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Janet D Rowley

Publications and source records attributed to Janet D Rowley.

At least 37 records · Page 2Linked to original sources
PubMed2003

Polymorphisms in the MLL breakpoint cluster region (BCR).

The MLL gene is involved in many chromosomal translocations leading to both acute myeloid and lymphoid leukemia. Some patients treated for primary malignancies with chemotherapeutic agents that inhibit DNA topoisomerase II (topo II) develop treatment-related leukemia (t-AML) caused by MLL gene rearrangement. Whether these patients are unusually susceptible to anti-topo II drugs, or whether this is a random adverse event is unknown. To discover genetic polymorphisms that may predispose patients to t-AML development, we sequenced the 8.3-kb MLL breakpoint cluster region (BCR) from 22 patients who had been treated with topo II inhibitors and who developed t-AML and from 37 patients who did not, and from eight infants and 20 normal individuals. Four polymorphic sites within Alu repetitive elements were identified; three affected the length of poly-A tracts and one altered the size of a trinucleotide repeat. The three poly-A tract polymorphisms occurred with equal frequency in leukemic patients and controls and hence are not predictors of risk. The trinucleotide GAA repeat has three alleles: (GAA)4, (GAA)5, and (GAA)6. The (GAA)6 allele is very rare. The adult t-AML patients are almost exclusively (GAA)4/5 heterozygotes (83%), whereas the normal population is only 55% (GAA)4/5 heterozygotic and is represented equally by (GAA)4 and (GAA)5 homozygotes (20% each). Only certain trends could be established because of the small sample size of these leukemic groups. Whereas adult t-AML patients are more likely to be (GAA)4/5 heterozygotes, this is not statistically significant, and this polymorphism within the MLL BCR has only a suggestive association with t-AML development.

Adolescent↗
PubMed2003

A t(11;15) fuses MLL to two different genes, AF15q14 and a novel gene MPFYVE on chromosome 15.

The mixed lineage leukemia gene (MLL, also known as HRX, ALL-1 and Htrx) located at 11q23 is involved in translocations with over 40 different chromosomal bands in a variety of leukemia subtypes. Here we report our analysis of a rare but recurring translocation, t(11;15)(q23;q14). This translocation has been described in a small subset of cases with both acute myeloblastic leukemia and ALL. Recent studies have shown that MLL is fused to AF15q14 in the t(11;15). Here we analyse a sample from another patient with this translocation and confirm the presence of an MLL-AF15q14 fusion. However, we have also identified and cloned another fusion transcript from the same patient sample. In this fusion transcript, MLL is fused to a novel gene, MLL partner containing FYVE domain (MPFYVE). Both MLL-AF15q14 and MLL-MPFYVE are in-frame fusion transcripts with the potential to code for novel fusion proteins. MPFYVE is also located on chromosome 15, approximately 170 kb telomeric to AF15q14. MPFYVE contains a highly conserved motif, the FYVE domain which, in other proteins, has been shown to bind to phosphotidyl-inositol-3 phosphate (PtdIns(3)P). The MLL-MPFYVE fusion may be functionally important in the leukemia process in at least some patients containing this translocation.

Amino Acid Motifs↗
PubMed2003

Clinical-cytogenetic associations in 306 patients with therapy-related myelodysplasia and myeloid leukemia: the University of Chicago series.

Therapy-related myelodysplasia and myeloid leukemia (t-MDS/t-AML) is a distinctive clinical syndrome occurring after exposure to chemotherapy (CT) or radiotherapy (RT). We report findings on 306 consecutive patients referred to our institution with morphologic review and cytogenetic analyses. Since 1972, 141 males and 165 females with a median age of 51 years (range, 3-83 years) at primary diagnosis and 58 years (range, 6-86 years) at secondary diagnosis were analyzed. Patients had been administered various cytotoxic agents, including alkylating agents (240 patients, 78%) and topoisomerase 2 inhibitors (115 patients, 39%). One hundred twenty-one (40%) had undergone CT alone, 43 (14%) had undergone RT alone, and 139 (45%) had undergone both modalities. At diagnosis of t-MDS/t-AML, 282 (92%) had clonal abnormalities involving chromosome 5 (n = 63), chromosome 7 (n = 85), chromosomes 5 and 7 (n = 66), recurring balanced rearrangements (n = 31), other clonal abnormalities (n = 39), or normal karyotype (n = 24). Abnormalities of chromosome 5, 7, or both accounted for 76% of all cases with an abnormal karyotype. Seventeen patients acquired t-MDS/t-AML after autologous stem cell transplantation, but no unique pattern of cytogenetic abnormalities was observed. Shorter latency was observed for patients with balanced rearrangements (median, 28 vs 67 months; P <.0001). Patients with acute leukemia were more likely to have balanced rearrangement than those with myelodysplasia (28% vs 4%; P <.0001). Median survival time after diagnosis of t-MDS/t-AML was 8 months; survival at 5 years was less than 10%. These data confirm and extend previous associations between clinical, morphologic, and cytogenetic findings in t-MDS/t-AML.

