Ribonuclease H-mediated antisense effects in intact human leukaemia cells.
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Biological Transport, Active↗
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Elevated platelet count features the variant type of BCR/ABL junction in chronic myelogenous leukaemia.
A variant form of BCR/ABL junction was identified in a patient with chronic myelogenous leukaemia (CML). The BCR/ABL fusion mRNA of this patient showed in-frame junction between BCR exon c3 and ABL exon 2. Although the diagnosis of CML was made, the patient showed clinical features of essential thrombocythaemia (ET) rather than that of typical CML. Treatment with interferon-alpha showed no cytogenetic response. The c3-a2 type of BCR/ABL junction seems to be associated with elevated platelet count and thus could form a novel clinical entity different from typical CML.
Adolescent↗
The biology of chronic myeloid leukemia.
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Cell Transformation, Neoplastic↗
Identification of a chromosomal gene controlling temperature-regulated expression of Shigella virulence.
Genes required for the full expression of Shigella virulence are on both the chromosome and a large virulence-associated plasmid. Expression of one or more virulence (vir) genes is temperature-regulated, wild-type strains being virulent (invasive) when grown at 37 degrees C but phenotypically avirulent (noninvasive) at 30 degrees C. A vir::lac operon fusion located on the virulence plasmid, which brings the lac genes under control of a temperature-regulated vir gene promoter, was used to select regulatory mutants constitutive for the Lac+ phenotype at the nonpermissive temperature. A transposon Tn10-induced mutant that was Lac+ at 30 degrees C and 37 degrees C was isolated, and the Tn10 insertion was transduced into a wild-type strain. The transductants all simultaneously became deregulated for virulence and invaded HeLa cells equally well at 30 degrees C and 37 degrees C. Other virulence-associated phenotypes were also deregulated and expressed at 30 degrees C. Southern hybridization with a probe for Tn10 determined the insertion to be on the chromosome. Fine mapping by transduction with phage P1L4 positioned the mutation between the galU and trp genes. A cosmid cloned fragment of Shigella chromosomal DNA containing the region around galU was used in complementation studies and showed that the closely linked regulatory gene was able to complement, in trans, the Tn10-induced mutation. We propose that this mutation defines a regulatory gene, virR, and that insertion of Tn10 into this gene inactivates a repressor that normally blocks expression of vir genes at 30 degrees C.
Bacterial Proteins↗
Involvement of the Haemophilus ducreyi gmhA gene product in lipooligosaccharide expression and virulence.
The lipooligosaccharide (LOS) present in the outer membrane of Haemophilus ducreyi is likely a virulence factor for this sexually transmitted pathogen. An open reading frame in H. ducreyi 35000 was found to encode a predicted protein that had 87% identity with the protein product of the gmhA (isn) gene of Haemophilus influenzae. In H. influenzae type b, inactivation of the gmhA gene caused the synthesis of a significantly truncated LOS which possessed only lipid A and a single 2-keto-3-deoxyoctulosonic acid molecule (A. Preston, D. J. Maskell, A. Johnson, and E. R. Moxon, J. Bacteriol. 178:396-402, 1996). The H. ducreyi gmhA gene was able to complement a gmhA-deficient Escherichia coli strain, a result which confirmed the identity of this gene. When the gmhA gene of H. ducreyi was inactivated by insertion of a cat cartridge, the resultant H. ducreyi gmhA mutant, 35000.252, expressed a LOS that migrated much faster than wild-type LOS in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the wild-type H. ducreyi strain and its isogenic gmhA mutant were used in the temperature-dependent rabbit model for dermal lesion production by H. ducreyi, the gmhA mutant was found to be substantially less virulent than the wild-type parent strain. The H. ducreyi gmhA gene was amplified by PCR from the H. ducreyi chromosome and cloned into the pLS88 vector. When the H. ducreyi gmhA gene was present in trans in gmhA mutant 35000.252, expression of the gmhA gene product restored the virulence of this mutant to wild-type levels. These results indicate that the gmhA gene product of H. ducreyi is essential for the expression of wild-type LOS by this pathogen.
