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Biomedical subjects

I D Hickson

Publications and source records attributed to I D Hickson.

At least 55 records · Page 3Linked to original sources
PubMed1999

Nuclear localization of human AP endonuclease 1 (HAP1/Ref-1) associates with prognosis in early operable non-small cell lung cancer (NSCLC).

The present study examined the immunohistochemical expression of human AP endonuclease 1 (HAP1/Ref-1), the major endonuclease in the repair of apurinic/apyrimidinic (AP) sites in cellular DNA, in normal lung and lung carcinomas. Cellular expression of HAP1 was determined using a standard avidin-biotin-peroxidase complex (ABC) technique and an anti-HAP1 rabbit polyclonal antibody on paraffin-embedded tissue sections from normal lung and in 103 primary non-small cell lung carcinomas (NSCLCs). In normal lung, the staining for HAP1 was found to be both nuclear and cytoplasmic in the pneumocytes of the alveoli. Superficial ciliated cells of the bronchial epithelium presented cytoplasmic staining, while staining for the basal cells was mostly nuclear. Bronchial glandular cells demonstrated mixed nuclear and cytoplasmic staining. Lung carcinomas showed all patterns of expression for HAP1. Loss of HAP1 expression was associated with low proliferation index (p=0.01) and with squamous histology (p=0.04). In squamous carcinomas, a significant correlation was observed between positive nuclear HAP1 and negative p53 expression (p=0.03). A survival benefit was seen in patients presenting nuclear HAP1 expression and those presenting the nuclear HAP1+/p53- phenotype (p=0.01 and 0.007, respectively). It is concluded that nuclear HAP1 localization may be relevant to its role as a DNA repair protein and/or to the recently proposed role as an activator of wild-type p53, and thus to the better outcome seen in this group of patients.

Aged↗
PubMed1999

Defending genome integrity during DNA replication: a proposed role for RecQ family helicases.

The RecQ family of DNA helicases have been shown to be important for the maintenance of genomic integrity in all organisms analysed to date. In human cells, representatives of this family include the proteins defective in the cancer predisposition disorder Bloom's syndrome and the premature ageing condition, Werner's syndrome. Several pieces of evidence suggest that RecQ family helicases form associations with one or more of the cellular topoisomerases, and together these heteromeric complexes manipulate DNA structure to effect efficient DNA replication, genetic recombination, or both. Here, we propose that RecQ helicases are required for ensuring that structural abnormalities arising during replication, such as at sites where replication forks encounter DNA lesions, are corrected with high fidelity. In mutants defective in these proteins, not only is replication abnormal, but cells display aberrant responses to DNA-damaging agents or inhibitors of DNA synthesis. We suggest that RecQ helicases may be important for the integration of cellular responses to these insults, such as by linking cell cycle checkpoint responses to recombinational repair.

Adenosine Triphosphatases↗
PubMed1999

The topoisomerase II-associated protein, Pat1p, is required for maintenance of rDNA locus stability in Saccharomyces cerevisiae.

The Pat1 protein of Saccharomyces cerevisiae was identified during a screen for proteins that interact with topoisomerase II. Previously, we have shown that pat1 delta mutants exhibit a slow-growth phenotype and an elevated frequency of both mitotic and meiotic chromosome mis-segregation. Here, we have studied the effects of deleting the PAT1 gene on chromosomal stability, with particular reference to rates of homologous recombination within the rDNA locus. This locus was analyzed because rDNA-specific hyperrecombination is known to occur in conditional top2 mutants. We show that pat1 delta strains mimic top2 mutants in displaying an elevated rate of intrachromosomal excision recombination at the rDNA locus, but not elsewhere in the genome. The elevated rate of recombination is dependent upon Rad52p, but not upon Rad51p or Rad54p. However, pat1 delta strains display additional manifestations of more general genomic instability, in that they show mild sensitivity to UV light and an increased incidence of interchromosomal recombination between heteroalleles.

Alleles↗
PubMed1999

Genetic disorders associated with cancer predisposition and genomic instability.

