Heisterkamp, Groffen, Arlinghaus, 1995). The two forms of the Bcr-Abl protein are very important factors in the induction and maintenance of leukemias, as confirmed by mouse studies.
The defect in the BCR-ABL gene is a translocation. Segments of chromosome 9 and 22 swap places which results in a gene (a fusion gene) between the BCR ( breakpoint cluster region) gene from chromosome 22 (region q11) and the Abl1 gene (Abelson, the name of a leukemia virus that carries a similar protein) located on chromosome 9.
Because of its discovery and the early recognition of its role in human cancer, BCR-ABL is one of the most highly studied oncogenes (Etten, 2004).
The translocation results in the oncogene, BCR-ABL gene which is located on the short derivative chromosome 22 and it encodes the BCR-ABL fusion protein with its molecular weight ranging from 185kDa to 210kDa. The Abl gene expresses a membrane-associated protein, tyrosine kinase and the bcr region expresses serinethreonine kinases but the tyrosine kinase function is of great clinical importance as it is the target for drug therapy.
Treatment of Chronic Myeloid Leukemia
Chronic myeloid leukemia (CML) is a pluripotent hematopoietic stem cell disorder characterized by accumulation of mature and immature granulocytes in peripheral blood and bone marrow due to uncontrolled growth and resistance to apoptosis (Rongzhen, Qinhua, Yingzi, Xiaoying, Xiaoxian, Dong, Qinghua, Xiaohua, Xiao-Fang, 2005).
A breakpoint cluster region (bcr) was identified on chromosome 22 the DNAs of all (over 30) Ph-positive CML patients examined to date have breakpoints in this region of up to 5.8 kb (Grosveld, Verwoerd, Agthoven, Klein, Ramachandran, Heisterkamp, Stam, Groffen, 1986).
Imatinib
In the late 1990s, Imatinib was first identified by Norvatis pharmaceuticals. Imatinib is a potent inhibitor of tyrosine kinase activity. Gleevec (also called Imatinib or STI571) is a small-molecule inhibitor that binds to the kinase domain of bcrabl oncoprotein and stabilizes the protein in its closed, inactive conformation, thereby inhibiting its activity, and is now a rst-line therapy for the majority of CML cases because of its high efficacy and relatively mild side-effects. (Rongzhen et al, 2005).
The Bcr-Abl fusion protein kinase causes chronic myeloid leukemia and is targeted by the signal transduction inhibitor STI-571Gleevecimatinib (STI-571). Sequencing of the BCR-ABL gene in patients who have relapsed after STI-571 chemotherapy has revealed a limited set of kinase domain mutations that mediate drug resistance (Azam, Latek, Daley, 2003).
In 80 of newly diagnosed cases of CML, imatinib induces the bone marrow to be totally free of the Philadephia chromosome(Levinson, Reid, Burt, Harrison, Fleming, 2008).
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