Search PubMed⌕ Search

Biomedical subjects

J D Rowley

Publications and source records attributed to J D Rowley.

At least 73 records · Page 4Linked to original sources
PubMed1996

Intergenic splicing of MDS1 and EVI1 occurs in normal tissues as well as in myeloid leukemia and produces a new member of the PR domain family.

The EVI1 gene, located at chromosome band 3q26, is overexpressed in some myeloid leukemia patients with breakpoints either 5' of the gene in the t(3;3)(q21;q26) or 3' of the gene in the inv(3)(q21q26). EVI1 is also expressed as part of a fusion transcript with the transcription factor AML1 in the t(3;21)(q26;q22), associated with myeloid leukemia. In cells with t(3;21), additional fusion transcripts are AML1-MDS1 and AML1-MDS1-EVI1. MDS1 is located at 3q26 170-400 kb upstream (telomeric) of EVI1 in the chromosomal region in which some of the breakpoints 5' of EVI1 have been mapped. MDS1 has been identified as a single gene as well as a previously unreported exon(s) of EVI1 We have analyzed the relationship between MDS1 and EVI1 to determine whether they are two separate genes. In this report, we present evidence indicating that MDS1 exists in normal tissues both as a unique transcript and as a normal fusion transcript with EVI1, with an additional 188 codons at the 5' end of the previously reported EVI1 open reading frame. This additional region has about 40% homology at the amino acid level with the PR domain of the retinoblastoma-interacting zinc-finger protein RIZ. These results are important in view of the fact that EVI1 and MDS1 are involved in leukemia associated with chromosomal translocation breakpoints in the region between these genes.

Amino Acid Sequence↗
PubMed1996

The chimeric genes AML1/MDS1 and AML1/EAP inhibit AML1B activation at the CSF1R promoter, but only AML1/MDS1 has tumor-promoter properties.

The (3;21)(q26;q22) translocation associated with treatment-related myelodysplastic syndrome, treatment-related acute myeloid leukemia, and blast crisis of chronic myeloid leukemia results in the expression of the chimeric genes AML1/EAP, AML1/MDS1, and AML1/EVI1. AML1 (CBFA2), which codes for the alpha subunit of the heterodimeric transcription factor CBF, is also involved in the t(8;21), and the gene coding for the beta subunit (CBFB) is involved in the inv(16). These are two of the most common recurring chromosomal rearrangements in acute myeloid leukemia. CBF corresponds to the murine Pebp2 factor, and CBF binding sites are found in a number of eukaryotic and viral enhancers and promoters. We studied the effects of AML1/EAP and AML1/MDS1 at the AML1 binding site of the CSF1R (macrophage-colony-stimulating factor receptor gene) promoter by using reporter gene assays, and we analyzed the consequences of the expression of both chimeric proteins in an embryonic rat fibroblast cell line (Rat1A) in culture and after injection into athymic nude mice. Unlike AML1, which is an activator of the CSF1R promoter, the chimeric proteins did not transactivate the CSF1R promoter site but acted as inhibitors of AML1 (CBFA2). AML1/EAP and AML1/MDS1 expressed in adherent Rat1A cells decreased contact inhibition of growth, and expression of AML1/MDS1 was associated with acquisition of the ability to grow in suspension culture. Expression of AML1/MDS1 increased the tumorigenicity of Rat1A cells injected into athymic nude mice, whereas AML1/EAP expression prevented tumor growth. These results suggest that expression of AML1/EAP and AML1/MDS1 can interfere with normal AML1 function, and that AML1/MDS1 has tumor-promoting properties in an embryonic rat fibroblast cell line.

Acute Disease↗
PubMed1996

Analysis of the t(6;11)(q27;q23) in leukemia shows a consistent breakpoint in AF6 in three patients and in the ML-2 cell line.

