CDKN1C expression in Beckwith-Wiedemann syndrome patients with allele imbalance.

In this study, we have examined CDKN1C expression in BWS patients with allele imbalance (AI) affecting the 11p15 region. Two of two informative patients with AI, attributable to mosaic paternal isodisomy, exhibited reduced levels of CDKN1C expression in the liver and kidney, respectively, relative to expression levels in the equivalent tissues in normal controls. Although overall expression was reduced, some expression from the paternally derived CDKN1C allele was evident, consistent with incomplete paternal imprinting of the gene. One patient showed evidence of maternal allele silencing in addition to AI. These findings show for the first time that CDKN1C expression is reduced in BWS patients with AI and suggest that CDKN1C haploinsufficiency contributes to the BWS phenotype in patients with mosaic paternal isodisomies of chromosome 11.

High frequency of t(12;21) in childhood acute lymphoblastic leukemia detected by RT-PCR.

Recently, a new recurrent translocation, t(12;21)(p13;q22), has been identified in B-cell lineage acute lymphoblastic leukemia (ALL). The translocation results in the fusion of two known genes, ETV6/TEL(12p13) and AML1(21q22), both of which have been shown to be involved in other hematological malignancies. The t(12;21) is virtually undetectable by routine cytogenetics, but the chimeric transcript ETV6-AML1 has been detected in childhood ALL by molecular techniques in up to 36% of cases, making it the most common genetic abnormality in these patients. It has been shown to be associated with a B-precursor phenotype and an excellent prognosis. We tested 66 diagnostic pediatric ALL samples by reverse transcription polymerase chain reaction (RT-PCR) and found evidence of the t(12;21) in 22 (33%). None of these had previously been identified as harboring the t(12;21), although six had karyotypic abnormalities involving either 12p13 or 21q22. ETV6-AML1 expression defined a subgroup of patients characterised by an age of between two and 12 years, B-lineage immunophenotype and non-hyperdiploid DNA content. Our data further support the importance of molecular diagnostic methods in the identification of clinically distinct subgroups of patients with ALL.

A WT1 antisense oligonucleotide inhibits proliferation and induces apoptosis in myeloid leukaemia cell lines.

The response of the CML-BC cell line, K562, the myelomonocytic cell line MM6 and the promyelocytic leukaemia cell line HL-60, to a 15 mer WT1 antisense oligonucleotide, targeted to the translation initiation site of the WT1 mRNA was examined. K562 cells exposed to 0.4 microM antisense oligonucleotide showed markedly reduced proliferation which was associated with reduced cell viability. Sense, scrambled and mutant antisense oligonucleotides had no effect on the proliferation of K562 cells. MM6 cells exposed to 0.4 microM antisense oligonucleotide also showed significantly reduced cellular proliferation which was also accompanied by loss of cell viability. In the K562 and MM6 antisense cultures that exhibited reduced cell viability, both DNA fragmentation and morphological features consistent with apoptosis could be identified. In contrast the growth of HL-60 cells was unaffected by exposure to 0.4 microM antisense oligonucleotide. In each of the cell lines examined, WT1 antisense oligonucleotide abrogated WT1 protein expression, and analysis of WT1 coding sequence in these cells showed that no oncogenic point mutations in the gene were present. We propose therefore that in some myeloid leukaemia cell lines, the expression of a normal WT1 protein is necessary for cell proliferation and that it plays a role in maintaining the viability of some leukaemia cells.

Splicing of exon 5 in the WT1 gene is disrupted in Wilms' tumour.

Using a reverse transcriptase polymerase chain reaction to examine alternate splicing at site I (exon 5) and site II (exon 9) in the Wilms' tumour suppressor gene, WT1, we found that in seven of the 10 Wilms' tumours examined, splicing at site I was disrupted. This is predicted to result in isoform imbalance in Wilms' tumours, with an increase in isoforms in which the 17 amino acids encoded by exon 5 are missing. These observations could not be explained by mutations or rearrangements in flanking introns. Disrupted alternate splicing of exon 5 may play a role in the aetiology of Wilms' tumour.

