Oncogenetic aspects of chronic myelocytic leukemia.
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DNA↗
Biomedical subjects
G Grosveld
Publications and source records attributed to G Grosveld.
HLA-DR genotyping by restriction fragment length polymorphism analyses.
We have established unique restriction fragment length polymorphism (RFLP) patterns characteristic of homozygous typing cells (HTCs) for HLA-DR-1 through HLA-DR-8 haplotypes. These RFLP patterns were found to segregate in family members and correlate 100% with HLA-DR antibody phenotyping. The RFLP patterns were used to type chronic myelocytic leukemic cells which have a Philadelphia translocation from 23 randomly selected Caucasoid patients. The results show an alternative method for the determination of the HLA-DR types without using live cells and to study disease association with the HLA-DR region.
Cell Line↗
Translocation of oncogene c-sis from chromosome 22 to chromosome 11 in a Ewing sarcoma-derived cell line.
Somatic cell hybrids, obtained after fusion of translocation (11;22)-positive Ewing sarcoma cells and Chinese hamster fibroblasts, were assayed for the presence of immunoglobulin C lambda, Philadelphia chromosome breakpoint cluster region, and c-sis oncogene sequences. It was found that c-sis was translocated from chromosome 22 to chromosome 11 in the Ewing sarcoma cells used, indicating that the breakpoint must be proximal to this locus. Moreover, we found that the chromosome 22-linked C lambda and breakpoint cluster region sequences are not translocated. This result confirms an earlier cytogenetic observation that the Ewing sarcoma-associated breakpoint in chromosome 22 is distal to those observed in translocation (8;22)-positive Burkitt lymphoma and in Philadelphia chromosome-positive chronic myeloid leukemia.
Aconitate Hydratase↗
Cloning a cDNA for the lysosomal alpha-glucosidase.
Messenger RNA was isolated from monkey testes and size-fractionated on sucrose gradients. In vitro translation of these mRNA fractions resulted in nascent, labeled alpha-glucosidase that could be precipitated with anti human alpha-glucosidase antiserum. A cDNA library was constructed from the most enriched fraction. The library was screened with cDNA made from mRNA obtained from immunoselected polysomes. Five cross-hybridizing clones were isolated and identified by their selection of alpha-glucosidase mRNA, as shown by hybrid released translation and further by their ability to hybridize with DNA from human chromosome 17, on which the gene coding for acid alpha-glucosidase is located.
Animals↗
The human c-abl oncogene in the Philadelphia translocation.
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Animals↗
Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22.
We have identified and molecularly cloned 46 kb of human DNA from chromosome 22 using a probe specific for the Philadelphia (Ph') translocation breakpoint domain of one chronic myelocytic leukemia (CML) patient. The DNAs of 19 CML patients were examined for rearrangements on chromosome 22 with probes isolated from this cloned region. In 17 patients, chromosomal breakpoints were found within a limited region of up to 5.8 kb, for which we propose the term "breakpoint cluster region" (bcr). The two patients having no rearrangements within bcr lacked the Ph' chromosome. The highly specific presence of a chromosomal breakpoint within bcr in Ph'-positive CML patients strongly suggests the involvement of bcr in this type of leukemia.
Bacterial Proteins↗
Regional mapping of the human immunoglobulin lambda light chain to the Philadelphia chromosome in chronic myeloid leukaemia.
The lambda light chain immunoglobulin constant region (C lambda) locus was mapped on human chromosome 22. A DNA probe containing part of the C lambda locus was isolated from a human chromosome 22 genomic library, and a series of rodent X human somatic cell hybrids (each of which contained different translocated parts of chromosome 22) were constructed and characterized. The hybridization of the C lambda probe to DNA from these cell hybrids was then studied by Southern blot analysis. The results demonstrates that the C lambda locus is situated very close to the translocation breakpoint on human chromosome 22 which is characteristic of chronic myeloid leukaemia, and at least part if not at all of the locus is situated on the Philadelphia chromosome.
Chromosome Mapping↗
Richter's syndrome with different immunoglobulin light chains and different heavy chain gene rearrangements.
In a patient with Richter's syndrome, the chronic lymphocytic leukemia (CLL) expressed lambda, mu, and delta immunoglobulin (lg) chains and the non-Hodgkin lymphoma (NHL) kappa, mu, and delta lg chains. The difference in lg light chain expression suggests that the CLL and NHL are independent malignancies, or that the oncogenic event occurred in a B cell differentiation stage after the heavy chain gene rearrangements but before the selection of the light chain. Analysis of DNA by Southern blotting revealed that the lg heavy chain genes of the two malignancies were rearranged in a different way. We therefore conclude that in this patient the NHL cannot be regarded as a progression of the CLL but should most likely be considered as an independent B cell malignancy, which arose in a susceptible host.
