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Biomedical subjects

N C Cross

Publications and source records attributed to N C Cross.

At least 109 records · Page 6Linked to original sources
PubMed1994

Retroviral transduction of Philadelphia-positive chronic myeloid leukemia cells with a human mutant p53 cDNA and its effect on in vitro proliferation.

Alterations in the tumor suppressor gene p53 are associated with the pathogenesis of blastic transformation of chronic myeloid leukemia (CML), but their exact role, particularly their relationship with the chimeric protein p210BCR/ABL, is poorly defined. Point mutations in p53 have been found in some cases of blast crisis and CML blastic cell lines, but it is not clear whether complete inactivation of p53 tumor suppressor function, with or without the production of a mutant protein, can by itself trigger the process of blastic transformation. By using retroviral gene transfer, we showed that the introduction of a mutant human p53 cDNA into hematopoietic progenitor cells from patients with CML in chronic phase, which already contain p210BCR/ABL, could promote their proliferation in vitro, and occasionally even lead to the growth of factor-independent colonies. We conclude that a mutant p53 may act in synergy with p210BCR/ABL and promote the survival and proliferation of CML hematopoietic stem and progenitor cells in vitro.

Cell Division↗
PubMed1994

An optimized multiplex polymerase chain reaction (PCR) for detection of BCR-ABL fusion mRNAs in haematological disorders.

A rapid and simple polymerase chain reaction (PCR) method is described that is capable of identifying any of the BCR-ABL transcripts that have yet been described in chronic myeloid or acute lymphoblastic leukaemia. Randomly primed cDNA is synthesized from leucocyte RNA and amplified in a single reaction containing four oligonucleotide primers (multiplex PCR). Different size products are generated from ela2 (p190) and b3a2 or b2a2 (p210) BCR-ABL transcripts which are readily and unambiguously distinguishable after agarose gel electrophoresis without the need for either nested PCR or hybridization. Chronic myeloid leukaemia cells are readily detectable even when diluted 1 in 1000 with normal blood. Samples which do not have BCR-ABL rearrangements produce a single band derived from the normal BCR gene, and the presence of this band controls for adequate RNA and cDNA preparation. Using this assay we have detected BCR-ABL transcripts in a variety of haematological disorders.

Base Sequence↗
PubMed1993

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↗
PubMed1993

Isolation and characterization of the genes encoding mouse and human type-5 acid phosphatase.

The gene (mT5AP) encoding murine type-5 acid phosphatase has been isolated and completely sequenced while the gene (hT5AP) encoding human T5AP has been partly sequenced. The murine gene spans 4 kb and contains five exons. Exon 1 is completely non-coding and exon 2 starts with the initiation codon in both mT5AP and hT5AP. The positions of the intron/exon boundaries are completely conserved between mT5AP and hT5AP, but are distinct from the gene encoding the related porcine protein, uteroferrin (Utf). There is strong homology at both the nucleotide (nt) and amino acid (aa) levels between the inferred mouse cDNA and the sequences of rat T5AP and hT5AP, and pig Utf. The mT5AP and hT5AP genes were found to have multiple transcription start points (tsp) by primer extension analysis, consistent with the absence of a consensus TATA box. The sequences for the 5'-flanking regions of mT5AP and hT5AP were determined to -1.6 and -1.0 kb, respectively, relative to the tsp. A 2-kb segment of the mT5AP 5' flanking region linked to a luciferase-encoding reporter gene (Luc) was sufficient to direct tissue-specific transcription in the mouse macrophage cell line, RAW264. Significant sequence similarity between the mT5AP and hT5AP promoters is restricted to the most proximal 200 bp, which also resembles the porcine Utf gene, and a 300-bp segment 700 bp upstream. A progesterone-response element is present only in the mouse promoter and the estrogen- and iron-response elements described previously in the pig gene are absent from both the mouse and human genes. These differences may result in distinctive regulation of T5AP and Utf expression.

Acid Phosphatase↗
PubMed1993

The ABL-BCR fusion gene is expressed in chronic myeloid leukemia.