Genomic instability in its broadest sense is a feature of virtually all neoplastic cells. In addition to the mutations and/or gene amplifications that appear to be a prerequisite for the acquisition of a neoplastic phenotype, human cancers exhibit other "markers" of genomic instability--in particular, a high degree of aneuploidy. Indeed, many studies have shown that aneuploidy is an almost invariant feature of cancer cells, and it has been argued by some that the emergence of aneuploid cells is a necessary step during tumorigenesis. The functional link between genomic instability and cancer is strengthened by the existence of several "genetic instability" disorders of humans that are associated with a moderate to severe increase in the incidence of cancers. These disorders include ataxia telangiectasia, Bloom's syndrome, Fanconi anemia, xeroderma pigmentosum, and Nijmegen breakage syndrome, all of which are very rare and are inherited in a recessive manner. Analysis of the cells from such cancer-prone individuals is clearly a potentially fruitful approach for delineating the genetic basis for instability in the genome. It is assumed that by identifying the underlying cause of genetic instability in these disorders, one can derive valuable information not only about the basis of particular genetic diseases, but also about the underlying causes of genomic instability in sporadic cancers in the general population. In this article, we review the clinical and cellular properties of genetic instability disorders associated with cancer predisposition. In particular, we focus on the rapid advances made in our understanding of these disorders that have derived from the cloning of the genes mutated in each case. Because in many instances the affected genes have analogs in lower eukaryotic species, we shall discuss how studies in yeasts in particular have proved valuable in our understanding of human diseases and predisposition to cancer.

Animals↗
PubMed1999

Expression levels of the DNA repair enzyme HAP1 do not correlate with the radiosensitivities of human or HAP1-transfected rat cell lines.

Apurinic/apyrimidinic (AP) sites in DNA are potentially lethal and mutagenic. They can arise spontaneously or following DNA damage from reactive oxygen species or alkylating agents, and they constitute a significant product of DNA damage following cellular exposure to ionizing radiation. The major AP endonuclease responsible for initiating the repair of these and other DNA lesions in human cells is HAP1, which also possesses a redox function. We have determined the cellular levels of this enzyme in 11 human tumour and fibroblast cell lines in relation to clonogenic survival following ionizing radiation. Cellular HAP1 levels and surviving fraction at 2 Gy (SF2) varied five- and tenfold respectively. However, no correlation was found between these two parameters following exposure to gamma-irradiation at low (1.1 cGy per min) or high (108 cGy per min) dose rates. To examine this further, wild-type and mutant versions of HAP1 were overexpressed, using an inducible HAP1 cDNA expression vector system, in the rat C6 glioma cell line which has low endogenous AP endonuclease activity. Induction of wild-type HAP1 expression caused a > fivefold increase in the capacity of cellular extracts to cleave an oligonucleotide substrate containing a single abasic site, but increased expression did not confer increased resistance to gamma-irradiation at high- or low-dose rates, or to the methylating agent methyl methanesulphonate (MMS). Expression in C6 cell lines of mutant forms of HAP1 deleted for either the redox activator or DNA repair functions displayed no apparent titrational or dominant negative effects. These studies suggest that the levels of endogenous AP endonuclease activities in the various cell lines examined are not limiting for efficient repair in cells following exposure to ionizing radiation or MMS. This contrasts with the correlation we have found between HAP1 levels and radiosensitivity in cervix carcinomas (Herring et al (1998) Br J Cancer 78: 1128-1133), indicating that HAP1 levels in this case assume a critical survival role and hence that established cell lines might not be a suitable model for such studies.

Animals↗
PubMed1999

Altered drug interaction and regulation of topoisomerase IIbeta: potential mechanisms governing sensitivity of HL-60 cells to amsacrine and etoposide.

