Approaches to the identification and molecular cloning of chromosome breakpoints.
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Animals↗
Biomedical subjects
C M Croce
Publications and source records attributed to C M Croce.
Translocation 11;14 in newly diagnosed chronic myelogenous leukemia.
In patients with chronic myelogenous leukemia (CML), the Philadelphia chromosome may be associated with a number of other cytogenetic lesions. However, t(11;14)(q13;q32), found mainly in B-cell lymphoproliferative disorders, has not been previously reported in Ph-positive CML. We describe a patient with hematologically typical chronic phase CML in whom both cytogenetic lesions were found at diagnosis.
Adult↗
A novel t(9;11)(p22;q23) with ALL-1 gene rearrangement associated with progression of a myeloproliferative disorder to acute myeloid leukemia.
We have analyzed genomic DNAs from a patient who developed acute myeloid leukemia 1 year after a myeloproliferative disorder was diagnosed. The development of the acute leukemia was associated with the acquisition of a t(9;11)(p22;q23) chromosome translocation. ALL-1 gene rearrangement, on chromosome 11, was present at the onset of the acute phase, but not during the chronic phase of the myeloproliferative disorder. The genomic rearrangement on chromosome 9 was within an unidentified region. By the use of polymerase chain reaction, we were able to determine that the chromosomal rearrangement was completely absent during the chronic phase of the myeloproliferative disorder, indicating that the ALL-1 gene rearrangement was causally related to the development of the acute phase. The rapid progression into the acute phase suggests that this case might be therapy related. This work provides a clear example of association of a molecular defect with the development of a specific clinical leukemic stage, and supports the indication that ALL-1 gene rearrangement is associated with poor clinical outcome in adult leukemias.
Adult↗
Molecular genetics of 11q23 chromosome translocations.
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Chromosomes, Human, Pair 11↗
Identification of the TCL1 gene involved in T-cell malignancies.
The TCL1 locus on chromosome 14q32.1 is frequently involved in chromosomal translocations and inversions with one of the T-cell receptor loci in human T-cell leukemias and lymphomas. The chromosome 14 region translocated or rearranged involves approximately 350 kb of DNA at chromosome band 14q32.1. Within this region we have identified a gene coding for a 1.3-kb transcript, expressed only in restricted subsets of cells within the lymphoid lineage and expressed at high levels in leukemic cells carrying a t(14;14)(q11;q32) chromosome translocation or a inv(14)(q11;q32) chromosome inversion. The cognate cDNA sequence reveals an open reading frame of 342 nt encoding a protein of 14 kDa. The TCL1 gene sequence, which, to our knowledge, shows no sequence homology with other human genes, is preferentially expressed early in T- and B-lymphocyte differentiation.
Amino Acid Sequence↗
Molecular characterization of prostate-specific antigen messenger RNA expressed in breast tumors.
Prostate-specific antigen (PSA) is considered a highly specific biochemical marker of the prostate gland and is currently used for prostate cancer diagnosis and monitoring of patients with prostate adenocarcinoma. We recently demonstrated, however, that about 30% of female breast tumors produce a M(r) 33,000 protein that has striking similarities to seminal PSA. In this study we characterized the presence of PSA in 6 breast tumors and in the testosterone-stimulated T47D breast cancer cell line at the mRNA level. Using reverse transcriptase-polymerase chain reaction and DNA sequencing techniques we identified PSA mRNA in immunoreactive PSA-positive breast tumors but not in immunoreactive PSA-negative breast tumors. The sequence of the generated polymerase chain reaction products was identical to the sequence of the PSA complementary DNA derived from prostate tissue. The data presented here support the notion that breast tumors produce a M(r) 33,000 protein which is identical to PSA produced by the prostate gland. Our study suggests that the presence of PSA in breast tumors may be used as a new additional biochemical marker for breast cancer prognosis, for the spreading of hematogenous micrometastases, and/or for response to adjuvant treatment.
Base Sequence↗
Characterization and localization of the TCL-1 oncogene product.
The TCL-1 gene maps at chromosome 14q32.1 and is activated in T cell leukemias and lymphomas by either chromosome translocations or inversions that juxtapose the TCL-1 gene to the alpha/delta or the beta locus of the T cell receptor. The open reading frame of the TCL-1 gene, coding for a protein of 114 amino acids, was expressed in bacteria and antisera were raised against it. The antibodies recognized the predicted TCL-1 M(r) 14,000 protein product in cells expressing TCL-1 mRNA. Cell fractionation experiments indicated that the TCL-1 protein is present in the microsomal fraction. These results were confirmed by confocal microscopy. The TCL-1 protein has considerable sequence similarities to the product of the MTCP-1 gene on chromosome Xq28, which is involved in T cell lympho-proliferative diseases. Thus, TCL-1 may represent a member of a novel family of genes involved in lymphoid proliferation and/or survival and in T cell malignancies.