The t(6;11)(q27;23) is one of the most common translocations observed in patients with acute myeloid leukemia (AML). The translocation breakpoint involves the MLL gene, which is the human homolog of the Drosophila trithorax gene, at 11q23 and the AF6 gene at 6q27. Reverse transcriptase-polymerase chain reaction (RT-PCR) using an MLL sense primer and an AF6 antisense primer detected the MLL/AF6 fusion cDNA from three leukemia patients with the t(6;11) [two AML and one T-acute lymphoblastic leukemia (ALL)] and one cell line. The fusion point in the AF6 cDNA from these cases is identical, regardless of the leukemia phenotype. The ML-2 cell line, which was established from a patient with AML that developed after complete remission of T-cell lymphoma, has retained an 11q23-24 deletion from the lymphoma stage and has acquired the t(6;11) with development of AML. The ML-2 cells have no normal MLL gene on Southern blot analysis, which indicates that an intact MLL gene is not necessary for survival of leukemic cells.

Adolescent↗
PubMed1996

Correlation between the ETV6/CBFA2 (TEL/AML1) fusion gene and karyotypic abnormalities in children with B-cell precursor acute lymphoblastic leukemia.

The recently identified ETV6/CBFA2 (formerly known as TEL/AML1) fusion gene occurs as a result of the t(12;21)(p12;q22). Initial reports have indicated that the fusion transcript occurs in up to 30% of children diagnosed with B-cell precursor (CD10+, CD19+) acute lymphoblastic leukemia (ALL). In order to characterize the incidence of the t(12;21) at both the chromosomal level as well as the RNA transcript level, we have used a combination of classical cytogenetics, reverse transcriptase-polymerase chain reaction (RT-PCR), and fluorescence in situ hybridization (FISH) to examine the bone marrow of 34 children diagnosed with B-cell precursor ALL. Nine of the 34 patient samples expressed the ETV6/CBFA2 transcript. When the results of RT-PCR were compared with the conventional karyotype, the fusion was present in 3 of 10 (33%) with chromosome 12 abnormalities, none of whom had an obvious t(12;21). The transcript was also detected in 5 of the 12 (41%) bone marrow samples with other abnormalities and in 1 of 12 (8%) samples with a normal karyotype. Seven of the 9 RT-PCR positive patient samples were studied with FISH. Of the 7, FISH confirmed the ETV6/CBFA2 fusion in 6. One other patient with a 12p abnormality had evidence for the fusion using FISH which was not detected by RT-PCR. Our results not only confirm that the frequency of the t(12;21) is unusually high in childhood B-cell precursor ALL, but also that none of the translocations in our series was detected with conventional cytogenetic techniques.

Burkitt Lymphoma↗
PubMed1996

Rearrangement of the AML1/CBFA2 gene in myeloid leukemia with the 3;21 translocation: expression of co-existing multiple chimeric genes with similar functions as transcriptional repressors, but with opposite tumorigenic properties.

