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C C Harris

Publications and source records attributed to C C Harris.

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

Mutational analysis of the p63/p73L/p51/p40/CUSP/KET gene in human cancer cell lines using intronic primers.

After the identification of p73, a second homologue of the human p53 tumor suppressor gene has been reported and named p63/p73L/p51/p40/CUSP/KET. We have investigated the hypotheses that: (a) p63 is mutated in diverse types of human cancers; and (b) p63 functions in the same pathway as p53 and p73 in the process of carcinogenesis; therefore, mutations in these three genes would be mutually exclusive. We have analyzed the genomic structure of the p63 gene and have performed mutational analyses on 54 human cell lines using intronic primers flanking each exon. We have confirmed that the human p63 open reading frame encodes the same length of protein as murine p63 that was initially reported to be 39 amino acids longer than human p63. By mutational analysis, we have shown that DLD1 and SKOV3 cells have either heterozygous mutations or polymorphisms in the putative DNA binding domain of p63. In these cell lines, p63 is biallelically expressed. We conclude that mutations in the p63 gene are rare in human cell lines. The fact that DLD1 is abnormal for both p63 and p53 genes suggests that they may not be involved in the same tumor suppressor pathway.

Amino Acid Sequence↗
PubMed1999

Drug-induced apoptosis is delayed and reduced in XPD lymphoblastoid cell lines: possible role of TFIIH in p53-mediated apoptotic cell death.

The tumor suppressor gene product p53 can bind to and inhibit the helicase activity of the multisubunit transcription-repair factor TFIIH. We previously reported that p53-mediated apoptosis is attenuated in primary human fibroblasts from individuals with Xeroderma Pigmentosum (XP) that harbor mutations in the TFIIH DNA helicases XPD or XPB. In this study we show that apoptosis is reduced and delayed in three XPD lymphoblastoid cell lines (LCLs), but not in an XPD heterozygote LCL, after exposure to doxorubicin, a DNA-damaging agent and topoisomerase II inhibitor frequently used in cancer therapy. Apoptosis was assessed by quantitation of Annexin V binding to exposed phosphatidylserine residues and by caspase-mediated cleavage of Poly(ADP)Ribose Polymerase (PARP). Apoptosis induced by doxorubicin was suppressed in LCLs retrovirally transduced with the Human Papillomavirus 16 E6 oncoprotein, consistent with the hypothesis that this is a p53-dependent process. PARP cleavage was not delayed in XPD LCLs in response to anti-Fas (CD95) antibody-mediated apoptosis, thus, the defect in the apoptotic pathway in these cells lies upstream of caspase activation. Similar changes in the expression of apoptosis-effector genes, p53, and p53-responsive genes p21Cip1/WAF-1/Sid1 (p21), gadd45, bcl-2 and bax were observed in normal and XPD LCLs after treatment with doxorubicin, indicating that delayed apoptosis was not a consequence of defective transcription of these genes. Thus, our studies provide further support to the hypothesis that XPD and p53 can functionally interact in a p53-mediated apoptotic pathway.

Antineoplastic Agents↗
PubMed1999

p53 mutation spectrum and load: the generation of hypotheses linking the exposure of endogenous or exogenous carcinogens to human cancer.

The activation of protooncogenes and inactivation of tumor suppressor genes in affected cells are considered as the core events that provide a selective growth advantage and clonal expansion during the multistep process of carcinogenesis. Somatic mutations, induced by exogenous or endogenous mechanisms, were found to alter the normal functions of the p53 tumor suppressor gene. p53 is the most prominent example of tumor suppressor genes because it is mutated in about half of all human cancer. In contrast to other tumor suppressor genes (like APC and RB), about 80% of p53 mutations are missense mutations that lead to amino acid substitutions in proteins and can alter the protein conformation and increase the stability of p53. These changes can also alter the sequence-specific DNA binding and transcription factor activity of p53. These abnormalities can abrogate p53 dependent pathways involved in important cellular functions like cell-cycle control, DNA repair, differentiation, genomic plasticity and programmed cell death. A number of different carcinogens have been found to cause different characteristic mutations in the p53 gene. For example, exposure to ultraviolet light is correlated with transition mutations at dipyrimidine sites; aflatoxin B(1) exposure is correlated with a G:C to T:A transversion that leads to a serine substitution at residue 249 of p53 in hepatocellular carcinoma; and exposure to cigarette smoke is correlated with G:C to T:A transversions in lung carcinoma. Therefore, measuring the characteristic p53 mutation load or frequency of mutated alleles in nontumorous tissue (before the clonal expansion of mutated cells), can generate hypotheses, e.g., providing a molecular linkage between exposure to a particular carcinogen and cancer, and identifying individuals at increased cancer risk.

