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Biomedical subjects

I D Hickson

Publications and source records attributed to I D Hickson.

At least 73 records · Page 4Linked to original sources
PubMed1997

Human apurinic endonuclease 1 expression in a colorectal adenoma-carcinoma sequence.

Human apurinic endonuclease 1 (HAP1) plays a key role in the repair of baseless sites in DNA. HAP1 is also known to be a potent regulator of the binding activity of a number of transcription factors. We have examined the immunohistochemical expression of the HAP1 protein in normal colorectal mucosa, hyperplastic polyps, tubulovillous adenomas, and carcinomas. In normal colonic mucosa, the predominant staining was nuclear in the less differentiated cells located at the lower part of the crypt, but it was cytoplasmic in the more differentiated superficial colonic epithelium. HAP1 expression was nuclear in 3 of 30 adenomas (10%) and 5 of 44 carcinomas (11%), but it was cytoplasmic in 11 of 30 adenomas (37%) and 22 of 44 carcinomas (50%) and both nuclear and cytoplasmic in 16 of 30 adenomas (53%) and 17 of 44 carcinomas (39%). The observed staining in stromal fibroblasts and endothelial cells was nuclear, whereas that in macrophages was cytoplasmic. Our data indicate that HAP1 is expressed in different subcellular compartments during normal differentiation and that this pattern is disrupted in adenomas and carcinomas. The differential localization may be relevant to the two different proposed functions of HAP1.

Adenocarcinoma↗
PubMed1997

Cell cycle-coupled relocation of types I and II topoisomerases and modulation of catalytic enzyme activities.

We visualized DNA topoisomerases in A431 cells and isolated chromosomes by isoenzyme-selective immunofluorescence microscopy. In interphase, topoisomerase I mainly had a homogeneous nuclear distribution. 10-15% of the cells exhibited granular patterns, 30% showed bright intranucleolar patches. Topoisomerase II isoenzymes showed spotted (alpha) or reticular (beta) nuclear patterns throughout interphase. In contrast to topoisomerase IIalpha, topoisomerase IIbeta was completely excluded from nucleoli. In mitosis, topoisomerase IIbeta diffused completely into the cytosol, whereas topoisomerases I and IIalpha remained chromosome bound. Chromosomal staining of topoisomerase I was homogeneous, whereas topoisomerase IIalpha accumulated in the long axes of the chromosome arms and in the centriols. Topoisomerase antigens were 2-3-fold higher in mitosis than in interphase, but specific activities of topoisomerase I and II were reduced 5- and 2.4-fold, respectively. These changes were associated with mitotic enzyme hyperphosphorylation. In interphase, topoisomerases could be completely linked to DNA by etoposide or camptothecin, whereas in mitosis, 50% of topoisomerase IIalpha escaped poisoning. Refractoriness to etoposide could be assigned to the salt-stable scaffold fraction of topoisomerase IIalpha, which increased from <2% in G1 phase to 48% in mitosis. Topoisomerases I and IIbeta remained completely extractable throughout the cell cycle. In summary, expression of topoisomerases increases towards mitosis, but specific activities decrease. Topoisomerase IIbeta is released from the heterochromatin, whereas topoisomerase I and IIalpha remain chromosome bound. Scaffold-associated topoisomerase IIalpha appears not to be involved in catalytic DNA turnover, though it may play a role in the replicational cycle of centriols, where it accumulates during M phase.

Antibody Specificity↗
PubMed1997

Cloning and characterization of a functional human homolog of Escherichia coli endonuclease III.

Repair of oxidative damage to DNA bases is essential to prevent mutations and cell death. Endonuclease III is the major DNA glycosylase activity in Escherichia coli that catalyzes the excision of pyrimidines damaged by ring opening or ring saturation, and it also possesses an associated lyase activity that incises the DNA backbone adjacent to apurinic/apyrimidinic sites. During analysis of the area adjacent to the human tuberous sclerosis gene (TSC2) in chromosome region 16p13.3, we identified a gene, OCTS3, that encodes a 1-kb transcript. Analysis of OCTS3 cDNA clones revealed an open reading frame encoding a predicted protein of 34.3 kDa that shares extensive sequence similarity with E. coli endonuclease III and a related enzyme from Schizosaccharomyces pombe, including a conserved active site region and an iron/sulfur domain. The product of the OCTS3 gene was therefore designated hNTH1 (human endonuclease III homolog 1). The hNTH1 protein was overexpressed in E. coli and purified to apparent homogeneity. The recombinant protein had spectral properties indicative of the presence of an iron/sulfur cluster, and exhibited DNA glycosylase activity on double-stranded polydeoxyribonucleotides containing urea and thymine glycol residues, as well as an apurinic/apyrimidinic lyase activity. Our data indicate that hNTH1 is a structural and functional homolog of E. coli endonuclease III, and that this class of enzymes, for repair of oxidatively damaged pyrimidines in DNA, is highly conserved in evolution from microorganisms to human cells.