Amino Acid Sequence↗
Loss of heterozygosity at 11q22-q23 in breast cancer.
Studies of loss of heterozygosity (LOH) in breast tumor DNA suggest that several tumor suppressor genes participate in the pathogenesis of breast cancer. Although the short arm of chromosome 11 has been implicated in breast cancer development, no previous LOH studies have indicated the involvement of a suppressor gene on 11q in breast carcinoma. To this end, tumor samples and corresponding normal tissue were collected from 62 unselected patients with primary breast cancer, and the extracted DNA was analyzed by polymerase chain reaction using microsatellite markers on chromosome 11. We found that 39% of the tumors (22 of 57 informative cases) revealed allelic loss in the region 11q22-23, and this loss was independent of LOH found to occur on 11p15. Interestingly, more than 90% of the tumors showed concordant loss of alleles at both 11q and 17p. The marker D11S528, showing LOH in 39% of informative cases, had the highest frequency of LOH among the markers that were used. The data presented indicate that the common overlapping region of LOH is between the loci D11S35 and D11S29, suggesting that this area contains a tumor suppressor gene frequently lost in breast cancer.
Ataxia Telangiectasia↗
Rarity of somatic and germline mutations of the cyclin-dependent kinase 4 inhibitor gene, CDK4I, in melanoma.
Evidence from cytogenetics, multipoint linkage analyses of familial melanoma, and loss of heterozygosity studies of familial and sporadic melanomas support localization of a melanoma susceptibility or tumor suppressor gene at chromosomal region 9p21-23. Recently, the inhibitor of cyclin-dependent kinase 4 (CDK4I; also known as p16INK4, multiple tumor suppressor 1, or CDKN2 gene) has been mapped to 9p21 and shown to be mutated or deleted in a large fraction of cell lines derived from many tumor types, including melanoma, suggesting that this gene could be a melanoma suppressor gene. In order to test for somatic mutations in the CDK4I gene in tumors, DNAs from 30 surgically resected melanomas of both cutaneous and uveal origins were sequenced. No mutations were detected in the coding region of the CDK4I gene, while mutations or deletions were detected in 60% (9 of 15) of the cultured melanoma cell line DNAs. Among presumptive familial cases, nine of which were members of families with one or two other documented melanoma cases, no germline mutations were detected by sequence analysis. A deletion in the second exon of the CDK4I gene was found in one germline allele of a familial melanoma patient from a family with eight affected first degree relatives. These results not only support the suggestion that the CDK4I gene is a familial malignant melanoma gene, they also suggest the presence of another suppressor gene locus within 9p21 which is the target of loss of heterozygosity in sporadic melanomas.
Base Sequence↗
Leucine-zipper dimerization motif encoded by the AF17 gene fused to ALL-1 (MLL) in acute leukemia.
Chromosome region 11q23 is involved in reciprocal chromosome translocations associated with human acute leukemias. These aberrations fuse the ALL-1 gene located at 11q23 to a series of partner genes positioned on a variety of human chromosomes. The fused genes encode chimeric proteins. Here we report the cloning and characterization of the ALL-1 partner at 17q21, the AF17 gene. The AF17 gene encodes a protein of 1093 amino acids, containing a leucine-zipper dimerization motif located 3' of the fusion point and a cysteine-rich domain at the N terminus. The latter can be arranged in three zinc fingers and shows homology to a domain within the protein Br140 (peregrin). AF17 contains stretches of amino acids previously associated with domains involved in transcriptional repression or activation. Based on features of AF17 and of the proteins encoded by the other partner genes analyzed and in conjunction with other recent studies, we propose a model in which ALL-1 rearrangements result in loss of function of the gene. In this model, the partner polypeptide plays an accessory role either by repressing activity of the truncated ALL-1 protein or by blocking the function of the normal protein presumably present in the leukemic cells.
Amino Acid Sequence↗
ALL-1 tandem duplication in acute myeloid leukemia with a normal karyotype involves homologous recombination between Alu elements.