Aflatoxin B1↗
PubMed1999

Mutational analysis of p73 and p53 in human cancer cell lines.

p73 is a candidate tumor suppressor gene with substantial DNA and protein homology to the p53 tumor suppressor gene. We have investigated two hypotheses: (a) p73 is mutated in diverse types of human cancer, and (b) p73 is functionally redundant with p53 in carcinogenesis so that mutations would be exclusive in these two genes. The entire coding region and intronic splice junctions of p73 were examined in 54 cancer cell lines. Three lung cancer cell lines contained mutations that affected the amino acid sequence. One amino acid substitution was in a region with homology to the specific DNA binding region of p53 and two microdeletions were outside the region of homology. Two of the cell lines with p73 mutations also carried p53 mutations. Although our results are inconsistent with the two hypotheses tested, p73 mutations may contribute infrequently to the molecular pathogenesis of human lung cancer.

DNA Mutational Analysis↗
PubMed1999

p53-mediated apoptosis is attenuated in Werner syndrome cells.

The WRN DNA helicase is a member of the DExH-containing DNA helicase superfamily that includes XPB, XPD, and BLM. Mutations in WRN are found in patients with the premature aging and cancer susceptibility syndrome known as Werner syndrome (WS). p53 binds to the WRN protein in vivo and in vitro through its carboxyl terminus. WS fibroblasts have an attenuated p53- mediated apoptotic response, and this deficiency can be rescued by expression of wild-type WRN. These data support the hypothesis that p53 can induce apoptosis through the modulation of specific DExH-containing DNA helicases and may have implications for the cancer predisposition observed in WS patients.

Animals↗
PubMed1999

Association with Cdc2 and inhibition of Cdc2/Cyclin B1 kinase activity by the p53-regulated protein Gadd45.

Recently Gadd45, a p53-regulated stress protein, has been implicated in the activation of a G2/M checkpoint after damage by UV radiation and alkylating agents. While inhibitory phosphorylation of Cdc2 and suppression of cyclin B1 levels are known to be involved in G2 delays after genotoxic stress, Gadd45 has now been found to directly inhibit the activity of Cdc2/Cyclin B1 complex, while it had no appreciable effect on Cdk2/ Cyclin E activity even at very high levels of Gadd45. In contrast, p21CiP1/Waf1 is an universal cdk/cyclin inhibitor and inhibited both of the cyclin complexes tested here. Gadd45 was also able to physically interact with Cdc2, but not Cyclin B1. Addition of Gadd45 to immunoprecipitated Cdc2/Cyclin B1 in vitro led to a dissociation of this complex, and thus may represent a new checkpoint mechanism whereby Cdc2/Cyclin B1 can be inhibited. With the use of an antisense approach, reduced Gadd45 expression attenuated the suppression of Cdc2/Cyclin B1 activity in UV-irradiated human cells. Taken together, these results implicate Gadd45 in the control of G2/M cell cycle progression after certain stresses.

Antibodies, Monoclonal↗
PubMed1999

GADD45 induction of a G2/M cell cycle checkpoint.

G1/S and G2/M cell cycle checkpoints maintain genomic stability in eukaryotes in response to genotoxic stress. We report here both genetic and functional evidence of a Gadd45-mediated G2/M checkpoint in human and murine cells. Increased expression of Gadd45 via microinjection of an expression vector into primary human fibroblasts arrests the cells at the G2/M boundary with a phenotype of MPM2 immunopositivity, 4n DNA content and, in 15% of the cells, centrosome separation. The Gadd45-mediated G2/M arrest depends on wild-type p53, because no arrest was observed either in p53-null Li-Fraumeni fibroblasts or in normal fibroblasts coexpressed with p53 mutants. Increased expression of cyclin B1 and Cdc25C inhibited the Gadd45-mediated G2/M arrest in human fibroblasts, indicating that the mechanism of Gadd45-mediated G2/M checkpoint is at least in part through modulation of the activity of the G2-specific kinase, cyclin B1/p34(cdc2). Genetic and physiological evidence of a Gadd45-mediated G2/M checkpoint was obtained by using GADD45-deficient human or murine cells. Human cells with endogenous Gadd45 expression reduced by antisense GADD45 expression have an impaired G2/M checkpoint after exposure to either ultraviolet radiation or methyl methanesulfonate but are still able to undergo G2 arrest after ionizing radiation. Lymphocytes from gadd45-knockout mice (gadd45 -/-) also retained a G2/M checkpoint initiated by ionizing radiation and failed to arrest at G2/M after exposure to ultraviolet radiation. Therefore, the mammalian genome is protected by a multiplicity of G2/M checkpoints in response to specific types of DNA damage.