Homozygous intragenic deletion in the WT1 gene in a sporadic Wilms' tumour associated with high levels of expression of a truncated transcript.

We have examined a panel of 21 sporadic Wilms' tumours for rearrangements in the Wilms' tumour suppressor gene, WT1. In one tumour with specific allele loss in chromosome 11p13, a homozygous deletion in the 3' end of the gene, encompassing exon 10 and the 3' untranslated region, was identified. High levels of a truncated WT1 transcript, predicted to encode a polypeptide missing the fourth zinc finger were expressed in this tumour. All other samples showed normal patterns of digestion on Southern blots. This observation confirms previous findings that large deletions in the gene occur infrequently in sporadic Wilms' tumours and that the zinc-finger region of the encoded polypeptide is critical for correct functioning of the gene.

The insulin-like growth factor 1 receptor gene is normally biallelically expressed in human juvenile tissue and tumours.

We have examined insulin-like growth factor 1 receptor (IGF1R) gene expression for evidence of imprinting in 15 informative patients with embryonal tumours. Biallelic expression was observed in all but one sample of normal juvenile kidney and liver, and in 9/10 associated Wilms' tumours, 3/3 hepatoblastomas and 2/2 adrenal tumours. A single patient with Beckwith-Wiedemann Syndrome (BWS) demonstrated monoallelic expression of the maternally derived IGF1R allele in normal kidney, associated Wilms' tumour and in peripheral blood lymphocytes. The observed biallelic expression of the IGF1R gene in all but one patient strongly suggests that the human gene is not normally imprinted.

Three non-overlapping regions of chromosome arm 11p allele loss identified in infantile tumors of adrenal and liver.

Tumor and constitutional chromosome arm 11p genotypes were compared in 6 hepatoblastoma (HB) patients and 2 adrenal adenoma (AA) patients, with one HB patient and both AA patients displaying clinical features associated with the Beckwith-Wiedemann syndrome (BWS). Using up to 14 chromosome 11 polymorphic markers, loss of constitutional heterozygosity (LOH) was demonstrated in both AA patients and in 4 of 6 HB patients. This identified three distinct and non-overlapping regions of 11p within which LOH occurred, which were defined as lying distal to the gamma-globin locus (11p15.5), proximal to the gamma-globin locus but distal to 11p13 (LOH being detected at 11p15.1), and restricted to the 11p13 region. Specific LOH within each 11p15 region was observed in HB, and this represents the first demonstration by a single study of LOH clearly affecting separate regions of chromosome band 11p15 in a particular tumor type. One AA showed LOH restricted to 11p13 loci, implicating the involvement of the WT1 gene. The second AA patient presented with genitourinary abnormalities and we therefore examined sequences coding for 3 zinc finger domains of WT1 in both AAs. No point mutations were identified in sequence from either patient. Nonetheless our results indicate that 3 separate 11p loci may be significant in the development of tumors which arise in association with BWS.

A gastric alcohol dehydrogenase in the baboon: purification and properties of a 'high-Km' enzyme, consistent with a role in 'first pass' alcohol metabolism.

The major isozyme of alcohol dehydrogenase in baboon stomach, ADH3, has been purified to homogeneity and characterized with a range of alcohol and aldehyde substrates. Using kcat/Km values as an indication of substrate efficacy, medium-chain length aliphatic alcohols and aldehydes were identified as the preferred substrates. ADH3 showed 'high-Km' properties with respect to ethanol, and is expected to significantly contribute to 'first-pass' metabolism of alcohol. The enzyme exhibited more than two orders of magnitude higher turnover of substrate than the baboon liver 'low-Km' ADH, and may play a role in the rapid metabolism of a wide range of ingested alcohols in the diet.

Bovine corneal aldehyde dehydrogenase: the major soluble corneal protein with a possible dual protective role for the eye.