Several recurring chromosomal translocations involve the AML1 gene at 21q22 in myeloid leukemias resulting in fusion mRNAs and chimeric proteins between AML1 and a gene on the partner chromosome. AML1 corresponds to CBFA2, one of the DNA-binding subunits of the enhancer core binding factor CBF. Other CBF DNA-binding subunits are CBFA1 and CBFA3, also known as AML3 and AML2. AML1, AML2 and AML3 are each characterized by a conserved domain at the amino end, the runt domain, that is necessary for DNA-binding and protein dimerization, and by a transactivation domain at the carboxyl end. AML1 was first identified as the gene located at the breakpoint junction of the 8;21 translocation associated with acute myeloid leukemia. The t(8;21)(q22;q22) interrupts AML1 after the runt homology domain, and fuses the 5' part of AML1 to almost all of ETO, the partner gene on chromosome 8. AML1 is an activator of several myeloid promoters; however, the chimeric AML1/ETO is a strong repressor of some AML1-dependent promoters. AML1 is also involved in the t(3;21)(q26;q22), that occurs in myeloid leukemias primarily following treatment with topoisomerase II inhibitors. We have studied five patients with a 3;21 translocation. In all cases, AML1 is interrupted after the runt domain, and is translocated to chromosome band 3q26. As a result of the t(3;21), AML1 is consistently fused to two separate genes located at 3q26. The two genes are EAP, which codes for the abundant ribosomal protein L22, and MDS1, which encodes a small polypeptide of unknown function. In one of our patients, a third gene EVI1 is also involved. EAP is the closest to the breakpoint junction with AML1, and EVI1 is the furthest away. The fusion of EAP to AML1 is not in frame, and leads to a protein that is terminated shortly after the fusion junction by introduction of a stop codon. The fusion of AML1 to MDS1 is in frame, and adds 127 codons to the interrupted AML1. Thus, in the five cases that we studied, the 3;21 translocation results in expression of two coexisting chimeric mRNAs which contain the identical runt domain at the 5' region, but differ in the 3' region. In addition, the chimeric transcript AML1/MDS1/EVI1 has also been detected in cells from one patient with the 3;21 translocation as well as in one of our patients. Several genes necessary for myeloid lineage differentiation contain the target sequence for AML1 in their regulatory regions. One of them is the CSF1R gene. We have compared the normal AML1 to AML1/MDS1, AML1/EAP and AML1/MDS1/EVI1 as transcriptional regulators of the CSF1R promoter. Our results indicate that AML1 can activate the promoter, and that the chimeric proteins compete with the normal AML1 and repress expression from the CSF1R promoter. AML1/MDS1 and AML1/EAP affect cell growth and phenotype when expressed in rat fibroblasts. However, the pattern of tumor growth of cells expressing the different chimeric genes in nude mice is different. We show that when either fusion gene is expressed, the cells lose contact inhibition and form foci over the monolayer. In addition, cells expressing AML1/MDS1 grow larger tumors in nude mice, whereas cells expressing only AML1/EAP do not form tumors, and cells expressing both chimeric genes induce tumors of intermediate size. Thus, although both chimeric genes have similar effects in transactivation assays of the CSF1R promoter, they affect cell growth differently in culture and have opposite effects as tumor promoters in vivo. Because of the results obtained with cells expressing one or both genes, we conclude that MDS1 seems to have tumorigenic properties, but that AML1/EAP seems to repress the oncogenic property of AML1/MDS1.

Animals↗
PubMed1996

The mixed lineage leukemia (MLL) protein involved in 11q23 translocations contains a domain that binds cruciform DNA and scaffold attachment region (SAR) DNA.

Translocations involving chromosome band 11q23, found in acute lymphoid and myeloid leukemias, disrupt the MLL gene. This gene encodes a putative transcription factor with regions of homology to several other proteins including the zinc fingers and other domains of the Drosophila trithorax gene product, and the "AT-hook" DNA-binding motif of high mobility group proteins. We have previously demonstrated that MLL contains transcriptional activation and repression domains using a GAL4 fusion protein system (21). The repression domain, which is capable of repressing transcription 3-5-fold, is located centromeric to the breakpoint region of MLL. The activation domain, located telomeric to the breakpoint region, activated transcription from a variety of promoters including ones containing only basal promoter elements. The level of activation was very high, ranging from 10-fold to more than 300-fold, depending on the promoter and cell line used for transient transfection. In translocations involving MLL, the protein produced from the der(11) chromosome which contains the critical junction for leukemogenesis includes the AT-hook domain and the repression domain. We assessed the DNA binding capability of the MLL AT-hook domain using bacterially expressed and purified AT-hook protein. In a gel mobility shift assay, the MLL AT-hook domain could bind cruciform DNA, recognizing structure rather than sequence of the target DNA. This binding could be specifically competed with Hoechst 33258 dye and with distamycin. In a nitrocellulose protein-DNA binding assay, the MLL AT-hook domain could bind to AT-rich SARs, but not to non-SAR DNA fragments. The role that the AT-hook binding to DNA may play in vivo is unclear, but it is likely that DNA binding could affect downstream gene regulation. The AT-hook domain retained on the der(11) would potentially recognize a different DNA target than the one normally recognized by the intact MLL protein. Furthermore, loss of an activation domain while retaining a repression domain on the der(11) chromosome could alter the expression of various downstream target genes, suggesting potential mechanisms of action for MLL in leukemia.

Amino Acid Sequence↗
PubMed1996

Scaffold attachment regions in centromere-associated DNA.