Although the BCR-ABL hybrid gene on chromosome 22q-plays a pivotal role in the pathogenesis of chronic myeloid leukemia (CML), little is known of the reciprocal chimeric gene, ABL-BCR, formed on chromosome 9q+. By reverse transcription/polymerase chain reaction amplification (RT/PCR) we have detected ABL-BCR mRNA in cells from 31 of 44 BCR-ABL positive CML patients and 3 of 5 CML cell lines. Of the 34 positive samples, 31 had classical t(9;22) (q34;q11) translocations; in 3 samples there was no Philadelphia (Ph) and/or 9q+ chromosomes. ABL-BCR expression consisted of ABL(Ib)-BCR mRNA in 26 patients and of both ABL(Ib)-BCR and ABL(Ia)-BCR mRNA species in 6 patients. The ABL-BCR transcripts encoded one or, more rarely, both of the two potential junctions, designated ABL-b3 and ABL-b4, which differed in size by 75 bp. In 2 patients, the BCR exon b3 was not present in either the BCR-ABL or the corresponding ABL-BCR transcript, whereas in 5 patients exon b3 was present in both transcripts. Direct sequencing of PCR fragments representing the full-length coding sequence of ABL-BCR cDNAs type Ib-b3, Ia-b3, Ib-b4, and Ia-b4 showed an open reading frame predicted to encode fusion proteins of 370 to 414 amino-acids. If an ABL-BCR gene product is produced in CML cells, it may be relevant as a mechanism for deregulating the GTPase activating protein (GAP) function of BCR.

Base Sequence↗
PubMed1993

Minimal residual disease after allogeneic bone marrow transplantation for chronic myeloid leukaemia in first chronic phase: correlations with acute graft-versus-host disease and relapse.

We have studied 61 patients who underwent allogeneic bone marrow transplantation (BMT) for chronic myeloid leukaemia (CML) in first chronic phase. Minimal residual disease was detected by the amplification of the leukaemia-specific BCR-ABL fusion mRNA with the polymerase chain reaction (PCR) using a highly sensitive nested primer strategy. As a general pattern, patients often had detectable BCR-ABL (PCR positive) for up to 6 or 9 months post BMT after which time BCR-ABL became undetectable (PCR negative). The conversion from PCR positive to PCR negative was not associated with the time at which cyclosporin A treatment was stopped. Six patients (10%) have relapsed during the period of this study, two within 1 year and four more than 1 year after transplant. The relationship between PCR positivity more than 1 year post transplant and relapse was significant (P = 0.036) but 15 patients who were PCR positive beyond 1 year remain in complete clinical and cytogenetic remission. Thus late positivity identifies a group of patients at increased risk of relapse but is of little predictive value for individual patients. Of the four late relapses, two had been persistently PCR positive and two were initially PCR positive, converted to negative and subsequently to positive again. Although all relapses were preceded by PCR positivity, relapse may occur only 12 months after a PCR negative result. The proportion of patients PCR negative at 3/4 months after BMT was found to increase significantly with the severity of acute GVHD (P = 0.002) but no relationship was found between acute GVHD and subsequent PCR results. There was no clear association between severity of chronic GVHD and PCR result.

Acute Disease↗
PubMed1993

Minimal residual disease after bone marrow transplant for chronic myeloid leukaemia detected by the polymerase chain reaction.

We describe the methodology and application of the polymerase chain reaction to detect BCR-ABL mRNA as a marker for CML cells. The technique is highly sensitive enabling the routine detection of 1 leukaemic cell in 10(5) or 10(6) normal cells and is therefore the most sensitive method available for detecting minimal residual disease. Analysis of marrow or blood from 80 patients after bone marrow transplantation for CML shows that residual leukemia is often detectable for several months but that most subsequently become PCR negative. Patients who relapsed were all PCR positive before the detection of Philadelphia positive metaphases in bone marrow aspirates.