Adolescent↗
PubMed2003

Duplexes of 21-nucleotide RNAs mediate RNA interference in differentiated mouse ES cells.

RNA interference (RNAi) is a novel phenomenon that can induce post-transcriptional gene silencing (PTGS) both in animals and plants. RNAi is effective in suppressing specific gene expression in the early mouse embryonic cells and in undifferentiated embryonic stem (ES) cells. In this study, we demonstrate that dsRNA is effective in inducing PTGS in differentiated ES cells: CD34+ embryoid body (EB) cells, as confirmed by western blot and immunocytochemical staining. PU.1 is a key transcription factor in myeloid differentiation. Undifferentiated ES cells do not express PU.1; however it is expressed when ES cells differentiate into EBs. PU.1 could be suppressed by the specific PU.1 dsRNA, but not non-specific Lamin A/C dsRNA, in the CD34+ EB cells when they were induced to myeloid differentiation in the presence of GM-CSF and IL-3. As a consequence, the level of expression of CD115 (M-CSF receptor), one of the downstream genes regulated by PU.1 is decreased in PU.1 dsRNA treated CD34+ EB cells, but not in Lamin A/C dsRNA treated cells. To explore this phenomenon in other myeloid gene, we also found that C/EBPalpha gene could be knocked down by C/EBPalpha dsRNA. Our finding demonstrates that RNAi is effective in inhibiting specific gene expression in differentiated ES cells. RNAi offers a new methodology for study of hematopoietic regulation using ES cell differentiation.

Animals↗
PubMed2003

Screening and quantification of multiple chromosome translocations in human leukemia.

BACKGROUND: Characterization of fusion gene transcripts in leukemia that result from chromosome translocations provides valuable information regarding appropriate treatment and prognosis. However, screening for multiple fusion gene transcripts is difficult with conventional PCR and state-of-the-art real-time PCR and high-density microarrays. METHODS: We developed a multiplex reverse transcription-PCR (RT-PCR) assay for screening and quantification of fusion gene transcripts in human leukemia cells. Chimeric primers were used that contained gene-specific and universal sequences. PCR amplification of fusion and control gene transcripts was achieved with use of an excess of universal primers to allow the ratio of abundance of fusion gene to endogenous or exogenous controls to be maintained throughout PCR. Multiplex RT-PCR products analyzed by an ABI 310 Genetic Analyzer were consistent with those of duplex RT-PCR (single analytical sample plus control). In addition, multiplex RT-PCR results were analyzed by an assay using an oligonucleotide microarray that contained probes for the splice-junction sequences of various fusion transcripts. RESULTS: The multiplex RT-PCR assay enabled screening of >10 different fusion gene transcripts in a single reaction. RT-PCR followed by analysis with the ABI Prism 310 Genetic Analyzer consistently detected 1 fusion-transcript-carrying leukemia cell in 100-10 000 cells. The assay covered a 1000-fold range. Preliminary results indicate that multiplex RT-PCR products can also be analyzed by hybridization-based microarray assay. CONCLUSIONS: The multiplex RT-PCR analyzed by either ABI Prism 310 Genetic Analyzer or microarray provides a sensitive and specific assay for screening of multiple fusion transcripts in leukemia, with the latter an assay that is adaptable to a high-throughput system for clinical screening.

Humans↗
PubMed2002

Silencing of B cell receptor signals in human naive B cells.