Animals↗
PubMed1999

Hepatitis B virus X protein inhibits nucleotide excision repair.

Human hepatitis B virus (HBV) is a major risk factor of human hepatocellular carcinoma. Both in vivo and in vitro studies have shown that HBV X protein (HBx) can bind to the p53 tumor-suppressor protein and interfere with the role that p53 plays in the cellular response to DNA damage. Our previous work has shown that HBx protein inhibits p53 sequence-specific transcriptional activation, p53-mediated apoptosis and p53 binding to the TFIIH transcription-nucleotide excision repair (NER) factors, including XPB and XPD. To investigate whether HBx interferes with the NER pathway, we utilized cell-proliferation and colony-formation assays to determine if cells expressing HBx are more sensitive to UVC-induced DNA damage. NER was also measured by a plasmid host cell re-activation assay using a vector containing a luciferase reporter gene. UV-irradiated plasmids were transfected into a human RKO colon carcinoma cell line that contains wild-type (wt) p53 as well as its derivatives, either mutant p53-143ala (RKO-143ala) or human papillomavirus E6 (RKO-E6, a wt p53 protein that is rapidly degraded and non-functional). We found that cells expressing HBx are more sensitive to UVC-induced killing. Moreover, expression of HBx resulted in a reduction of NER efficiency in RKO cells to 52 +/- 2% (compared with control), RKO-143a1a cells to 46 +/- 3% and RKO-E6 cells to 60 +/- 3%. Similar results were also obtained with a HepG2 hepatoblastoma cell line carrying wt p53. In addition, we found that HBx bound directly to either XPB or XPD DNA helicase in vitro. Thus, our data indicate that HBx may interfere with the NER pathway through both p53-dependent and p53-independent mechanisms. Because HBx binds to TFIIH-associated proteins, we propose that HBx may interfere with the NER pathway also through binding to and altering the activities of helicases necessary for NER and, thereby, increase the mutation rate induced by chemical carcinogens, such as aflatoxin B1, during human liver carcinogenesis.

Apoptosis↗
PubMed1999

COOH-terminal domain of p53 modulates p53-mediated transcriptional transactivation, cell growth, and apoptosis.

The tumor suppressor protein p53 contributes to the control of cell cycle checkpoints and stress-induced apoptosis and is frequently mutated in many different types of human cancers. The COOH terminus of p53 modulates the transcriptional and apoptotic activities of the protein. Although COOH-terminal mutants of p53 are uncommon, we proposed that these p53 mutants nevertheless contributed to the selective clonal expansion of the cancer cells. Therefore, we analyzed the tumor-derived p53 COOH-terminal domain (CTD) mutants (352D/H, 356G/W, 342-stop, 360-del, and 387-del) functionally. The results have revealed that all mutants have impaired apoptotic activity when compared with wild-type p53. However, some of these mutants still transcriptionally transactivate p21Waf/Cip1 and inhibit cell growth. Interestingly, of the tumor-derived CTD mutants, oligomerization-defective mutant 342-stop was the only one that did not exhibit sequence-specific DNA binding or failed to transactivate p21Waf1/Cip1, Bax, and IGF-BP3 transcriptionally. The failure to inhibit cell growth by this tumor-derived CTD mutant supports the hypothesis that p53 sequence-specific transcriptional transactivity to p21Waf1/Cip1 is correlated with induction of cell cycle arrest and that the p53 transcriptional transactivity requires oligomerization of the p53 protein. These and other data indicate that the CTD of p53 is an important component of p53-mediated apoptosis and cell growth arrest and that inactivation of the apoptotic function, but not the inhibition of growth, is an important step during human tumorigenesis.

Apoptosis↗
PubMed1999

Evolutionary conservation and somatic mutation hotspot maps of p53: correlation with p53 protein structural and functional features.