Base Sequence↗
PubMed1993

Molecular evidence of minimal residual disease after treatment for leukaemia and lymphoma: an updated meeting report and review.

The very rapid development in the last few years of techniques based on use of the polymerase chain reaction (PCR) for characterizing molecular lesions in leukaemia and lymphoma now offers the opportunity for monitoring residual disease at a sensitivity of one malignant cell in 10(5) or 10(6) normal cells. Maximal specificity is presumably achieved when the DNA sequences amplified are truly leukaemia-specific, such as BCR/ABL in chronic myelogenous leukemia, RARA PML/RARA in t(15;17) acute myelogenous leukemia, DEK/CAN in t(6;9) AML, PBX1/E2A in t(1;19) acute lymphoblastic leukemia (ALL), or TAL-1 deletions in other T-ALLs. Comparable sensitivity may be achieved by using immunoglobulin heavy chain (IGH) and T-cell receptor (TCR) gene rearrangements if a clonospecific probe can be generated. However, the presence of similar sequences in IgH genes from normal B lymphocytes may decrease the specificity. For clinical purposes the crucial issues are the following. Can PCR techniques be used for confirmation of diagnosis and evaluation of extent of disease? Can PCR data obtained in remission provide information about the probability of cure or of relapse? Can techniques be developed to quantitate the PCR product and thereby increase its predictive value? These and other issues were addressed at the 4th Workshop of the Molecular Biology/BMT Study Group that took place in Bristol UK on 9-10 May 1992.

Humans↗
PubMed1993

Matched unrelated donor bone marrow transplantation for chronic myeloid leukaemia in chronic phase: comparison of ex vivo and in vivo T-cell depletion.

Between January 1985 and March 1992, 48 patients with chronic phase CML underwent BMT from volunteer unrelated donors (MUD) serologically identical at HLA-A, B and DR loci. 19 patients received donor marrow ex vivo T-cell depleted (EX-TCD) with Campath monoclonal antibodies. 29 patients received unmanipulated donor marrow with CsA/MTX GVHD prophylaxis; 28 received additional intravenous antilymphocyte therapy from day +1 to +5 (IN-TCD). Overall 26 patients survive at median follow up of 362 days; actuarial survival at 3 years is 50%. 3 patients have sustained haematological relapse; actuarial leukaemia-free survival is 38%. There is no difference in overall survival between the EX-TCD and IN-TCD groups, but primary graft failure (n = 4) occurred only in the EX-TCD group, while GVHD (grade II or greater) occurred more frequently in the IN-TCD group (61% vs. 29%, p = 0.084). The optimum method for GVHD prophylaxis in MUD BMT remains uncertain.

Adolescent↗
PubMed1991

Aldolase B mutations in Italian families affected by hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is an inborn error of metabolism caused by aldolase B deficiency. The aldolase B gene has been cloned and the following mutations causing HFI have been identified: A149P (a G----C transversion in exon 5), A174D (a C----A transversion in exon 5), L288 delta C (a base pair deletion in exon 8), and N334K (a G----C transversion in exon 9). We have investigated the occurrence of these mutations in 11 Italian patients affected by HFI using PCR and hybridisation to specific oligomers. We found that four patients were homozygous for the A149P mutation, two patients were homozygous for the A174D mutation, three patients were compound heterozygotes for both the A149P and A174D mutations, one patient was homozygous for the N334K mutation, and one patient did not show any of the reported mutations (HFI diagnosis carried out by aldolase B assay). The L288 delta C mutation has not been found in this survey.

Base Sequence↗
PubMed1991

Regulation and expression of type V (tartrate-resistant) acid phosphatase in human mononuclear phagocytes.