Rearrangements of the ALL-1 gene by reciprocal translocations involving chromosome band 11q23 are frequently associated with human acute leukemia. We have previously reported the detection of ALL-1 gene rearrangements in adult patients with acute myeloid leukemia lacking cytogenetic evidence of 11q23 translocations. These included 2 of 19 patients with normal karyotypes as well as 3 of 4 patients with trisomy 11 as a sole cytogenetic abnormality. Rearrangement of the ALL-1 genes in two of the patients with trisomy 11 was shown to result from a direct tandem duplication of a portion of the gene spanning exons 2-6. Here we report the characterization of the ALL-1 gene rearrangement in one of the previously reported acute myeloid leukemia patients with a normal karyotype. ALL-1 rearrangement in this patient results from a direct tandem duplication of a portion of the gene spanning exons 2-8. RNA polymerase chain reaction and DNA sequence analysis show that the partially duplicated ALL-1 gene is transcribed into mRNA capable of encoding a partially duplicated protein. Sequence analysis of the genomic fusion region provides evidence for Alu-mediated homologous recombination as a mechanism for partial duplication of the ALL-1 gene.
Acute Disease↗
The c-kit ligand suppresses apoptosis of human natural killer cells through the upregulation of bcl-2.
The bcl-2 protein plays a central role in the regulation of programmed cell death in a variety of tissues and is pivotal to the survival of lymphocytes in vivo. The growth factors responsible for survival of normal lymphocytes are unknown but are likely to maintain viability in part through the regulation of bcl-2 expression. A subset of human natural killer (NK) cells (CD3-CD56bright) are unique among lymphocytes in their constitutive expression of c-kit, a tyrosine kinase cell surface receptor that binds c-kit ligand (KL). Alone, KL does not promote proliferation or further differentiation of CD56bright NK cells. We now report that, in the absence of serum or additional growth factors, KL prevents apoptosis of cultured CD56bright NK cells, as assessed by DNA fragmentation studies, and maintains viability, as measured by biologic responses (i.e., proliferation and cytotoxicity) to the subsequent addition of other cytokines. Furthermore, we demonstrate that KL induces CD56bright NK cells to express the bcl-2 protein. In the presence of anti-c-kit antibody, the tyrosine kinase inhibitor genistein, or bcl-2 antisense oligonucleotide, the protective effect of KL on the survival of CD56bright NK cells is dramatically reduced. These data demonstrate that the binding of KL to its tyrosine kinase receptor results in the upregulation of bcl-2, thereby preventing apoptosis in this subset of normal human lymphocytes. As soluble KL is plentiful in normal human serum, this survival mechanism may be operative for CD56bright NK cells in vivo.
Antigens, CD↗
ALL-1 partial duplication in acute leukemia.
The ALL-1 gene, located on chromosome band 11q23, is fused to a variety of other genes by reciprocal chromosomal translocations present in 5-10% of human acute leukemias. We have recently reported the detection by Southern blot of ALL-1 gene rearrangements in adult patients with acute myeloid leukemia lacking cytogenetic evidence of 11q23 translocations. These include 2 of 19 patients with normal karyotypes as well as 3 of 4 patients with trisomy 11. To characterize the abnormal ALL-1 genes, we cloned the ALL-1 rearrangements from two patients with trisomy 11. Characterization of the clones, together with Southern blot analysis, indicates that the ALL-1 rearrangement in both patients is the result of a direct tandem duplication of a portion of the ALL-1 gene spanning exons 2-6. The partial ALL-1 duplication is also detected by Southern blot analysis in a patient with a normal karyotype. RNA PCR and DNA sequence analysis show that the partially duplicated ALL-1 gene is transcribed into mRNA capable of encoding a partially duplicated protein. Partial duplication of ALL-1, in which a portion of a putative protooncogene is fused with itself, represents an additional genetic mechanism for leukemogenesis. Our findings suggest that the presence of trisomy in malignancy may sometimes indicate the partial duplication of a cellular protooncogene.
Acute Disease↗
Nucleophosmin (NPM) gene rearrangements in Ki-1-positive lymphomas.
The (2;5)(p23;q35) translocation which results in the fusion of the NPM (nucleophosmin) gene on chromosome 5q35 with the novel ALK (anaplastic lymphoma kinase) gene on chromosome 2p23 [S.W. Morris et al., Science (Washington DC), 263: 1281-1284, 1994] is associated with Ki-1 (CD30)-positive anaplastic large cell lymphomas (ALCL); a group of morphologically and immunophenotypically heterogenous high grade large cell lymphomas (LCL), which share many characteristics with Hodgkin's disease (HD), including the presence of variable numbers of Reed-Sternberg-like cells and the expression of CD30 antigen. Using a DNA probe immediately 5' to the NPM coding sequences, we have examined NPM gene rearrangements by Southern blotting in 5 Ki-1-positive lymphoma cell lines carrying a translocation involving the 5q35 breakpoint and in 25 Ki-positive lymphoma tumors, including 9 HD. Using this method, we detected rearrangements in all cell lines with apparent clustering of the breakpoints. Analysis of 25 Ki-1-positive lymphomas indicated that only 4 neoplasms, including two HD, had NPM gene rearrangements. Thus, our findings suggest that only a subset of ALCL has detectable involvement of the NPM gene. In addition, the presence of NPM gene rearrangements in HD indicates the involvement of this gene in a fraction of HD. Thus, NPM gene rearrangements may identify a certain subtype in ALCL and HD which may be closely related.