Aged↗
Localization of the human c-sis oncogene in Ph1-positive and Ph1-negative chronic myelocytic leukemia by in situ hybridization.
Oncogenes are a group of evolutionary conserved cellular genes (c-onc) homologous to the transforming genes of oncogenic retroviruses (v-onc). Some of them are localized near the breakpoints of specific chromosomal aberrations occurring in various neoplasms, as for example the Philadelphia translocation, t(9;22)(q34;q11), in chronic myelocytic leukemia (CML). Recently, we localized the human c-abl oncogene to chromosome region 9q34 and demonstrated a translocation of this gene to the Philadelphia chromosome (Ph1,22q-) in various forms of Ph1-positive, but not Ph1-negative, chronic myelocytic leukemia (CML). Another human oncogene, c-sis, is located on chromosome 22 and was recently reported to be transferred to chromosome 9q+ in one CML patient. We have now studied 2 CML patients with classic and variant types of Ph1 translocation, one Ph1-negative case, and a healthy control using in situ hybridization of a c-sis probe to metaphase chromosomes. These studies show that c-sis: (1) is localized to region 22q12.3-q13.1, far away from the breakpoint region 22q11 in CML, (2) segregates with the translocated part of chromosome 22 to different chromosomes in Ph1-positive patients, and (3) remains on chromosome 22 in the Ph1-negative case. Therefore, these data give no support for an active role of the c-sis gene in the generation of CML. Thus, if either of these two oncogenes is involved in the development of Ph1-positive CML, c-abl appears to be the more important one.
Chromosomes, Human, 21-22 and Y↗
c-sis is translocated from chromosome 22 to chromosome 9 in chronic myelocytic leukemia.
By analysis of a series of somatic cell hybrids derived by fusion of either mouse or Chinese hamster cells with leukocytes from different chronic myelocytic leukemia (CML) patients or from normal donors, we have localized the human oncogene, c-sis, on the q11 to qter segment of chromosome 22 and demonstrated its translocation from chromosome 22 to chromosome 9 (q34) in CML.
Animals↗
A cellular oncogene is translocated to the Philadelphia chromosome in chronic myelocytic leukaemia.
The transforming genes of oncogenic retroviruses are homologous to a group of evolutionary conserved cellular onc genes. The human cellular homologue (c-abl) of the transforming sequence of Abelson murine leukaemia virus (A-MuL V) was recently shown to be located on chromosome 9. The long arm of this chromosome is involved in a specific translocation with chromosome 22, the Philadelphia translocation (Ph1), t(9; 22) (q34, q11), which occurs in patients with chronic myelocytic leukaemia (CML)3-5. Here we investigate whether the c-abl gene is included in this translocation. Using c-abl and v-abl hybridization probes on blots of somatic cell hybrids, positive hybridization is found when the 22q- (the Philadelphia chromosome), and not the 9q+ derivative of the translocation, is present in the cell hybrids. From this we conclude that in CML, c-abl sequences are translocated from chromosome 9 to chromosome 22q-. This finding is a direct demonstration of a reciprocal exchange between the two chromosomes and suggests a role for the c-abl gene in the generation of CML.
Chromosomes, Human, 21-22 and Y↗
The structure and expression of the haemoglobin genes.
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Animals↗
Comparison of cloned rabbit and mouse beta-globin genes showing strong evolutionary divergence of two homologous pairs of introns.
Cloned beta-globin genes of both mouse and rabbit each contain a large and a small intervening sequence (intron) of about equal length at precisely the same positions relative to the coding sequence. The homologous introns show some sequence similarity, particularly at the junctions with the coding sequence. They most probably arose from a common ancestral sequence and diverged substantially during evolution.
Animals↗
The binding of activated C3 to polysaccharides and immunoglobulins.
The nature of the acceptors for activated C3 present on immunoglobulins was studied by using the fixation of C3 to Sepharose beads after activation with trypsin as a model system. C3 fixation to Sepharose is a property exclusively linked to the short active state of C3. This binding could be inhibited by various carbohydrates and their affinity for C3 was calculated from the extent of the inhibition of the binding of C3 to Sepharose. Mono-, di-tri- and tetrasaccharides showed on a molar basis an increasing but still weak affinity for active C3. The C3 fixation of Sepharose could be inhibited by immunoglobulins and the degree of inhibition was proportional to the hexose content of the immunoglobulin preparations. The isolated polysaccharide moiety of IgG gave the same inhibition as the intact IgG. In addition cell wall polysaccharides of Salmonella abortus equi were found to have a high affinity for active C3. Thus, the more complex polysaccharides such as those present on immunoglobulins and cell walls might function as acceptor for activated C3.
Acetylglucosamine↗
Localization of the c-ab1 oncogene adjacent to a translocation break point in chronic myelocytic leukaemia.