Missense mutations in p53 frequently occur at 'hotspot' amino acids which are highly conserved and represent regions of structural or functional importance. Using the p53 mutation database and the p53 DNA sequences for 11 species, we more precisely defined the relationships among conservation, mutation frequency and protein structure. We aligned the p53 sequences codon-by-codon and determined the degree of substitution among them. As a whole, p53 is evolving at an average rate for a mammalian protein-coding gene. As expected, the DNA binding domain is evolving more slowly than the carboxy and amino termini. A detailed map of evolutionary conservation shows that within the DNA binding domain there are repeating peaks and valleys of higher and lower evolutionary constraint. Mutation hotspots were identified by comparing the observed distribution of mutations to the pattern expected from a random multinomial distribution. Seventy-three hotspots were identified; these 19% of codons account for 88% of all reported p53 mutations. Both high evolutionary constraint and mutation hotspots are noted at amino acids close to the protein-DNA interface and at others more distant from DNA, often buried within the core of the folded protein but sometimes on its surface. The results indicate that targeting highly conserved regions for mutational and functional analysis may be efficient strategies for the study of cancer-related genes.

Chromosome Mapping↗
PubMed1999

Molecular epidemiology of human cancer risk: gene-environment interactions and p53 mutation spectrum in human lung cancer.

The p53 tumour suppressor gene is at the crossroads of a network of cellular pathways including cell cycle checkpoints, DNA repair, chromosomal segregation, and apoptosis. These pathways have evolved to maintain the stability of the genome during cellular stress from DNA damage, hypoxia, and activated oncogenes. The high frequency of p53 mutations in human cancer is a reflection of the importance of p53 involvement in this network of pathways during human carcinogenesis. An electronic database containing p53 mutations from more than 9000 cancers (http:/(/)www.iarc.fr/p53/homepage.html) can be used to generate hypotheses for further clinical, epidemiological, and laboratory investigations. For example, one can hypothesize that (a) p53 mutations vary in their pathobiological significance; (b) cellular content influences the selection of p53 mutations in clonally derived cancers; (c) the location and type of mutation within the p53 gene provide clues to functional domains in the gene product; and (d) the p53 mutation spectrum can be a molecular link between aetiological agents and human cancer. This review will focus on the role of p53 and cancer susceptibility genes in the molecular pathogenesis and epidemiology of human lung cancer.

Cocarcinogenesis↗
PubMed1999

Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells.

Germline mutations of the Brca1 tumor suppressor gene predispose women to breast and ovarian cancers. To study mechanisms underlying BRCA1-related tumorigenesis, we derived mouse embryonic fibroblast cells carrying a targeted deletion of exon 11 of the Brca1 gene. We show that the mutant cells maintain an intact G1-S cell cycle checkpoint and proliferate poorly. However, a defective G2-M checkpoint in these cells is accompanied by extensive chromosomal abnormalities. Mutant fibroblasts contain multiple, functional centrosomes, which lead to unequal chromosome segregation, abnormal nuclear division, and aneuploidy. These data uncover an essential role of BRCA1 in maintaining genetic stability through the regulation of centrosome duplication and the G2-M checkpoint and provide a molecular basis for the role of BRCA1 in tumorigenesis.

Alternative Splicing↗
PubMed1999

Molecular epidemiological study of non-small-cell lung cancer from an environmentally polluted region of Poland.

The p53 mutation spectrum can generate hypotheses linking carcinogen exposure to human cancer. Although it is well-documented that tobacco smoking is a major cause of lung cancer, the contribution of air pollution is less well-established. We determined the molecular and immunohistochemical changes (p53 gene mutations, p53 protein accumulation and WAF1 protein expression) and genetic polymorphisms of GSTM1, CYP1A1 and CYP2D6 genes in a case series of non-small-cell lung cancers from Silesia. This region of southern Poland is highly industrialized with considerable environmental pollution. More than 50% of lung cancers (90/164) contained p53 mutations and 75% showed the combined alteration of the p53 gene and protein accumulation. Males occupationally exposed to coal-derived substances showed a relatively high frequency of squamous and large-cell carcinomas, relatively frequent mutations in codon 298 of p53 and a low frequency of p53 immunohistochemically positive tumours. Codon 298 GAG-->TAG mutations have rarely been found in lung cancers in other populations. We found no correlation between WAF1 protein expression and mutations in the p53 gene or p53 protein accumulation. No statistically significant relationship was found between p53 mutations and GSTM1, CYP1A1, CYP2D6 genotypes. Never smokers with lung cancers from Silesia had a higher frequency of G:C-->T:A transversions than previously reported of the p53 mutation spectrum in never smokers (6/15 vs 4/34; P = 0.06 by chi2). These data are a tentative indication that occupational and environmental exposure to polycyclic aromatic hydrocarbons, such as benzo(a)pyrene, in polluted air contributes to the molecular pathogenesis of lung cancer in never smokers.