Due to indications that kinetochore proteins are an integral part of the protein scaffold component of the chromosome (Earnshaw et al. 1984), we chose to map the distribution of scaffold attachment regions (SARs) at centromeres. Using the SAR mapping assay of Mirkovitch et al., Southern blots were prepared and probed with 32P-labeled fragments from the human 1.9 kb centromeric alpha-satellite repeat unit of chromosome 1 or the 1.7 kb centromeric alpha-satellite repeat unit of chromosome 16. Our results demonstrated the presence of one SAR site per 1.9 kb repeat unit in chromosome 1, and every 1.7 kb repeat unit in chromosome 16, separated by regions of small DNA loops over the length of the alpha-satellite regions. We also identified several in vitro vertebrate topoisomerase II and cenP-B consensus sequences throughout the chromosome 1 alpha-satellite region using computer and base ratio analysis, to address the question as to why some alpha-satellite regions are SAR related and others are not. To provide in situ indications of SAR localization in the human genome, SAR DNA and non-SAR DNA were prepared following lithium 3,5-di-iodosalicylate extraction. Sequences protected from DNAse I digestion by SAR proteins, as compared with unprotected DNA that was digested by the enzyme, was labeled with biotin-UTP, hybridized to chromosomal DNA in situ, and then detected with fluorescein-avidin-DCS. Both SAR and non-SAR DNA selectively labeled virtually all centromeric regions of the human metaphase karyotype. Chromosomal arms were less strongly bound by SAR DNA, with a pattern that followed the chromosomal axis. In the more condensed chromosomes an R-banding pattern was evident. In general, labeling patterns produced by both SAR and non-SAR fractions were similar, as expected from the indications that SAR DNAs are heterogenous in sequence and do not form a specific class of sequences. We conclude that centromeric regions of several, possibly all, human metaphase chromosomes are also regions where the chromosomal axis contains loops, smaller in size than in the arms and where attachment sites are concentrated. This clustering of SARs may be responsible in part for the tight chromatin packing associated with the primary constriction of the centromeric region.

Blotting, Southern↗
PubMed1996

Oligomerization of the ABL tyrosine kinase by the Ets protein TEL in human leukemia.

TEL is a member of the Ets family of transcription factors which are frequently rearranged in human leukemia. The mechanism of TEL-mediated transformation, however, is unknown. We report the cloning and characterization of a chromosomal translocation associated with acute myeloid leukemia which fuses TEL to the ABL tyrosine kinase. The TEL-ABL fusion confers growth factor-independent growth to the marine hematopoietic cell line Ba/F3 and transforms Rat-1 fibroblasts and primary murine bone marrow cells. TEL-ABL is constitutively tyrosine phosphorylated and localizes to the cytoskeleton. A TEL-ABL mutant containing an ABL kinase-inactivating mutation is not constitutively phosphorylated and is nontransforming but retains cytoskeletal localization. However, constitutive phosphorylation, cytoskeletal localization, and transformation are all dependent upon a highly conserved region of TEL termed the helix-loop-helix (HLH) domain. TEL-ABL formed HLH-dependent homo-oligomers in vitro, a process critical for tyrosine kinase activation. These experiments suggest that oligomerization of TEL-ABL mediated by the TEL HLH domain is required for tyrosine kinase activation, cytoskeletal localization, and transformation. These data also suggest that oligomerization of Ets proteins through the highly conserved HLH domain may represent a previously unrecognized phenomenon.

Aged↗
PubMed1996

Myeloid leukemia after hematotoxins.

One of the most serious consequences of cancer therapy is the development of a second cancer, especially leukemia. Several distinct subsets of therapy-related leukemia can now be distinguished. Classic therapy-related myeloid leukemia typically occurs 5 to 7 years after exposure to alkylating agents and/or irradiation, has a myelodysplastic phase with trilineage involvement, and is characterized by abnormalities of the long arms of chromosomes 5 and/or 7. Response to treatment is poor, and allogenic bone marrow transplantation is recommended. Leukemia following treatment with agents that inhibit topoisomerase II, however, has a shorter latency, no preleukemic phase, a monoblastic, myelomonocytic, or myeloblastic phenotype, and balanced translocations, most commonly involving chromosome bands 11q23 or 21q22. The MLL gene at 11q23 or the AML1 gene at 21q22 are almost uniformly rearranged. MLL is involved with many fusion gene partners. Therapy-related acute lymphoblastic leukemia also occurs with 11q23 rearrangements. Therapy-related leukemias with 11q23 or 21q22 rearrangements, inv(16) or t(15;17), have a more favorable response to treatment and a clinical course similar to their de novo counterparts.