The human c-ab1 oncogene maps within the region (q34-qter) of chromosome 9 which is translocated to chromosome 22, the Philadelphia (Ph') chromosome, in chronic myelocytic leukaemia (CML). The position of the Ph' chromosomal break point is shown to be variable and, in one CML patient, has been localized immediately 5' of, or within, the c-ab1 oncogene. A DNA restriction fragment corresponding to this site has been molecularly cloned and shown to represent a chimaeric fragment of DNA from chromosomes 9 and 22.
Abelson murine leukemia virus↗
Translocation of c-ab1 oncogene correlates with the presence of a Philadelphia chromosome in chronic myelocytic leukaemia.
The localization of cellular oncogenes near the break points of tumour-specific chromosomal aberrations suggests an involvement of these genes in the generation of neoplasms. Recently, we demonstrated the translocation of the human cellular homologue (c-ab1) of the transforming sequence of Abelson murine leukaemia virus (A-MuLV) from chromosome 9 to the Philadelphia chromosome (Ph1) in chronic myelocytic leukaemia (CML). In an attempt to investigate the significance of this translocation in the pathogenesis of CML, we have now studied two CML patients with complex translocations, t(9; 11; 22) and t(1; 9; 22), and two CML Ph1-negative patients with apparently normal karyotypes. In addition to using blot hybridization with human c-ab1 probes and DNA from rodent: CML cell hybrids as before, we have used in situ hybridization of these probes directly to metaphase chromosomes of CML patients. These studies show that the c-ab1 gene is translocated in Ph1-positive but not in Ph1-negative CML patients. CML without the Ph1 chromosome seems to be a distinct entity with a different origin, and this view is supported by clinical observations including correlations which reveal a poorer prognosis.
Abelson murine leukemia virus↗
Structural organization of the bcr gene and its role in the Ph' translocation.
The Philadelphia (Ph') chromosome, an abnormal chromosome 22 (ref. 1), is one of the best-known examples of a specific human chromosomal abnormality strongly associated with one form of human leukaemia, chronic myelocytic leukaemia (CML). The finding that a small region of chromosome 9 which includes the c-abl oncogene is translocated to chromosome 22 prompted studies to elucidate the molecular mechanisms involved in this disease. We have demonstrated previously that the chromosome 9 of one patient with CML contains a breakpoint 14 kilobases (kb) 5' of the most 5' v-abl-homologous exon. These data suggest a role for c-abl in CML, a theory supported by the presence of an abnormally sized abl messenger RNA and protein in the CML cell line K562. The region involved in the translocation on chromosome 22 has also been identified: all Ph'-positive patients examined to date have a breakpoint within a 5.8-kb region, for which we have proposed the name 'breakpoint cluster region' (bcr). To determine whether bcr contains protein-encoding regions, probes from bcr were tested for their ability to hybridize to complementary DNA sequences. A 0.6-kb HindIII/BamHI bcr restriction enzyme fragment proved suitable for isolating several cDNA clones from a human fibroblast cDNA library. Using bcr cDNA sequences, we obtained data strongly suggesting the presence of a chimaeric bcr/abl mRNA in the leukaemic cells of Ph'-positive CML patients. The recent isolation of cDNA clones containing bcr and abl sequences confirms this finding. Because the bcr part of the chimaeric mRNA could be required to induce the transforming activity of the human c-abl oncogene, we have now initiated studies to characterize the normal 'bcr gene' and to determine the effect of a translocation within its coding domain. We demonstrate that as a result of the Ph' translocation, a variable number of bcr exons are included in the chimaeric bcr/abl mRNA. The bcr gene sequences in this mRNA could be responsible for the transition of the abl cellular proto-oncogene into an oncogene.
Base Sequence↗
[Philadelphia translocation and the human c-abl oncogene--relations in the light of molecular genetics].
Application of recombinant DNA techniques led to the characterization of a heterogenous group of evolutionary conserved genes with potential transforming activity, called oncogenes. Regularly they seem to be involved in normal cell proliferation and differentiation. Various mechanisms including an increased dosage of gene product as well as subtile point mutations activate these sequences to oncogenes sensu strictu. Molecular analysis of the Philadelphia translocation in leukemic cells of CML-patients revealed a consistent translocation of the human c-abl-oncogene from chromosome 9 to the Ph1-chromosome, regardless of the cytogenetic subtype. Moreover we could demonstrate individual breakpoints for every patient investigated so far. However, these breakpoints are clustered on chromosome 22 within sequences of the bcr-gene. In leukemic cells containing the rearranged-c-abl/bcr sequences a new transcript is detected which is possibly the mRNA for an altered c-abl-protein that unmasks associated tyrosine specific kinase activity. These gene rearrangements were not detected in Ph1-negative CML-patients. Another human oncogene, c-sis, is located on chromosome 22, but seems not to play a crucial role in the generation of CML. These results are discussed in the context of recent advances in oncogene-research.
Child↗