Topoisomerase II (topo II), an enzyme essential for cell viability, is present in mammalian cells as the alpha- and beta-isoforms. In human leukemia HL-60/S or HL-60/doxorubicin (DOX)0.05 cells, the levels of topo IIalpha- or beta-protein were similar in either asynchronous exponential or synchronized cultures. Although topo IIalpha was hypophosphorylated in HL-60/DOX0.05 compared with HL-60/S cells, both overall and site-specific hyperphosphorylation of topo IIbeta was apparent in HL-60/DOX0.05 compared with HL-60/S cells. The phosphorylation of topo IIalpha and not beta was enhanced in the S and G(2) + M phases of HL-60/S cells. In contrast, an increase in the phosphorylation of topo IIbeta compared with alpha was apparent in the G(1) and S phases of HL-60/DOX0.05 cells. The cytotoxicity and depletion of topo IIalpha or beta in cells treated with drug for 1 h revealed that mole-for-mole, amsacrine was 2-fold more effective than etoposide in killing HL-60/S or HL-60/DOX0.05 cells and in depleting the beta versus alpha topo II protein. Present results demonstrate that: 1) hyperphosphorylation of topo IIbeta in HL-60/DOX0.05 cells may be a compensatory consequence of the hypophosphorylation of topo IIalpha to maintain normal topo II function during proliferation, and 2) enhanced sensitivity of HL-60/S or HL-60/DOX0.05 cells to amsacrine may be due to the preferential interaction and depletion of topo IIbeta.

Amsacrine↗
PubMed1998

Attenuation of drug-stimulated topoisomerase II-DNA cleavable complex formation in wild-type HL-60 cells treated with an intracellular calcium buffer is correlated with decreased cytotoxicity and site-specific hypophosphorylation of topoisomerase IIalpha.

Topoisomerase II (topo II), an essential enzyme for cell viability, is also the target for clinically important anti-neoplastic agents that stimulate topo II-mediated DNA scission. The role of alterations in topo IIalpha phosphorylation and its effect on drug-induced DNA damage and cytotoxicity were investigated. Following loading of HL-60 cells with the calcium buffer 1, 2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetra(acetoxymethyl) ester (BAPTA-AM), which abrogates intracellular Ca2+ transients, a significant decrease in etoposide (VP-16)- or amsacrine (m-AMSA)-stabilized topo II-DNA cleavable complex formation and a corresponding decrease in cytotoxicity was observed. In a cell-free system, nuclear extracts from BAPTA-AM-treated cells exhibited markedly less activity when assayed for VP-16-stabilized topo II-DNA complex formation, but not decatenation of kinetoplast DNA. In contrast, the loading of HL-60 cells with N,N,N', N'-tetrakis-(2-pyridyl)ethylenediamine (TPEN), which binds heavy metals without disturbing calcium or magnesium concentrations, did not significantly affect VP-16-stimulated topo II-DNA cleavable complex formation or cytotoxicity. In HL-60 cells the accumulation of BAPTA, but not TPEN, also led to the hypophosphorylation of topo IIalpha. Tryptic phosphopeptide mapping of topo IIalpha protein from HL-60 cells revealed: (a) eight major phosphorylation sites in untreated cells; (b) hypophosphorylation of two out of eight sites in BAPTA-AM-treated cells; and (c) hypophosphorylation of between two and four out of eight sites in topo II-poison-resistant HL-60 cells. The two hypophosphorylated sites present following BAPTA-AM treatment of wild-type cells were identical with the hypophosphorylated sites in the resistant cells, but were not the same as the sites that are substrates for casein kinase II [Wells, Addison, Fry, Ganapathi and Hickson (1994) J. Biol. Chem. 269, 29746-29751]. In summary, changes in intracellular Ca2+ transients that lead to the site-specific hypophosphorylation of topo IIalpha are possibly involved in regulating the DNA damage caused by and the cytotoxic potential of topo II poisons.

Antigens, Neoplasm↗
PubMed1998

The Bloom's syndrome helicase unwinds G4 DNA.

BLM, the gene that is defective in Bloom's syndrome, encodes a protein homologous to RecQ subfamily helicases that functions as a 3'-5' DNA helicase in vitro. We now report that the BLM helicase can unwind G4 DNA. The BLM G4 DNA unwinding activity is ATP-dependent and requires a short 3' region of single-stranded DNA. Strikingly, G4 DNA is a preferred substrate of the BLM helicase, as measured both by efficiency of unwinding and by competition. These results suggest that G4 DNA may be a natural substrate of BLM in vivo and that the failure to unwind G4 DNA may cause the genomic instability and increased frequency of sister chromatid exchange characteristic of Bloom's syndrome.