Antineoplastic Agents, Alkylating↗
PubMed1996

Lineage involvement by BCR/ABL in Ph+ lymphoblastic leukemias: chronic myelogenous leukemia presenting in lymphoid blast vs Ph+ acute lymphoblastic leukemia.

Chronic myelogenous leukemia (CML) can sometimes present in lymphoid blast phase (L-BP), and can be difficult to distinguish from Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). Some have suggested that the determination of cell lineages involved by the Ph chromosome may be used for distinguishing CML presenting in L-BP (presumably multilineage disease) from Ph+ ALL (presumably lymphoid-restricted), although others have suggested the term 'stem cell ALL' for the multilineage process. Because it has been difficult to perform lineage studies of the Ph chromosome, we investigated the use of fluorescence in situ hybridization (FISH) with probes for BCR (on chromosome 22) and ABL (on chromosome 9) to study lineage involvement in Ph+ lymphoblastic malignancies. We analyzed routine blood and marrow specimens from eight patients who presented with Ph+ lymphoblastic leukemia and found that FISH recognized the 9;22 translocation, distinguished between the two common molecular variants, and readily identified multilineage vs lymphoblast-restricted disease. In our series, four patients had multilineage and four had lymphoblast-restricted disease. Multilineage disease was associated with morphologic features of CML at diagnosis and/or reversion to chronic phase CML after treatment leading us to consider it as CML presenting in L-BP. Patients with lymphoid-restricted disease lacked such findings. The survival of three of our four patients with multilineage disease was prolonged, at 25, 28+, and 126+ months, and when data from our entire series are added to those of 18 previously reported cases that were studied for lineage involvement (reviewed in Leukemia 1993; 7: 147), the difference in overall survival between patients with multilineage and lymphoblast-restricted disease is significant (median overall survival of 47 months vs 8 months, respectively; P=0.013, log rank). Our findings illustrate that FISH analysis can be used to recognize lineage involvement in patients presenting with Ph+ lymphoblastic malignancies, and they provide further support to the notion that multilineage and lymphoblast-restricted disease are distinct clinically as well as biologically.

Adolescent↗
PubMed1995

TEL and KIP1 define the smallest region of deletions on 12p13 in hematopoietic malignancies.

Unbalanced translocations as well as interstitial deletions of the short arm of chromosome 12 [del(12p)] are found as recurring chromosomal changes in a broad spectrum of hematopoietic malignancies. These changes result in the hemizygous deletion of genetic material from 12p. We mapped a yeast artificial chromosome containing the TEL gene, a cosmid contig containing part of TEL and a P1 contig containing the KIP1 gene to 12p13. These probes were used for fluorescence in situ hybridization to analyze samples from 47 patients with various hematologic malignancies who had unbalanced translocations (25 patients) leading to loss of 12p or deletions (22 patients) involving 12p13. The patients had acute lymphoblastic leukemia (8 cases), myelodysplastic syndrome (MDS; 11 cases), acute myeloid leukemia (AML; 10 cases), myeloproliferative disorders (4 cases), therapy-related MDS or AML (7 cases), non-Hodgkin's lymphoma (2 cases), and other hematopoietic malignancies (5 cases). All three probes were hemizygously detected in 26 cases and were completely retained in only 9 cases. In 12 cases probes for one of the two genes were deleted, allowing us to map the smallest region of overlap of these deletions to a small genomic region that is bordered on the telomeric side by the TEL gene and on the centromeric side by KIP1. The genomic distance between TEL and KIP1 is estimated to be about 1 to 2 Mbp.

Adolescent↗
PubMed1995

Fusion of the TEL gene on 12p13 to the AML1 gene on 21q22 in acute lymphoblastic leukemia.