Air Pollution↗
PubMed1999

GSTM1, GSTP1, CYP1A1 and CYP2D6 polymorphisms in lung cancer patients from an environmentally polluted region of Poland: correlation with lung DNA adduct levels.

The CYP and GST genetic polymorphisms, controlling metabolism of xenobiotics, are considered to influence an individual's susceptibility to environmental and occupational carcinogens and predisposition to cancer. In the study, the effect of the GSTM1, GSTP1, CYP1A1 and CYP2D6 polymorphisms was investigated in relation to PAH-DNA adduct levels in non-tumourous lung tissue from non-small cell lung cancer (NSCLC) patients living in the industrialized region of Upper Silesia, Poland. The level of adducts among smokers was significantly elevated when compared to non-smokers (P = 0.0005). Adduct levels correlated inversely with age of patients (P = 0.00001). The GSTP1 and CYP2D6 polymorphisms had no influence on DNA adduct levels. There was a significant relationship between high adduct levels and the combined GSTM1 (null)/CYP1A1-Ile/Val genotype in the squamous cell carcinoma group (P = 0.028). An elevated number of adducts was found in patients with the GSTM1 (null)/CYP1Al-Ile/Val genotype compared to the GSTM1 (null)/CYP1A1-Ile/Ile carriers (P = 0.043). A higher frequency of the CYP1A1-Ile/Val and GSTM1 (null)/CYP1A1-Ile/Val genotypes was observed in patients with high adduct levels (P = 0.05 and P = 0.009, respectively). A significant prevalence of the GSTM1(null)/CYP1A1-Ile/Val carriers in the adenocarcinoma group was found (P = 0.003). Thus, our findings imply that the GSTMI and CYP1A1 exon 7 polymorphisms may influence PAH-DNA adduct levels in target tissue from NSCLC patients, especially in the squamous cell carcinoma group. Moreover, individuals carrying the GSTM1(null)/CYP1A1-Ile/Val genotype might exhibit a greater predisposition to a peripheral type of lung cancer.

Adult↗
PubMed1999

Cancer-prone oxyradical overload disease.

Oxyradical overload disease develops in conditions involving chronic inflammation and may be of inherited etiology, e.g. haemochromatosis and Wilson disease, be acquired, e.g. infection with hepatitis B or C virus or Helicobactor pylori, or be chemically induced, e.g. acid reflux in Barrett oesophagus. Susceptibility to cancer is frequently a pathological consequence of extensive oxyradical damage that leads to a cycle of cell death and regeneration and causes mutations in cancer-related genes. In this brief review, we focus on the possible interactive effects of nitric oxide and the p53 tumour suppressor gene in human carcinogenesis.

Apoptosis↗
PubMed1998

Molecular epidemiology of human cancer.

A challenging goal of molecular epidemiology is to identify an individual's risk of cancer. Molecular epidemiology integrates molecular biology, in vitro and in vivo laboratory models, biochemistry, and epidemiology to infer individual cancer risk. Molecular dosimetry of carcinogen exposure is an important facet of molecular epidemiology and cancer risk assessment. Carcinogen macromolecular adduct levels, cytogenetic alterations and somatic cell mutations can be measured to determine the biologically-effective doses of carcinogens. Molecular epidemiology also explores host cancer susceptibilities, such as carcinogen metabolism, DNA repair, and epigenetic and genetic alterations in tumor suppressor genes. p53 is a prototype tumor suppressor gene and is well suited for analysis of mutational spectrum in human cancer. The analyses of germline and somatic mutation spectra of the p53 tumor suppressor gene provide important clues for cancer risk assessment in molecular epidemiology. For example, characteristic p53 mutation spectra have been associated with: dietary aflatoxin B1 exposure and hepatocellular carcinoma; sunlight exposure and skin carcinoma; and cigarette smoking and lung cancer. The mutation spectrum also reveals those p53 mutants that provide cells with a selective clonal-expansion advantage during the multistep process of carcinogenesis. The p53 gene encodes a multifunctional protein involved in the cellular response to stress including DNA damage and hypoxia. Certain p53 mutants lose tumor suppressor activity and gain oncogenic activity, which is one explanation for the commonality of p53 mutations in human cancer. Molecular epidemiological results can be evaluated for causation by inference of the Bradford-Hill criteria, i.e. strength of association (consistency, specificity and temporality) and biological plausibility, which utilizes the 'weight of the evidence principle'.

Animals↗