Adenosine Triphosphatases↗
PubMed1998

Altered expression and activity of topoisomerases during all-trans retinoic acid-induced differentiation of HL-60 cells.

Regulation of topoisomerase II (TOPO II) isozymes alpha and beta is influenced by the growth and transformation state of cells. Using HL-60 cells induced to differentiate by all-trans retinoic acid (RA), we have investigated the expression and regulation of TOPO II isozymes as well as the levels of topoisomerase I (TOPO I). During RA-induced differentiation of human leukemia HL-60 cells, levels of TOPO I remained unchanged, whereas the levels and phosphorylation of TOPO IIalpha and TOPO IIbeta proteins were increased twofold to fourfold and fourfold to eightfold, respectively. The elevation of TOPO II (alpha and beta) protein levels and phosphorylation was apparent at 48 hours of treatment with RA and persisted through 96 hours. The increased level of TOPO IIbeta protein was also detected in differentiated cells subsequently cultured for 96 hours in RA-free medium. Pulse chase experiments in cells labeled with 35S-methionine showed that the rate of degradation of TOPO IIbeta protein in control cells was about twofold faster than that in the differentiated RA-treated cells. The level of decatenation activity of kDNA was comparable in nuclear extracts from control or RA-treated cells. Whereas etoposide (1 to 10 micromol/L) -induced DNA cleavage was not significantly different, apoptosis was significantly lower (P = .012) in RA-treated versus control cells after exposure to 10 micromol/L etoposide. Consistent with unaltered levels of TOPO I, camptothecin (CPT) -induced DNA cleavage was similar in control or RA-treated cells. However, apoptosis after exposure to 1 to 10 micromol/L CPT was significantly lower (P = .003 to P < .001) in RA-treated versus control cells. Results suggest that TOPO IIbeta protein levels are posttranscriptionally regulated and that degradation of TOPO IIbeta is decreased during RA-induced differentiation. Furthermore, whereas the total level of TOPO II (alpha + beta) is increased with RA, the level of TOPO II catalytic activity and etoposide-stabilized DNA cleavage activity remains unaltered. Thus, TOPO IIbeta may have a specific role in transcription of genes involved in differentiation with RA treatment.

Cell Differentiation↗
PubMed1998

Physiological regulation of eukaryotic topoisomerase II.

Topoisomerase II is an essential enzyme in all organisms with several independent roles in DNA metabolism. In this article we review our knowledge on the regulation of the expression and catalytic activity of topoisomerase II in both lower and higher eukaryotes. Current data indicate that the regulation of topoisomerase II gene expression is complex, with positive and negative controls in evidence at the level of both promoter activity and mRNA stability. Similarly, the activity of the mature enzyme can be regulated by the action of several different protein kinases. Of particular interest is the cell cycle-dependent phosphorylation of topoisomerase II, including multiple, mitosis-specific modifications, which are proposed to regulate the essential chromosome decatenation activity of the enzyme.

Animals↗
PubMed1998

Casein kinase II stabilizes the activity of human topoisomerase IIalpha in a phosphorylation-independent manner.

Previous reports have indicated that topoisomerase II (topo II) co-purifies with and is a substrate for casein kinase II. We have carried out a detailed study of the effect that purified casein kinase II has on the activity of purified recombinant human topo IIalpha. Co-incubation of topo IIalpha and casein kinase II led to an apparent activation of the topo IIalpha; however, in experiments in which topo IIalpha was preincubated at 37 degrees C with or without native casein kinase II prior to assaying for decatenation activity, it emerged that the kinase was exerting its "activating" function via a decrease in the rate of topo IIalpha enzyme inactivation during the incubation period. This stabilization of topo IIalpha by casein kinase II was ATP-independent and was observed in both mutated and truncated derivatives of topo IIalpha lacking the major casein kinase II phospho-acceptor sites, indicating the lack of a requirement for phosphorylation. Consistent with a nonenzymatic role for casein kinase II, stoichiometric quantities of kinase were required for topo IIalpha stabilization. These data indicate that casein kinase II plays a significant role in regulating human topo IIalpha protein action via stabilization against thermal inactivation.