Chromosomes, Human, Pair 2↗
Sequence analysis of the breakpoint cluster region in the ALL-1 gene involved in acute leukemia.
DNA rearrangements caused by chromosome translocations between band 11q23 and various chromosomes can be detected by a single probe, B859, an 859-base pair complementary DNA fragment derived from the human ALL-1 gene. To try to understand why band 11q23 becomes a frequent target of the translocations, we have sequenced the entire breakpoint cluster region, a 8342-base pair BamHI genomic fragment delineated by B859. We found eight Alu repeats located within this region in the same orientation as the ALL-1 gene. We have also analyzed the sequences of the breakpoints in 10 patients with 6 different types of 11q23 aberration. In five patients the breaks coincided with Alu sequences on chromosome 11, but not on the partner chromosomes. Also, seven of the breaks occurred in the region delineated by exons 6 and 7, which is composed mainly of Alu sequences. In three patients topoisomerase II recognition site-like sequences, at different stringency levels, were identified at the breakpoints on chromosome 11. We conclude that while there is no specific sequence element present at all the breakpoints, the high density of Alu sequences in the breakpoint cluster region possibly makes the latter more prone to recombination events.
Acute Disease↗
The human homologue of the retroviral oncogene qin maps to chromosome 14q13.
Chromosomal mapping of the human QIN gene (renamed FKH2 by the Human Genome Organization Nomenclature Committee) was initially accomplished by correlation of the presence of the QIN locus with specific chromosome regions in a rodent-human hybrid panel. This analysis revealed that the human QIN gene maps to chromosome region 14q11.2-->14q32, between the TCR and IGH loci. Further analysis by fluorescence in situ hybridization techniques with a human QIN genomic clone refined the human QIN gene localization to 14q13.
Chromosome Mapping↗
Down-regulation of bcl-2 by p53 in breast cancer cells.
bcl-2 and p53 gene products have been both linked to programmed cell death pathways. We have analyzed several human breast cancer cell lines for the expression of bcl-2 and p53. We found an inverse correlation between the expression of the two proteins. The result suggested that mutant p53 could substitute for bcl-2 function in breast cancer cells and that could also down-regulate bcl-2 expression. We found, indeed, that overexpression of a mutant p53 (mut 175) in MCF-7 cells could induce down-regulation of bcl-2 both at protein and mRNA level. However, the promoter region of the human bcl-2 gene does not contain the negative regulatory element responsible for the down-regulation. If this mechanism will be proved also for the wild-type p53 allele, it may disclose a possible mechanism for p53-induced apoptosis: down-regulation of bcl-2.
Alleles↗
Tumor and growth suppression of breast cancer cells by chromosome 17-associated functions.
Losses of functions from chromosome 17 are the most frequent genetic abnormalities in human breast cancer. To assess the biological role of chromosome 17 in the development of breast cancer, we transferred a normal human chromosome 17 to two breast cancer cell lines. No viable clone maintaining an intact chromosome was obtained in either MDA-MB-231 or MCF-7. Only one MDA-231/H17 clone contained the long arm of the transferred chromosome 17. Interestingly, this clone lost the ability to induce tumors in nude mice, indicating that at least one gene mapping to the long arm of chromosome 17 could suppress the tumorigenic phenotype. The p53 protein most likely was responsible for the selective loss of the short arm of the chromosome. Both cell lines have no wild-type p53 activity. MDA-MB-231 carries a single mutant TP53 allele, while MCF-7 carries two wild-type alleles, but p53 protein is excluded from the nucleus. Transfection in both cell lines of vectors expressing wild-type p53 produced only clones with rearrangements of the transfected TP53 complementary DNA. Thus, nonregulated expression of the p53 protein driven by the strong cytomegalovirus promoter may have triggered a rapid process of cell death. Stable expression of a mutant p53 in MCF-7 cells proved that nuclear localization of the protein was possible; however, no progression toward an estrogen-independent tumorigenic phenotype was induced. This work indicates that functional inactivation of the wild-type p53 protein and of the product of a gene located on 17q are essential to the development of breast neoplasms.
Amino Acid Sequence↗