Human type V (tartrate-resistant) acid phosphatase belongs to a unique group of iron-binding proteins that includes uteroferrin and other purple phosphatases. The enzyme is normally restricted to osteoclasts and certain phagocytic cells but its rôle is unknown. We show that phosphatase mRNA is abundant in cells of monohistiocytic phenotype and that enzyme expression in cultured human monocyte-derived macrophages is depressed by gamma-interferon and bacterial lipopolysaccharide, agents that promote functional differentiation in these cells. In contrast, phorbol ester, which stimulates intracellular calcium-mediated events, greatly enhances type V phosphatase expression and mRNA abundance. Lymphokine and phorbol ester-modulated expression of type V acid phosphatase expression thus represents a model system for investigating proliferative responses that are specific to cells of the mononuclear macrophage system.

Acid Phosphatase↗
PubMed1990

Type 5 acid phosphatase. Sequence, expression and chromosomal localization of a differentiation-associated protein of the human macrophage.

The purple acid phosphatases and uteroferrin belong to a diverse multifunctional class of binuclear iron-containing proteins that includes haemerythrin and ribonucleotide reductase. In the pig, uteroferrin has been implicated in the delivery of iron to the foetus, but the role of the related human type 5 acid phosphatase that is principally found in resident tissue macrophages is not yet clear. To define further the function of this metalloenzyme, we have isolated and sequenced a cDNA clone for type 5 acid phosphatase and investigated expression of its gene in human tissues. The phosphatase clone contains an open reading frame of 975 bp and encodes a protein of 325 amino acids, including a signal peptide of 19 residues and two potential sites for N-glycosylation. The type 5 acid phosphatase gene mapped to the short arm of human chromosome 19 and was found to have a restriction fragment length polymorphism on digestion with XbaI. Expression of phosphatase mRNA was restricted to mononuclear phagocytes and the enzyme was induced greater than 20-fold on transformation of normal human monocytes to macrophages by culture in serum-supplemented medium. Type 5 acid phosphatase thus represents a tightly regulated system for the study of molecular events in the differentiation programme of the normal macrophage.

Acid Phosphatase↗
PubMed1990

Molecular analysis of aldolase B genes in hereditary fructose intolerance.

The molecular basis of hereditary fructose intolerance (HFI) was studied in 50 subjects (41 pedigrees, 82 apparently independent mutant alleles of aldolase B) by direct analysis of aldolase B genes amplified by means of the polymerase chain reaction. The mutation A149P (ala 149----pro) was found in 67% of alleles but was significantly more common in patients from northern than from southern Europe. Two other point mutations of aldolase B were identified. A174D (C----A; ala 174----asp) was found in subjects from Italy, Switzerland, and Yugoslavia (overall frequency 16%) but not in those from the United Kingdom, France, or the United States. L288 delta C carried a single base-pair deletion causing frameshift at codon 288 and was restricted to Sicilian subjects. By testing for these mutations in amplified DNA with a limited panel of allele-specific oligonucleotides, more than 95% of HFI patients will be susceptible to genetic diagnosis.

Alleles↗
PubMed1990

Partial aldolase B gene deletions in hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is an autosomal recessive condition caused by a deficiency of aldolase B. We have recently shown that three point mutations in this gene account for approximately 85% of HFI alleles in Europe and the United States and are thus of diagnostic importance. In this paper we define three new lesions in the aldolase B gene: two are large deletions, one of 1.65 kb and one of 1.4 kb; the third is a small deletion of 4 bp. We have determined the breakpoints of these deletions and have demonstrated that the presence of such lesions may complicate the genotyping of individuals for diagnosis of HFI.

Base Sequence↗
PubMed1989

Molecular analysis of aldolase B genes in the diagnosis of hereditary fructose intolerance in the United Kingdom.

To investigate the molecular basis of hereditary fructose intolerance, we have studied 12 British patients, all of whom were found to carry a single mutation in the gene coding for aldolase B. We have estimated the frequency of this lesion, termed A149P, amongst affected individuals in the population and predict that a diagnosis may be made non-invasively in more than 83 per cent of cases by demonstrating the presence of this allele. Genetic diagnosis and detection of asymptomatic carriers of the disease may be achieved by the specific amplification of DNA derived from mouthwash samples followed by hybridization to allele-specific oligonucleotides.

Adolescent↗