Antigens, Neoplasm↗
PubMed1998

Human AP endonuclease 1 (HAP1) protein expression in breast cancer correlates with lymph node status and angiogenesis.

Human AP endonuclease (HAP1) plays a major role in the repair of apurinic/apyrimidinic (AP) sites in cellular DNA. We used immunohistochemistry to examine the expression of HAP1 in normal breast and in 102 primary breast carcinomas. In normal breast epithelium, HAP1 had a uniformly nuclear localization. However, in lactating glandular epithelium, the expression of HAP1 was predominantly cytoplasmic. In carcinomas, both nuclear and cytoplasmic (44%), cytoplasmic (28%) or nuclear staining (24%) were observed. In four cases (4%), no HAP1 expression was detected. All patterns of expression for HAP1 were demonstrated for ductal carcinomas in situ (DCIS), although comedo-type DCIS were usually accompanied by mostly cytoplasmic staining. Similarly, the HAP1 expression in regions of invasive tumour necrosis was cytoplasmic. Pure nuclear HAP1 expression was significantly correlated with low angiogenesis (P = 0.007) and negative lymph node status (P = 0.001). In contrast, cases with cytoplasmic as well as nuclear staining were associated with poor prognostic factors, such as high angiogenesis (P = 0.03) and node positivity (P = 0.03). The pure nuclear staining may be related to better differentiation, as in normal breast, and hence better prognostic features, and cytoplasmic staining to a more metabolically active phenotype with high protein synthesis, as in lactating breast.

Adult↗
PubMed1998

Mapping of topoisomerase II alpha epitopes recognized by autoantibodies in idiopathic pulmonary fibrosis.

Autoantibodies against DNA topoisomerase II alpha have been identified in the sera of patients with idiopathic pulmonary fibrosis (IPF). To map topoisomerase II autoepitopes, we tested by ELISA and immunoblotting the IPF anti-topoisomerase II-positive sera against a series of recombinant proteins which covered the full length of topoisomerase II alpha. Specific patterns of reactivity were observed, indicating the existence of multiple epitopes on topoisomerase II, either highly complex or conformational/discontiguous or conformational/contiguous ones. The latter resided in amino acid residues 854-1147 and 1370-1447. A detailed analysis of these regions was undertaken, but we were not able to pinpoint a sequential peptide-sized epitope, or any significant homology with foreign pathogens. Further, we observed a significant correlation between the progression from a contiguous to a quaternary/tertiary structure-dependent autoepitope and the disease duration but not with the disease severity. Therefore, this result supports the hypothesis that anti-topoisomerase II autoreactivity evolves following an antigen-driven process.

Adult↗
PubMed1998

Expression and subcellular localization of human AP endonuclease 1 (HAP1/Ref-1) protein: a basis for its role in human disease.

AIMS: Human AP endonuclease 1 (HAP1) plays a major role in the repair of apurinic/apyrimidinic (AP) sites in cellular DNA by catalysing hydrolytic cleavage of the phosphodiester backbone 5' to the site. HAP1 is also known to be a potent reduction-oxidation (redox) factor, regulating the binding activity of a number of transcription factors. The purpose of the present study was to examine the expression of HAP-1 in a wide range of human tissues. METHODS AND RESULTS: Using a recently developed specific rabbit polyclonal antibody, we performed immunohistochemistry on paraffin-embedded tissue material. Nuclear staining was detected in crypt cells of the small and large intestine, epithelial cells of breast ducts, basal cells of the skin, alveolar cells of the lung, lymphocytes of the marginal zone of the spleen, in the surface epithelium and stromal cells of the ovary and the transitional epithelium of the bladder. Unexpectedly for a presumed nuclear protein, the staining pattern in some cell populations was mainly cytoplasmic (e.g. superficial cells of gastrointestinal tract, Langerhans cells, Leydig cells and spermatocytes, epithelium of the prostate glands), or both cytoplasmic and nuclear (e.g. epithelial cells of thymus, follicular thyroid cells, parietal cells of the stomach, glandular epithelial cells of the cervix, epithelial cells of exocrine pancreas). CONCLUSION: This differential expression in a wide spectrum of cells is indicative of a potential multifunctional action of HAP1, not necessarily restricted to a role in the nucleus.