Chromosomal rearrangements involving band 12p13 are found in a wide variety of human leukemias but are particularly common in childhood acute lymphoblastic leukemia. The genes involved in these rearrangements, however, have not been identified. We now report the cloning of a t(12;21) translocation breakpoint involving 12p13 and 21q22 in two cases of childhood pre-B acute lymphoblastic leukemia, in which t(12;21) rearrangements were not initially apparent. The consequence of the translocation is fusion of the helix-loop-helix domain of TEL, an ETS-like putative transcription factor, to the DNA-binding and transactivation domains of the transcription factor AML1. These data show that TEL, previously shown to be fused to the platelet-derived growth factor receptor beta in chronic myelomonocytic leukemia, can be implicated in the pathogenesis of leukemia through its fusion to either a receptor tyrosine kinase or a transcription factor. The TEL-AML1 fusion also indicates that translocations affecting the AML1 gene can be associated with lymphoid, as well as myeloid, malignancy.

Amino Acid Sequence↗
PubMed1995

Assignment of the human p27Kip1 gene to 12p13 and its analysis in leukemias.

The p27Kip1 (p27) gene encodes an inducible inhibitor of cyclin-dependent kinase activity. Using a murine p27 cDNA as probe, we obtained a human cDNA clone and subsequently used it to isolate a genomic clone of this gene. The coding region of the human p27 gene was contained in two exons. Both the amino acid sequence and intron-exon organization of p27 were similar to those previously found for the related cyclin-dependent kinase inhibitor p21Waf1 (p21). The p27 gene was localized to chromosome band 12p13 by a combination of somatic cell hybrid and fluorescence in situ hybridization analyses. The p27 gene product is thought to control the leukocyte cell cycle and the 12p13 chromosomal band is known to be deleted in leukemias, suggesting that the p27 gene may act as a tumor suppressor gene in leukemias. Although p27 was found to reside in the minimal region of chromosomal loss in hematological malignancies, no mutations of p27 were observed in leukemia samples. Haploinsufficiency of p27 may confer a growth advantage to leukemia cells.

Base Sequence↗
PubMed1995

Molecular characterization of 16p deletions associated with inversion 16 defines the critical fusion for leukemogenesis.

The inversion of chromosome 16 [inv(16)] in acute myeloid leukemia (AML) is associated with a p-arm deletion in a subset of patients. The inversion results in two fusion genes: 5'-CBFB/MYH11-3' on 16p and 5'-MYH11/CBFB-3' on 16q. We have studied cells from 42 patients with inv(16) (38 patients) or t(16;16) (four patients) to define the frequency and characteristics of the deletion further. Using fluorescence in situ hybridization (FISH) with probes from cosmids, cosmid contigs, and yeast artificial chromosomes (YACs), we found that six patients with inv(16) had a deletion of between 150 and 350 kb centromeric to the p-arm inversion breakpoint cluster region (p-ibc). This region was shown to contain the 5' portion of the myosin heavy chain (MYH11) gene. YACs containing the p-ibc, which had been useful as FISH probes in the diagnosis of inv(16), detected the inversion in deletion as well as nondeletion patient cells. Thus, the deleted region identified in patients is entirely contained within the human genomic content of the YACs. Southern blot experiments using probes flanking the p-ibc indicated that the deletion removes segments within 10 kb centromeric of the p-ibc. Reverse transcription-polymerase chain reaction (RT-PCR) using primers from the 5' region of CBFB and the 3' region of MYH11 (distal to the p-ibc) produced the 5'-CBFB/MYH11-3' chimeric transcript in inv(16)/del patients. These data confirm that the 5'-CBFB/MYH11-3' chimeric transcript, rather than the reciprocal 5'-MYH11/CBFB-3', is the critical product for chromosome 16-related leukemogenesis.

Acute Disease↗
PubMed1995

Identification of cytogenetically undetected 12p13 translocations and associated deletions with fluorescence in situ hybridization.

We performed fluorescence in situ hybridization (FISH) on bone marrow or peripheral blood cells thought to contain a del(12p) or an unbalanced 12p11-12 translocation from 17 patients who had various hematologic malignant diseases. We used 11 cosmid, phage, and plasmid probes which we had previously ordered on 12p. Cells from three patients with myeloid disorders were shown to have 12p13 translocations that involved chromosome 2 in two of them. Moreover, in all patients, FISH showed that the translocations were associated with proximal interstitial deletions which contributed to the difficulty in identifying these translocations. Our data suggest that some rearrangements of 12p which have been described previously as deletions or unbalanced translocations may, in fact, represent 12p13 translocations accompanied by an interstitial deletion.

Acute Disease↗