Bovine corneal aldehyde dehydrogenase was purified to homogeneity and characterized with aldehyde substrates at pH 7.4. The enzyme was a dimer with a subunit size of 65 kDa. Using kcat/Km values as an indication of substrate efficacy, aldehyde products of lipid peroxidation were recognized as the likely 'natural' substrates. Protein yields from enzyme purification, as well as electrophoretic analyses of crude and purified enzyme preparations, demonstrated that this enzyme is the major soluble protein in bovine cornea, and constitutes around 0.5% wet weight of tissue. A dual role in protecting the eye against UV-B light is proposed--oxidation of aldehydes generated by light induced lipid peroxidation, and the direct absorption of UV-B light by bovine corneal ALDH.

Purification of hamster dihydroorotate synthetase using Procion blue-Sepharose.

Dihydroorotate (DHO) synthetase is a trifunctional protein that catalyzes the first three reactions of de novo pyrimidine biosynthesis. A single-step procedure for purification of DHO synthetase from mutant hamster cells that overproduce this protein has been developed. The synthetase is adsorbed from a postmitochondrial supernatant to a column of Procion blue-Sepharose 4B and, after the column is washed, the synthetase is eluted as a single peak with 0.4 M KCl. Pooled fractions from the trailing side of this peak yield DHO synthetase with a specific activity for aspartate transcarbamylase of 14 mumol/min/mg protein, representing a purification factor of 8.5-fold and a recovery of 28% from the postmitochondrial supernatant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the DHO synthetase was of high purity. A further 34% of the DHO synthetase from the leading side of the eluted peak contained a minor proportion of a proteolytic fragment. Similar results were obtained with an established four-step purification procedure.

Purification and properties of mouse stomach aldehyde dehydrogenase. Evidence for a role in the oxidation of peroxidic and aromatic aldehydes.

The major isozyme of aldehyde dehydrogenase in mouse stomach, AHD-4, has been purified to homogeneity and characterized with a range of aldehyde substrates at pH 7.4. The enzyme was a dimer with a subunit size of 65 kDa. Using V/Km values as an indication of substrate efficacy, aromatic aldehydes were the preferred substrates. The enzyme used either NAD+ or NADP+ as cofactor, but showed a preference for NAD+. AHD-4 showed 'high-Km' properties with respect to acetaldehyde, but differed from the 'high-Km' liver mitochondrial enzyme (AHD-1), in that it was not a semialdehyde dehydrogenase. The enzyme was significantly active towards the peroxidic aldehyde, 4-hydroxynonenal, and may play a role in vivo in the detoxification of aromatic aldehydes and the aldehyde products of lipid peroxidation.

Mercaptan and dicarboxylate inhibitors of hamster dihydroorotase.

In mammals, dihydroorotase is part of a trifunctional protein, dihydroorotate synthetase, which catalyzes the first three reactions of de novo pyrimidine biosynthesis. Dihydroorotase catalyzes the formation of a peptide-like bond between the terminal ureido nitrogen and the beta-carboxyl group of N-carbamyl-L-aspartate to yield heterocyclic L-dihydroorotate. A variety of evidence suggests that dihydroorotase may have a catalytic mechanism similar to that of a zinc protease [Christopherson, R. I., & Jones, M. E. (1980) J. Biol. Chem. 255, 3358-3370]. Tight-binding inhibitors of the zinc proteases, carboxypeptidase A, thermolysin, and angiotensin-converting enzyme have been synthesized that combine structural features of the substrates with a thiol or carboxyl group in an appropriate position to coordinate a zinc atom bound at the catalytic site. We have synthesized (4R)-2-oxo-6-thioxohexahydropyrimidine-4-carboxylate (L-6-thiodihydroorotate) and have found that this analogue is a potent competitive inhibitor of dihydroorotase with a dissociation constant (Ki) in the presence of excess Zn2+ ion of 0.17 +/- 0.02 microM at pH 7.4. The potency of inhibition by L-6-thiodihydroorotate in the presence of divalent metal ions decreases in the order Zn2+ greater than Ca2+ greater than Co2+ greater than Mn2+ greater than Ni2+; L-6-thiodihydroorotate alone is less inhibitory and has a Ki of 0.85 +/- 0.14 microM. 6-Thioorotate has a Ki of 82 +/- 8 microM which decreases to 3.8 +/- 1.4 microM in the presence of Zn2+. Zn2+ alone is a moderate inhibitor of dihydroorotase and does not enhance the potency of other inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Liver cytosolic aldehyde dehydrogenases from "alcohol-drinking" and "alcohol-avoiding" mouse strains: purification and molecular properties.