Adenocarcinoma↗
PubMed1997

Response to ICRF-159 in cell lines resistant to cleavable complex-forming topoisomerase II inhibitors.

We have studied the relationship between expression of genes implicated in mediating resistance to cleavable complex-forming topoisomerase II (topo II) inhibitors and cellular sensitivity to ICRF-159, a 'catalytic' inhibitor of topo II. Overexpression of the membrane transporters, P-glycoprotein and multidrug resistance-related protein (MRP), or down-regulation of topo IIalpha and/or -beta, did not confer ICRF-159 resistance. Indeed, marked topo IIalpha down-regulation appeared to be associated with collateral sensitivity to ICRF-159. Our results indicate that the resistance mechanisms that pertain to cleavable complex-forming topo II inhibitors and ICRF-159 are distinct. The evidence presented here suggests that topo IIalpha, not topo IIbeta, is more likely to be the major in vivo target for ICRF-159.

Antineoplastic Agents↗
PubMed1997

The distribution and expression of the two isoforms of DNA topoisomerase II in normal and neoplastic human tissues.

In mammalian cells, there are two isoforms of DNA topoisomerase II, designated alpha (170-kDa form) and beta (180-kDa form). Previous studies using cell lines have shown that the topoisomerase IIalpha and beta isoforms are differentially regulated during the cell cycle and in response to changes in growth state. Moreover, both isoforms can act as targets for a range of anti-tumour drugs. Here, we have analysed the normal tissue distribution in humans of topoisomerase IIalpha and beta using isoform-specific antibodies. In addition, we have studied expression of these isoforms in 69 primary tumour biopsies, representative either of tumours that are responsive to topoisomerase II-targeting drugs (breast, lung, lymphoma and seminoma) or of those that show de novo drug resistance (colon). Topoisomerase IIalpha was expressed exclusively in the proliferating compartments of all normal tissues, and was detectable in both the cell nucleus and cytoplasm. In biologically aggressive or rapidly proliferating tumours (e.g. high-grade lymphomas and seminomas), there was a high level of topoisomerase IIalpha, although expression was still detectable in colon tumours, indicating that expression of this isoform is not sufficient to explain the intrinsic drug resistance of colon tumours. Topoisomerase IIbeta was expressed ubiquitously in vivo and was localized in both the nucleoli and the nucleoplasm. This isoform was present in quiescent cell populations, but was expressed at a generally higher level in all tumours and proliferating cells than in normal quiescent tissues. We conclude that topoisomerase IIalpha is a strict proliferation marker in normal and neoplastic cells in vivo, but that topoisomerase IIbeta has a much more general cell and tissue distribution than has topoisomerase IIalpha. The apparent up-regulation of topoisomerase IIbeta in neoplastic cells has implications for the response of patients to anti-tumour therapies that include topoisomerase II-targeting drugs.

Antigens, Neoplasm↗
PubMed1997

The structure and functions of the HAP1/Ref-1 protein.

The HAP1/Ref-1 (hereafter referred to as HAP1) protein is a nuclear enzyme that apparently performs two distinct roles in the cellular defense against oxidative stress. One well-established role is in the repair of a variety of lesions induced in DNA either by spontaneous hydrolysis or by reactive oxygen species (ROS). This function has been characterized in great detail and the roles played by individual active site amino acid residues have been defined. The second role, which was identified only relatively recently and is still not characterized in detail, is to regulate the DNA binding activity of a group of nuclear factors. This second role proceeds via the modification of the oxidation/reduction (redox) state of a cysteine residue in the target protein, although the mechanism by which this is achieved remains to be elucidated. In this article, we shall review the latest knowledge on the relationship between structure and the dual functions of HAP1, and we will seek to explain in detail the roles played by several individual amino acid residues in the catalytic function of the HAP1 protein.

Animals↗
PubMed1996

Pat1: a topoisomerase II-associated protein required for faithful chromosome transmission in Saccharomyces cerevisiae.