Aldose-Ketose Isomerases↗
Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa.
Nitrosoguanidine-induced Pseudomonas aeruginosa mutants which were unable to utilize glycerol as a carbon source were isolated. By utilizing PAO104, a mutant defective in glycerol transport and sn-glycerol-3-phosphate dehydrogenase (glpD), the glpD gene was cloned by a phage mini-D3112-based in vivo cloning method. The cloned gene was able to complement an Escherichia coli glpD mutant. Restriction analysis and recloning of DNA fragments located the glpD gene to a 1.6-kb EcoRI-SphI DNA fragment. In E. coli, a single 56,000-Da protein was expressed from the cloned DNA fragments. An in-frame glpD'-'lacZ translational fusion was isolated and used to determine the reading frame of glpD by sequencing across the fusion junction. The nucleotide sequence of a 1,792-bp fragment containing the glpD region was determined. The glpD gene encodes a protein containing 510 amino acids and with a predicted molecular weight of 56,150. Compared with the aerobic sn-glycerol-3-phosphate dehydrogenase from E. coli, P. aeruginosa GlpD is 56% identical and 69% similar. A similar comparison with GlpD from Bacillus subtilis reveals 21% identity and 40% similarity. A flavin-binding domain near the amino terminus which shared the consensus sequence reported for other bacterial flavoproteins was identified.
Amino Acid Sequence↗
Gene expression of parathyroid tumors: molecular subclassification and identification of the potential malignant phenotype.
Parathyroid tumors are heterogeneous, and diagnosis is often difficult using histologic and clinical features. We have undertaken expression profiling of 53 hereditary and sporadic parathyroid tumors to better define the molecular genetics of parathyroid tumors. A class discovery approach identified three distinct groups: (1) predominantly hyperplasia cluster, (2) HRPT2/carcinoma cluster consisting of sporadic carcinomas and benign and malignant tumors from Hyperparathyroidism-Jaw Tumor Syndrome patients, and (3) adenoma cluster consisting mainly of primary adenoma and MEN 1 tumors. Gene sets able to distinguish between the groups were identified and may serve as diagnostic biomarkers. We demonstrated, by both gene and protein expression, that Histone 1 Family 2, amyloid beta precursor protein, and E-cadherin are useful markers for parathyroid carcinoma and suggest that the presence of a HRPT2 mutation, whether germ-line or somatic, strongly influences the expression pattern of these 3 genes. Cluster 2, characterized by HRPT2 mutations, was the most striking, suggesting that parathyroid tumors with somatic HRPT2 mutation or tumors developing on a background of germ-line HRPT2 mutation follow pathways distinct from those involved in mutant MEN 1-related parathyroid tumors. Furthermore, our findings likely preclude an adenoma to carcinoma progression model for parathyroid tumorigenesis outside of the presence of either a germ-line or somatic HRPT2 mutation. These findings provide insights into the molecular pathways involved in parathyroid tumorigenesis and will contribute to a better understanding, diagnosis, and treatment of parathyroid tumors.
Gene Expression Profiling↗
Aberrant BCR-ABL transcript with intronic insertion in a patient with philadelphia chromosome-positive chronic myeloid leukemia: implications for disease progression.
The BCR-ABL fusion gene is important for the leukemogenesis of chronic myeloid leukemia (CML). A relationship between types of BCR-ABL transcripts in CML and clinical features has been proposed. We present here a patient with CML who carried an aberrant BCR-ABL transcript with an intronic sequence insert. A 26-year-old woman was diagnosed as having Philadelphia chromosome (Ph) positive CML. Reverse transcription polymerase chain reaction detected an atypically large BCR-ABL mRNA transcript. Sequencing revealed a 589bp insertion consisting of a 5' portion of BCR intron b2 and a 3' portion of ABL intron 1b between BCR exon b2 and ABL exon a2. Although the typical b2a2 transcript was undetectable initially, it appeared after intensive chemotherapy. The aberrant transcript presumably arose as a result of a lack of splicing, and chemotherapy might modify the disease course by selecting the subpopulation of the CML clone expressing typical BCR-ABL mRNA dominantly.