Liver cytosolic aldehyde dehydrogenases (AHD-2) have been isolated in a highly purified state from "alcohol-drinking" (C57BL/6J) and "alcohol-avoiding" (DBA/2J) strains of mice. The purified enzymes were resolved into three major and one minor form of activity by isoelectric focusing (IEF) techniques and showed similar zymogram patterns. The enzymes had identical subunit sizes on SDS-polyacrylamide gels: 53,000. Gel exclusion chromatography, using Ultrogel AcA34, indicated that the enzymes were dimers. The enzymes exhibited biphasic kinetic characteristics and were readily distinguished from each other. The purified forms of AHD-2 from C57BL/6J and DBA/2J mice exhibited two apparent Km values in each case: 10 microM/100 microM and 30 microM/330 microM respectively. AHD-2 exhibited a broad pH optimum in the range 7.0-9.0 and was very sensitive towards disulphuram inhibition, with 50% inhibition occurring at 0.17 microM. The kinetic results support proposals that AHD-2 may be the primary enzyme for oxidizing acetaldehyde during ethanol oxidation in vivo.

Simultaneous purification and characterization of glucokinase, fructokinase and glucose-6-phosphate dehydrogenase from Zymomonas mobilis.

The three enzymes glucokinase (EC 2.7.1.2), fructokinase (EC 2.7.1.4) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were isolated in high yield from extracts of Zymomonas mobilis. The principal steps in the isolation procedures involved the use of selected dye-ligand adsorbent columns, with affinity elution of two of the three enzymes. Glucokinase and fructokinase are dimeric proteins (2 X 33000 Da and 2 X 28000 Da respectively) and glucose-6-phosphate dehydrogenase is a tetramer (4 X 52000 Da). Some similarities in the structural and kinetic parameters of the two kinases were noted, but they have absolute specificity for their substrates. Fructokinase is strongly inhibited by glucose; otherwise non-substrate sugars had little effect on any of the three enzymes.

Biochemical and genetic studies on mouse aldehyde dehydrogenases.

Aldehyde dehydrogenase (AHD) exists as isozymes which are differentially distributed among tissues and subcellular fractions of mouse tissues. Genetic variants for liver mitochondrial (AHD-1) and cytoplasmic (AHD-2) isozymes have been used to map the responsible loci (Ahd-1 and Ahd-2) on chromosomes 4 and 19 respectively. Evidence for a regulatory locus (Ahd-3r) controlling the inducibility of the mouse liver microsomal isozyme (AHD-3) has also been obtained. More recent studies have described genetic and biochemical evidence for three additional AHD isozymes: a stomach isozyme (AHD-4); another liver mitochondrial enzyme (AHD-5); and a testis isozyme (AHD-6). Genetic analyses have indicated that AHD-4 and AHD-6 are encoded by distinct but closely linked loci on the mouse genome (Ahd-4 and Ahd-6), which segregate independently of Ahd-1 and Ahd-2. Liver mitochondrial isozymes, AHD-1 and AHD-5, have been purified to homogeneity using affinity chromatography. The very high affinity of AHD-5 for acetaldehyde suggests that this enzyme is predominantly responsible for acetaldehyde oxidation in mouse liver mitochondria.