Saccharomyces cerevisiae top2 mutants deficient in topoisomerase II activity are defective in chromosome segregation during both mitotic and meiotic cell divisions. To identify proteins that act in concert with topoisomerase II during chromosome segregation in S.cerevisiae, we have used a two-hybrid cloning approach. We report the isolation of the PAT1 gene (for protein associated with topoisomerase II), which encodes a novel 90 kDa proline- and glutamine-rich protein that interacts with a highly conserved, leucine-rich region of topoisomerase II in vivo. Strains lacking Pat1p exhibit a slow growth rate and a phenotype reminiscent of conditional top2 mutants grown at the semi-permissive temperature; most notably, a reduced fidelity of chromosome segregation during both mitosis and meiosis. These findings indicate that the PAT1 gene is necessary for accurate chromosome transmission during cell division in eukaryotic cells and suggest that the interaction of Pat1p and topoisomerase II is an important component of this function.

Binding Sites↗
PubMed1996

p53 regulates the minimal promoter of the human topoisomerase IIalpha gene.

DNA topoisomerase IIalpha is an essential enzyme for chromosome segregation during mitosis. Consistent with a cell division-specific role, the expression of the topoisomerase IIalpha gene is strongly influenced by the proliferation status of cells. The p53 protein is one of the most important regulators of cell cycle progression in mammals, with an apparent dual role in the induction of cell cycle arrest following cytotoxic insults and in the regulation of the apoptotic cell death pathway. We have analysed whether p53 plays a role in regulating expression of the human topoisomerase IIalpha gene. We show that wild-type, but not mutant, p53 is able to decrease substantially the activity of the full length topoisomerase IIalpha gene promoter. Using a series of constructs comprising various deleted or mutated versions of the promoter lacking critical cis-acting elements, we show that this p53-specific regulation of the topoisomerase IIalpha promoter is independent of all characterised transcription factor binding sites and is directed at the minimal gene promoter. We conclude that expression of wild-type p53 induces downregulation of the human topoisomerase IIalpha promoter by acting on the basal transcription machinery. These findings implicate topoisomerase II as one of the downstream targets for p53-dependent regulation of cell cycle progression in human cells.

Antigens, Neoplasm↗
PubMed1996

Asparagine 212 is essential for abasic site recognition by the human DNA repair endonuclease HAP1.

HAP1 is a divalent cation-dependent endonuclease from human cells with specificity for apurinic/apyrimidinic (AP) sites in DNA. Extraction of the essential metal ion from purified HAP1 stabilized its binding to an oligonucleotide containing a single AP site, permitting AP site binding studies to be undertaken using gel retardation assays. Binding of HAP1 to such an oligonucleotide was dependent upon the presence of an AP site. Previous structural and modelling studies have suggested a role for Asn212 (Asn153 in exonuclease III, the bacterial homologue of HAP1) in substrate recognition. Substitution of alanine for Asn212 abolished the AP endonuclease activity of purified recombinant HAP1 protein. More conservative substitutions of aspartate or glutamine for Asn212 still led to a reduction in specific activity of at least 300-fold. Moreover, none of the three Asn212 substitution mutants of HAP1 possessed detectable AP site binding activity in vitro. This study indicates that chelation of the active site metal ion in HAP1 stabilizes the complex of the protein with AP sites and identifies an active site asparagine residue as an important component of AP site recognition by the HAP1 protein.

Asparagine↗
PubMed1996

Phorbol ester-induced down-regulation of topoisomerase II alpha mRNA in a human erythroleukemia cell line. Evidence for a post-transcriptional mechanism.

Tumor-promoting phorbol esters such as phorbol 12-myristate 13-acetate (PMA) are reported to induce megakaryocyte terminal differentiation of the erythroleukemia cell line K562. This differentiation is accompanied by the regulation of various gene products such as gamma-globin (Lumelsky and Forget, Mol Cell Biol 11: 3528-3536, 1991) and platelet-derived growth factor-beta (PDGF-beta) (Mäkelä et al., Mol Cell Biol 7: 3656-3662, 1987). PMA has also been found to regulate topoisomerase (topo) II alpha in other myeloid leukemia lines. The purpose of this study was to investigate whether PMA regulates topo II alpha in K562 cells and, if so, to identify the mechanisms responsible for this regulation. Northern blot analysis revealed that topo II alpha mRNA is down-regulated as is gamma-globin. This activity was not due to a generalized decrease in mRNA, as PDGF-beta message actually increased in response to PMA treatment. RNase protection assays confirmed the decline in the topo II alpha message. Transfection experiments with various topo II promoter CAT constructs extending to 2200 bp upstream of the ATG start site revealed regions that enhance and regions that inhibit CAT expression in the absence of PMA However, PMA did not affect this CAT expression. Run-on experiments using 5' and 3' human topo II cDNA probes confirmed that transcriptional initiation of the topo II gene was not affected by PMA, whereas that of c-myc did decrease. Therefore, the apparent decrease in topo II alpha mRNA in K562 cells upon their treatment with PMA appeared to be the result of a post-transcriptional mechanism.