Adult↗
Philadelphia negative BCR-ABL positive chronic myeloid leukemia mimicking juvenile chronic myeloid leukemia in a 2-year-old child.
We present the case of a two-year-old child with an atypical presentation of chronic myeloid leukemia. At diagnosis, he showed clinical and biological features of juvenile chronic myeloid leukemia (CML). However, eosinophilia was observed in blood and bone marrow. The bone marrow karyotype did not demonstrate the Philadelphia chromosome but BCR-ABL rearrangement was shown to be present by reverse transcriptase polymerase chain reaction (RT-PCR) analysis and confirmed by fluorescent in situ hybridization (FISH) analysis. Discussion centres on the differentiation between juvenile CML and childhood chronic myelogenous Leukemia and the importance of carrying out RT PCR for all juvenile CML cases.
Child, Preschool↗
Identification of putative programmed -1 ribosomal frameshift signals in large DNA databases.
The cis-acting elements that promote efficient ribosomal frameshifting in the -1 (5') direction have been well characterized in several viral systems. Results from many studies have convincingly demonstrated that the basic molecular mechanisms governing programmed -1 ribosomal frameshifting are almost identical from yeast to humans. We are interested in testing the hypothesis that programmed -1 ribosomal frameshifting can be used to control cellular gene expression. Toward this end, a computer program was designed to search large DNA databases for consensus -1 ribosomal frameshift signals. The results demonstrated that consensus programmed -1 ribosomal frameshift signals can be identified in a substantial number of chromosomally encoded mRNAs and that they occur with frequencies from two- to sixfold greater than random in all of the databases searched. A preliminary survey of the databases resulting from the computer searches found that consensus frameshift signals are present in at least 21 homologous genes from different species, 2 of which are nearly identical, suggesting evolutionary conservation of function. We show that four previously described missense alleles of genes that are linked to human diseases would disrupt putative programmed -1 ribosomal frameshift signals, suggesting that the frameshift signal may be involved in the normal expression of these genes. We also demonstrate that signals found in the yeast RAS1 and the human CCR5 genes were able to promote significant levels of programmed -1 ribosomal frameshifting. The significance of these frameshifting signals in controlling gene expression is not known, however.
Amino Acid Sequence↗
Karyotype and BCR studies after bone marrow transplantation for chronic myeloid leukaemia.
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Biomarkers, Tumor↗
Significance of the P210 versus P190 molecular abnormalities in adults with Philadelphia chromosome-positive acute leukemia.
We investigated the significance of p210 and p190 molecular abnormalities in 32 adults with Philadelphia chromosome (Ph)-positive acute leukemia. p210 was detected in 15 patients (47%), p190 in 16 (50%), and both in one (3%). p210 was noted in 11 of 24 patients (46%) with acute lymphocytic leukemia, and in four of eight patients (50%) with acute myelogenous or undifferentiated leukemia. Among 29 patients with untreated disease (p210, 14 patients; p190, 15 patients), no significant differences in the two molecularly distinct groups were observed by pretreatment characteristics including age, degree of organomegaly, anemia, leukocytosis, thrombocytopenia, occurrence of karyotypic abnormalities in addition to Ph, or residual diploid metaphases. Complete response (CR) rates were also similar. Although the remission duration tended to be longer with p190 (P = .08), the differences were minor (median duration 29 v 20 weeks) and not paralleled by differences in survival rate. In 10 patients studied by karyotypic analysis in remission, two of four patients with p190 and two of six patients with p210 showed 100% normal metaphases. One of the seven patients (14%) with p210 who achieved CR manifested a morphologic picture of second chronic-phase chronic myelogenous leukemia lasting for 1 month. We conclude that the molecular studies in Ph-positive acute leukemia are not associated with significantly different clinico-laboratory, karyotypic, or prognostic implications.