To identify changes in the regulation of B cell receptor (BCR) signals during the development of human B cells, we generated genome-wide gene expression profiles using the serial analysis of gene expression (SAGE) technique for CD34(+) hematopoietic stem cells (HSCs), pre-B cells, naive, germinal center (GC), and memory B cells. Comparing these SAGE profiles, genes encoding positive regulators of BCR signaling were expressed at consistently lower levels in naive B cells than in all other B cell subsets. Conversely, a large group of inhibitory signaling molecules, mostly belonging to the immunoglobulin superfamily (IgSF), were specifically or predominantly expressed in naive B cells. The quantitative differences observed by SAGE were corroborated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. In a functional assay, we show that down-regulation of inhibitory IgSF receptors and increased responsiveness to BCR stimulation in memory as compared with naive B cells at least partly results from interleukin (IL)-4 receptor signaling. Conversely, activation or impairment of the inhibitory IgSF receptor LIRB1 affected BCR-dependent Ca(2+) mobilization only in naive but not memory B cells. Thus, LIRB1 and IL-4 may represent components of two nonoverlapping gene expression programs in naive and memory B cells, respectively: in naive B cells, a large group of inhibitory IgSF receptors can elevate the BCR signaling threshold to prevent these cells from premature activation and clonal expansion before GC-dependent affinity maturation. In memory B cells, facilitated responsiveness upon reencounter of the immunizing antigen may result from amplification of BCR signals at virtually all levels of signal transduction.

Antigens, CD34↗
PubMed2002

Identifying novel transcripts and novel genes in the human genome by using novel SAGE tags.

The number of genes in the human genome is still a controversial issue. Whereas most of the genes in the human genome are said to have been physically or computationally identified, many short cDNA sequences identified as tags by use of serial analysis of gene expression (SAGE) do not match these genes. By performing experimental verification of more than 1,000 SAGE tags and analyzing 4,285,923 SAGE tags of human origin in the current SAGE database, we examined the nature of the unmatched SAGE tags. Our study shows that most of the unmatched SAGE tags are truly novel SAGE tags that originated from novel transcripts not yet identified in the human genome, including alternatively spliced transcripts from known genes and potential novel genes. Our study indicates that by using novel SAGE tags as probes, we should be able to identify efficiently many novel transcripts/novel genes in the human genome that are difficult to identify by conventional methods.

Base Pair Mismatch↗
PubMed2002

Molecular portraits of B cell lineage commitment.

In an attempt to characterize early B cell development including the commitment of progenitor cells to the B cell lineage, we generated and compared genomewide gene expression profiles of human hematopoietic stem cells (HSCs) and pre-B cells (PBCs) by using serial analysis of gene expression. From more than 100,000 serial analysis of gene expression tags collected from human CD34(+) HSCs and CD10(+) CD19(+) PBCs, 42,399 unique transcripts were identified in HSCs but only 16,786 in PBCs, suggesting that more than 60% of transcripts expressed in HSCs were silenced during or after commitment to the B cell lineage. On the other hand, mRNAs of pre-B cell receptor (pre-BCR)-associated genes are virtually missing in HSCs but account for more than 10% of the transcriptome of PBCs, which also show increased expression of apoptosis-related genes. Both concentration of the transcriptional repertoire on pre-BCR-related genes together with marked up-regulation of apoptosis mediators in PBC might reflect selection for the expression of a functional pre-BCR within the bone marrow. Besides known regulator genes of early B cell development such as PAX5, E2A, and EBF, the most abundantly expressed genes in PBCs include ATM, PDGFRA, SIAH1, PIM2, C/EBPB, WNT16, and TCL1, the role of which has not been established yet in early B cell development.

Antigens, CD↗
PubMed2002

The t(8;21) fusion protein, AML1 ETO, specifically represses the transcription of the p14(ARF) tumor suppressor in acute myeloid leukemia.

The t(8;21) is one of the most frequent chromosomal translocations associated with acute leukemia. This translocation creates a fusion protein consisting of the acute myeloid leukemia-1 transcription factor and the eight-twenty-one corepressor (AML1 ETO), which represses transcription through AML1 (RUNX1) DNA binding sites and immortalizes hematopoietic progenitor cells. We have identified the p14(ARF) tumor suppressor, a mediator of the p53 oncogene checkpoint, as a direct transcriptional target of AML1 ETO. AML1 ETO repressed the p14(ARF) promoter and reduced endogenous levels of p14(ARF) expression in multiple cell types. In contrast, AML1 stimulated p14(ARF) expression and induced phenotypes consistent with cellular senescence. Chromatin immunoprecipitation assays demonstrated that AML1 ETO was specifically bound to the p14(ARF) promoter. In acute myeloid leukemia samples containing the t(8;21), levels of p14(ARF) mRNA were markedly lower when compared with other acute myeloid leukemias lacking this translocation. Repression of p14(ARF) may explain why p53 is not mutated in t(8;21)-containing leukemias and suggests that p14(ARF) is an important tumor suppressor in a large number of human leukemias.

Antigens, CD↗
PubMed2002

Oligo(dT) primer generates a high frequency of truncated cDNAs through internal poly(A) priming during reverse transcription.