Animals↗
PubMed1996

Human DNA topoisomerases II alpha and II beta can functionally substitute for yeast TOP2 in chromosome segregation and recombination.

The ability of the human DNA topoisomerase II alpha and II beta isozymes to complement functional defects conferred by conditional top2 mutations in Saccharomyces cerevisiae has been investigated. At the restrictive temperature, top2 strains show multiple abnormalities, including an inability to complete mitotic and meiotic division owing to a defect in chromosome segregation, and hyper-recombination within the repetitive rDNA gene cluster. We show that the human topoisomerases II alpha and II beta can each support both vegetative growth and the production of viable spores in a top2-4 mutant at the restrictive temperature. Similarly, both human isozymes can rescue a strain carrying a top2 gene disruption, and suppress hyper-recombination within the rDNA gene cluster. We conclude that the human topoisomerase II alpha and II beta isozymes are functionally interchangeable with yeast topoisomerase II and suggest that any isozyme-specific roles in human cells are likely to be dependent upon factors other than inherent differences in catalytic ability between the alpha and beta isozymes.

Antigens, Neoplasm↗
PubMed1996

Regulation of the human topoisomerase IIalpha gene promoter in confluence-arrested cells.

Expression of DNA topoisomerase IIalpha mRNA and protein reflects the proliferative state of mammalian cell lines and tissues with high levels in actively cycling cells but marked down-regulation during serum deprivation or cell density-induced growth arrest. Using stably integrated gene fusions comprising the human topoisomerase IIalpha promoter with a growth hormone reporter gene, we have localized elements required for the differential activity of the topoisomerase IIalpha promoter in proliferating and confluence-arrested cells. Deletion analysis localized the region of the promoter that responded to changes in the cellular growth state to between -101 and -144 base pairs. Mutation analysis identified an inverted CCAAT box (ICB) located at -108 to -104 as necessary for promoter down-regulation in confluence-arrested cells, while several other potential cis-acting elements, including four additional ICBs, were shown not to be required. The critical ICB was recognized in vitro by the CCAAT box binding factor, NF-Y, with levels of binding activity higher in extracts from proliferating cells than from confluence-arrested cells. We conclude that the differential regulation of topoisomerase IIalpha gene expression in cycling and confluence-arrested cells is mediated, at least in part, through proliferation-specific binding of factors to an ICB element in the gene promoter.

3T3 Cells↗
PubMed1996

Response to epirubicin in patients with superficial bladder cancer and expression of the topoisomerase II alpha and beta genes.

Biopsies of superficial bladder cancer were analysed to study the relationship between response to epirubicin and the expression of the human topoisomerase II alpha and beta genes. Tissue samples were obtained prior to treatment and a marker tumour was left in the bladder. Transcript levels of both genes were generally lower in biopsies taken following treatment failure. Levels of topoisomerase II mRNA were uniformly lower in tumour tissue than in biopsies of normal tissue.

Aged↗
PubMed1996

Differential expression of the topoisomerase II alpha and beta genes in human breast cancers.