Adult↗
Different suppression of Ph1 positive hemopoiesis induced by intensive chemotherapy in lymphoid and myeloid blast crisis of CML.
BACKGROUND: The suppression of Ph1-positive hemopoiesis is a major goal in the treatment of CML; in this context the CML patients in blast crisis obtaining a complete clinical remission represent a useful model to investigate the behavior of the Ph1-positive and negative clones during bone marrow repopulation after ablative therapy. METHODS: Seven CML patients in blast crisis (four lymphoid and three myeloid) who obtained a complete clinical remission after an intensive polychemotherapy treatment were evaluated by cytogenetic and molecular analysis both in blast and remission phases. Standard cytogenetic and Southern techniques were employed; in addition, minimal residual disease status (MRD) was ascertained by amplification (PCR) of the specific bcr-abl chimeric transcripts. RESULTS: After a single cycle of induction, all lymphoid cases displayed a complete restoration of Ph1-negative hemopoiesis; by contrast, one myeloid blast crisis showed a partial suppression of Ph1-positive hemopoiesis only after two cycles of chemotherapy, and in the remaining two cases the hematological remission was indeed a reversion to the chronic phase. CONCLUSIONS: Ph1-positive chronic clones present in lymphoid blast crisis showed a higher degree of sensitivity to intensive chemotherapy than those present in the myeloid cases. This observation further suggests that the growth properties of the Ph1-positive clones are highly variable from case to case and probably tend to progress during the time-course of the disease.
Antineoplastic Combined Chemotherapy Protocols↗
Non-radioactive detection of the rearranged BCR/ABL sequences amplified by polymerase chain reaction.
The polymerase chain reaction (PCR) is a powerful technique for the detection of the bcr/abl rearrangement in chronic myelogenous leukemia (CML). It allows the exponential amplification of the rearranged region, thus facilitating its detection. The specificity of the bcr/abl cDNA sequence amplified by PCR is most commonly verified by hybridization to a 32P-labeled probe. In this paper, an assay for the colorimetric detection of the amplified bcr/abl fragment is described, which offers several advantages over the use of radioactive probes. We adapted the PCR for synthesis and simultaneous labeling of DNA fragments with a non-radioactive steroid compound called digoxigenin. This labeling procedure was used to generate a digoxigenin-labeled internal probe for the chimeric bcr/abl mRNA. The assay described is based on the hybridization of the amplified bcr/abl sequence to the non-radioactively labeled probe and on the subsequent detection by an enzyme-linked immunoassay and enzyme-catalyzed color reaction. Using this protocol, we investigated 20 patients with CML along with six healthy individuals and two cell lines derived from patients with CML for the presence of the bcr/abl rearrangement. It is shown that the assay is both highly sensitive and specific and that it is readily applicable to the routine diagnosis of CML. In addition, the assay could be adapted to a number of clinical diagnostic uses.
Colorimetry↗
Application of a new protocol for nested PCR to the detection of minimal residual bcr/abl transcripts.
Nested PCR (NPCR), a two-step procedure in which the products of a first PCR using 'outer' primers are reamplified using 'inner primers', has been successfully used to test for the chronic myeloid leukemia (CML)-specific bcr-abl transcripts. A major drawback of the conventional nesting strategy is linked to the opening of the reaction tube between the two successive PCR reactions, giving a risk of contaminating the second mix with amplicons. In this paper, the application of a new protocol for NPCR without reopening the reaction tube between the two steps of the procedure is described for the research of residual leukemic cells in the peripheral blood of 14 CML patients treated by bone marrow transplantation (BMT) or interferon (IFN). This assay which is both highly specific and sensitive, offers several advantages over the use of conventional NPCR: it is more sensitive, faster and decreases the risk of false-positive results. In addition, chemiluminescent detection of amplified DNA after transfer onto a nylon membrane, although comparable with radioactive hybridization in terms of sensitivity and speed, is more advantageous in safety and convenience. In conclusion, this assay could be adapted to a number of clinical diagnostic uses.