We have analyzed a systematic flaw in the current system of gene identification: the oligo(dT) primer widely used for cDNA synthesis generates a high frequency of truncated cDNAs through internal poly(A) priming. Such truncated cDNAs may contribute to 12% of the expressed sequence tags in the current dbEST database. By using a synthetic transcript and real mRNA templates as models, we characterized the patterns of internal poly(A) priming by oligo(dT) primer. We further demonstrated that the internal poly(A) priming can be effectively diminished by replacing the oligo(dT) primer with a set of anchored oligo(dT) primers for reverse transcription. Our study indicates that cDNAs designed for genomewide gene identification should be synthesized by use of the anchored oligo(dT) primers, rather than the oligo(dT) primers, to diminish the generation of truncated cDNAs caused by internal poly(A) priming.

Animals↗
PubMed2002

Genomic DNA breakpoints in AML1/RUNX1 and ETO cluster with topoisomerase II DNA cleavage and DNase I hypersensitive sites in t(8;21) leukemia.

The translocation t(8;21)(q22;q22) is one of the most frequent chromosome translocations in acute myeloid leukemia (AML). AML1/RUNX1 at 21q22 is involved in t(8;21), t(3;21), and t(16;21) in de novo and therapy-related AML and myelodysplastic syndrome as well as in t(12;21) in childhood B cell acute lymphoblastic leukemia. Although DNA breakpoints in AML1 and ETO (at 8q22) cluster in a few introns, the mechanisms of DNA recombination resulting in t(8;21) are unknown. The correlation of specific chromatin structural elements, i.e., topoisomerase II (topo II) DNA cleavage sites, DNase I hypersensitive sites, and scaffold-associated regions, which have been implicated in chromosome recombination with genomic DNA breakpoints in AML1 and ETO in t(8;21) is unknown. The breakpoints in AML1 and ETO were clustered in the Kasumi 1 cell line and in 31 leukemia patients with t(8;21); all except one had de novo AML. Sequencing of the breakpoint junctions revealed no common DNA motif; however, deletions, duplications, microhomologies, and nontemplate DNA were found. Ten in vivo topo II DNA cleavage sites were mapped in AML1, including three in intron 5 and seven in intron 7a, and two were in intron 1b of ETO. All strong topo II sites colocalized with DNase I hypersensitive sites and thus represent open chromatin regions. These sites correlated with genomic DNA breakpoints in both AML1 and ETO, thus implicating them in the de novo 8;21 translocation.

Adult↗
PubMed2002

Unique balanced chromosome abnormalities in treatment-related myelodysplastic syndromes and acute myeloid leukemia: report from an international workshop.

A total of 123 balanced rearrangements, including 26 occurring as a sole anomaly, not known to be recurrent in myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) prior to the Workshop, were ascertained retrospectively from 104 patients with treatment-related MDS/AML (t-MDS/t-AML). Thirteen of the aberrations were reported previously in single cases and hence may be classified as recurrent as a result of the Workshop. Patients with Unique aberrations had complex karyotypes more often (P < 0.001 for all pairwise comparisons) than did other Workshop subgroups, with 72% having 3 or more aberrations. Among 85 cases with secondary chromosomal abnormalities, -5, -7, del(5q), and del(7q) were observed in 76%, which is significantly higher (P < or = 0.007 for all pairwise comparisons) than the frequencies found in the Workshop subgroups of patients with previously known recurring aberrations. The chromosome bands most often involved in balanced aberrations were 1p36 and 3q26-27. Treatment exposure was significantly different (less topoisomerase II inhibitor exposure, more radiotherapy-only exposure) than for patients with 11q23 (P < 0.001 and P = 0.002, respectively) and 21q22 (P = 0.007 and P = 0.002, respectively) abnormalities. The median time from the first toxic exposure to secondary disease, 59 months, was significantly longer (P < or = 0.016 for all significant pairwise comparisons) than the median latency of all other patients except those in the Rare subgroup, and the median survival time, 7 months, was significantly shorter than for patients in the 21q22, inv(16), and t(15;17) subgroups (P < or = 0.002 for all pairwise comparisons), but similar to patients in the 11q23 and Rare subgroups. In contrast to known recurring abnormalities, significantly more patients (61%, all P < 0.001) presented with t-MDS, with over one-third of these patients progressing to t-AML. Thus, this group of patients appears to be more similar to the typical t-MDS/t-AML patients, with complex karyotypes as well as chromosome 5 and 7 abnormalities, than to those with recurrent balanced rearrangements.

Adolescent↗