Antibodies↗
PubMed1997

Evidence for a common active site for cleavage of an AP site and the benzene-derived exocyclic adduct, 3,N4-benzetheno-dC, in the major human AP endonuclease.

We have previously reported that the 3,N4-benzetheno-dC (p-BQ-dC) endonuclease activity found in HeLa cells is a novel function of the major human AP endonuclease (HAP1) [Hang et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 13737-13741]. In this study, we compare the enzymatic and biochemical properties of the enzyme toward p-BQ-dC and an AP site in a defined oligonucleotide. A comparative analysis of the specificity constants (Kcat./Km) for p-BQ-dC and an AP site indicates that the AP site is the preferred substrate. The enzyme does not cleave other structurally related exocyclic adducts and modified nucleosides such as 1,N6-etheno-dA, 3,N4-etheno-dC, 1, N2-etheno-dG, 1,N2-propano-dG, 8-oxo-dG, and thymine glycol. The p-BQ-dC activity requires a double-stranded DNA substrate and is affected by the base in the opposite strand, with maximal activity for a p-BQ-dC.G pair and minimal activity for a p-BQ-dC.C pair. The p-BQ-dC activity also requires Mg2+, Mn2+, or Zn2+ with optimal concentration spectra similar to those for the AP function. The optimal pH ranges for these two functions are also similar to each other (5.5-6.5). Six mutant HAP1 proteins containing single amino acid substitutions were assayed in parallel for comparison of their activities toward p-BQ-dC and the AP site. These mutants either concomitantly lost (N212A, D210N) or had reduced (D219A, E96A, and N212Q) or unchanged (H116N) p-BQ-dC and AP activities. This parallelism strongly supports the hypothesis that cleavage of p-BQ-dC requires the same catalytic active site as that proposed for the AP function. This dual activity toward two structurally unrelated substrates, an AP site and a bulky exocyclic adduct, has implications for substrate recognition. The AP site and p-BQ-dC cause different changes in the local conformation around the lesion as it is visualized by molecular modeling.

Binding Sites↗
PubMed1997

The Bloom's syndrome gene product is a 3'-5' DNA helicase.

Bloom's syndrome (BS) is an autosomal recessive condition characterized by short stature, immunodeficiency, and a greatly elevated frequency of many types of cancer. The gene mutated in BS, BLM, encodes a protein containing seven "signature" motifs conserved in a wide range of DNA and RNA helicases. BLM is most closely related to the subfamily of DEXH box-containing DNA helicases of which the prototypical member is Escherichia coli RecQ. To analyze its biochemical properties, we have overexpressed an oligohistidine-tagged version of the BLM gene product in Saccharomyces cerevisiae and purified the protein to apparent homogeneity using nickel chelate affinity chromatography. The recombinant BLM protein possesses an ATPase activity that is strongly stimulated by either single- or double-stranded DNA. Moreover, BLM exhibits ATP- and Mg2+-dependent DNA helicase activity that displays 3'-5' directionality. Because many of the mutations in BS individuals are predicted to truncate the BLM protein and thus eliminate the "helicase" motifs or map to conserved positions within these motifs, our data strongly suggest that these mutations will disable the 3'-5' helicase function of the BLM protein.

Adenosine Triphosphatases↗
PubMed1997

The crystal structure of the human DNA repair endonuclease HAP1 suggests the recognition of extra-helical deoxyribose at DNA abasic sites.

The structure of the major human apurinic/ apyrimidinic endonuclease (HAP1) has been solved at 2.2 A resolution. The enzyme consists of two symmetrically related domains of similar topology and has significant structural similarity to both bovine DNase I and its Escherichia coli homologue exonuclease III (EXOIII). A structural comparison of these enzymes reveals three loop regions specific to HAP1 and EXOIII. These loop regions apparently act in DNA abasic site (AP) recognition and cleavage since DNase I, which lacks these loops, correspondingly lacks AP site specificity. The HAP1 structure furthermore suggests a mechanism for AP site binding which involves the recognition of the deoxyribose moiety in an extrahelical conformation, rather than a 'flipped-out' base opposite the AP site.

Amino Acid Sequence↗