Base Sequence↗
Competitive polymerase chain reaction to estimate the number of BCR-ABL transcripts in chronic myeloid leukemia patients after bone marrow transplantation.
We have developed a competitive polymerase chain reaction (PCR) titration assay that estimates the number of BCR-ABL transcripts in chronic myeloid leukemia patients to monitor minimal residual disease after bone marrow transplantation (BMT). The assay gave reproducible results and allowed differences in BCR-ABL message levels of half an order of magnitude to be distinguished. Of 91 patients studied by nonquantitative PCR, 28 who had a positive PCR result on at least one occasion posttransplant were analyzed by competitive PCR. Seventeen patients had no evidence in their marrow of cytogenetic relapse during the period of observation; BCR-ABL transcript numbers in these cases ranged from approximately 10 to 800/micrograms RNA. Ten of the 11 patients who relapsed cytogenetically were studied when Philadelphia-positive metaphases were first detected in their marrow; transcript numbers ranged from 1,600 to 7 x 10(5)/micrograms RNA. Patients in hematologic relapse had between 9 x 10(4) and 10(6) BCR-ABL transcripts/micrograms RNA. Patients who progressed from cytogenetic remission to cytogenetic relapse and then to hematologic relapse had increasing numbers of BCR-ABL transcripts in their blood. Three patients had clear evidence of rising numbers of BCR-ABL transcripts before routine detection of cytogenetic relapse. Conversely patients without cytogenetic relapse generally had low or falling numbers of transcripts. We conclude that serial monitoring of residual disease post-BMT by estimating the number of BCR-ABL transcripts provides more information than conventional cytogenetics or nonquantitative PCR and may identify patients in need of therapeutic intervention before the onset of overt relapse.
Base Sequence↗
Chronic myelogenous leukemia: too much or too little growth, or both?
CML, characterized by the BCR/ABL gene rearrangement has been more extensively studied than any other malignancy. Over the last decade, significant progress has been made in our understanding of BCR-ABL-induced alterations in intracellular signaling. Unfortunately, we still only poorly understand the correlation between the clinical symptoms of chronic phase CML and the BCR-ABL oncoprotein. This is in part due to lack of a good in vivo animal model of chronic phase CML. In vivo and in vitro studies from the Clarkson group, recently reviewed in this journal (Leukemia 1997; 11: 1404-1428), have significantly enhanced our understanding of the pathophysiology of CML. However, further characterization of the effect of the BCR-ABL oncoprotein on signal molecules involved with cell differentiation, cell proliferation, cell survival and cell adhesion in primary Ph+ CML progenitors or in vivo models of CML will be needed to provide a full understanding of the pathophysiology of chronic phase CML.
Animals↗
Three suppressor mutations which cure a mitochondrial RNA maturase deficiency occur at the same codon in the open reading frame of the nuclear NAM2 gene.
Dominant mutations of the nuclear NAM2 gene are able to compensate for a deficiency of the maturase encoded by the fourth intron of the mitochondrial cytochrome b gene. We have determined the complete nucleotide sequence of the NAM2-1 suppressor allele. The results of S1 nuclease protection experiments show that two overlapping poly(A)+ RNAs are transcribed from the gene using different promoters. The longer transcript contains two open reading frames (ORFs), a long ORF which could encode a protein of 894 amino acids, mol. wt 102,000 daltons, and a short ORF of 51 codons which is omitted from the shorter transcript. The wild-type nam2+ and two other suppressor alleles, NAM2-6 and NAM2-7, have been cloned. A comparison of the sequence of the wild-type and the three suppressor alleles shows that on three separate occasions the same codon specifying glycine was mutated (once to serine and twice to cysteine). Finally sequence comparisons identified two regions in the long ORF, distinct from the position of the suppressor mutations, that could correspond to binding domains for a nucleotide and a nucleic acid.
Alleles↗