Topoisomerase II is a key target for several anti-cancer drugs used for breast cancer therapy, including doxorubicin, epirubicin and mitoxantrone. Two isoforms of topoisomerase II (alpha and beta) have been described in human cells which differ in their subcellular localisation, biochemical properties and susceptibility to inhibition by anti-cancer drugs. The relative level of expression of the alpha and beta isoforms may contribute to the degree of tumour responsiveness to different chemotherapeutic agents. To assess the relationship between expression of topoisomerase II isoforms and established prognostic factors and pathological variables, 56 primary breast tumour samples were studied. The expression of the two topoisomerase II genes was apparently not co-ordinately regulated in these tissue samples. There was no relationship between any of the commonly used pathological variables [tumour size, lymph node status, S-phase fraction (SPF)] and the level of expression of topoisomerase II beta mRNA. However, high topoisomerase II alpha gene expression was significantly associated with a high SPF (sign-rank test; P = 0.01). Moreover, the ratio of mRNA levels for topoisomerase II alpha and beta showed a stronger relationship to SPF (median raito 0.62 for tumours with SPF < 10, and 1.64 for SPF > 10; P = 0.0021, sign-rank test). As expected from previous studies, an SPF > 10 was associated with poor overall survival (P = 0.01). Immunohistochemical analysis revealed that topoisomerase II beta was widely distributed ( > 90% positive tumour cells), but that topoisomerase II alpha expression was less widely expressed, with a pattern of expression similar to that of the proliferation-dependent antigen recognised by Ki67. Because topoisomerase II gene expression showed a log-normal distribution, log-transformed data were used in multivariate analysis of relapse-free survival. This showed that lymph node status and topoisomerase II beta mRNA expression were the only significant survival factors (P = 0.001 and 0.05, respectively, with relative risks of 1.3 and 1.8). These results indicate that topoisomerase II alpha, but not beta, expression is dependent upon cellular proliferation status, but that the more widely expressed topoisomerase II beta protein may play a significant role as a target for anti-tumour therapy.

Antigens, Neoplasm↗
PubMed1996

SGS1, a homologue of the Bloom's and Werner's syndrome genes, is required for maintenance of genome stability in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae SGS1 gene is homologous to Escherichia coli RecQ and the human BLM and WRN proteins that are defective in the cancer-prone disorder Bloom's syndrome and the premature aging disorder Werner's syndrome, respectively. While recQ mutants are deficient in conjugational recombination and DNA repair, Bloom's syndrome cell lines show hyperrecombination. Bloom's and Werner's syndrome cell lines both exhibit chromosomal instability, sgs1 delta strains show mitotic hyperrecombination, as do Bloom's cells. This was manifested as an increase in the frequency of interchromosomal homologous recombination, intrachromosomal excision recombination, and ectopic recombination. Hyperrecombination was partially independent of both RAD52 and RAD1. Meiotic recombination was not increased in sgs1 delta mutants, although meiosis I chromosome missegregation has been shown to be elevated sgs1 delta suppresses the slow growth of a top3 delta strain lacking topoisomerase III. Although there was an increase in subtelomeric Y' instability in sgs1 delta strains due to hyperrecombination, no evidence was found for an increase in the instability of terminal telomeric sequences in a top3 delta or a sgs1 delta strain. This contrasts with the telomere maintenance defects of Werner's patients. We conclude that the SGS1 gene product is involved in the maintenance of genome stability in S. cerevisiae.

Adenosine Triphosphatases↗
PubMed1995

Cell cycle phase-specific phosphorylation of human topoisomerase II alpha. Evidence of a role for protein kinase C.

Type II topoisomerases are essential for faithful cell division in all organisms. In human cells, the alpha isozyme of topoisomerase II has been implicated in catalyzing mitotic chromosome segregation via its action as a DNA unlinking enzyme. Here, we have shown that the enzymatic activity of topoisomerase II alpha protein purified from HeLa cell nuclei was strongly enhanced following phosphorylation by protein kinase C. We have investigated the possibility that this kinase is involved in cell cycle phase-specific phosphorylation of topoisomerase II alpha in HeLa cells. Two-dimensional tryptic phosphopeptide mapping revealed that topoisomerase II alpha protein immunoprecipitated from metabolically labeled HeLa cells was differentially phosphorylated during the G2/M phases of the cell cycle. To identify sites of phosphorylation, and the kinase(s) responsible for this modification, oligohistidine-tagged recombinant domains of topoisomerase II alpha protein were overexpressed in Escherichia coli and purified by affinity chromatography. Phosphorylation of a short fragment of the N-terminal ATPase domain of topoisomerase II alpha by protein kinase C in vitro generated two phosphopeptides that co-migrated with prominent G2/M phase-specific phosphopeptides from the HeLa cell-derived topoisomerase II alpha protein. Site-directed mutagenesis studies indicated that phosphorylation of serine 29 generated both of these phosphopeptides. Our results implicate protein kinase C in the cell cycle phase-dependent modulation of topoisomerase II alpha enzymatic activity in human cells.

Amino Acid Sequence↗