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PubMed2004

Genetic susceptibility to tobacco-related cancer.

Although cigarette smoking is the dominant risk factor for several epithelial cancers, only a small fraction of individuals with tobacco exposure develop cancer. The underlying hypothesis is that genetic factors may render certain smokers more susceptible to cancer than others. Genetic alterations in critical regulatory pathways may predispose cells to carcinogenesis. These pathways include regulation of xenobiotic metabolism; control of genomic stability, including DNA repair mechanisms, cell-cycle checkpoints, apoptosis and telomere length; and control of microenvironmental factors, such as matrix metalloproteinases, inflammation and growth factors. In addition, epigenetic events, such as promoter hypermethylation and loss of imprinting, are also involved in carcinogenesis. In this review, we will summarize recent advances in genetic susceptibility to tobacco-related cancer. Emphasizing on risk assessment, we will describe how genetic variations in the above-mentioned genetic pathways modify the tobacco-related cancer risk. In addition, we will discuss how genetic variations may assist in predicting clinical outcome, such as the natural history of cancer and treatment response. The measurements of genetic susceptibility by both genotypic and phenotypic assays are covered in the text. Finally, we present a number of current concerns that need to be addressed as the exciting field of molecular cancer epidemiology advances rapidly.

Cytochrome P-450 Enzyme System↗
PubMed2002

UV-induced DNA damage and repair: a review.

Increases in ultraviolet radiation at the Earth's surface due to the depletion of the stratospheric ozone layer have recently fuelled interest in the mechanisms of various effects it might have on organisms. DNA is certainly one of the key targets for UV-induced damage in a variety of organisms ranging from bacteria to humans. UV radiation induces two of the most abundant mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) and their Dewar valence Isomers. However, cells have developed a number of repair or tolerance mechanism to counteract the DNA damage caused by UV or any other stressors. Photoreactivation with the help of the enzyme photolyase is one of the most important and frequently occurring repair mechanisms in a variety of organisms. Excision repair, which can be distinguished into base excision repair (BER) and nucleotide excision repair (NER), also plays an important role in DNA repair in several organisms with the help of a number of glycosylases and polymerases, respectively. In addition, mechanisms such as mutagenic repair or dimer bypass, recombinational repair, cell-cycle checkpoints, apoptosis and certain alternative repair pathways are also operative in various organisms. This review deals with UV-induced DNA damage and the associated repair mechanisms as well as methods of detecting DNA damage and its future perspectives.

DNA Damage↗
PubMed2000

Replicative senescence as a barrier to human cancer.

There is evidence that one critically short telomere may be recognized as DNA damage and, as a consequence, induce a p53/p21WAF- and p16INK4A-dependent G1 cell cycle checkpoint to cause senescence. Additionally, senescence via a p53- and p16(INK4A)-dependent mechanism can be induced by the over- or under-stimulation of certain signalling pathways that are involved in cancer. Central to this alternative senescence mechanism is the p14ARF protein, which connects oncogene activation, but not DNA damage, to p53 activation and senescence. We find that immortal keratinocytes almost invariably have dysfunctional p53 and p16 and have high levels of telomerase, but very often express a wild-type p14(ARF). Furthermore, when normal keratinocytes senesce they show a striking elevation of p16 protein, but not of p14(ARF) or its downstream targets p53 and p21(WAF). These results suggest that p16, rather than p14(ARF), is the more important gene in human keratinocyte senescence, but do not exclude a co-operative role for p14(ARF), perhaps in the induction of senescence by activated oncogenes in neoplasia. Regardless of mechanism, these results suggest that replicative senescence acts as a barrier to human cancer development.

Carrier Proteins↗
PubMed1999

Heat-induced cell cycle arrest of Saccharomyces cerevisiae: involvement of the RAD6/UBC2 and WSC2 genes in its reversal.

The Saccharomyces cerevisiae RAD6 (UBC2 ) gene encodes a ubiquitin-conjugating enzyme that is involved in a wide range of cellular processes including DNA repair, sporulation and N-end rule protein degradation. Under mild heat stress conditions (37-38 degrees C) rad6 null and rad6-149 mutant cells are unable to grow. The molecular basis for this failure to grow is unknown. Here we show that the heat sensitivity of rad6 mutants is not due to cell death but to an inability to progress in the cell cycle. The temperature-induced cell cycle arrest of these mutants is due to a block in a branch of the RAD6 pathway distinct from the DNA repair and the N-end rule protein degradation pathways. Wild-type cells heated to 38 degrees C arrest transiently in the late G1 phase and then resume growth. At 38 degrees C rad6 mutant cells arrest in late G1 but, unlike wild-type cells, are unable to resume cell cycle progression. In both wild-type and in rad6 mutant cells, CLN1 and CLN2 transcript levels fall sharply upon temperature increase. In wild-type cells levels of these transcripts recover rapidly, whereas in the rad6 mutant they recover slowly. As rad6 cells remain arrested even after CLN1 and CLN2 mRNAs regain their preheat stress levels, factors additional to reduced G1 cyclin gene expression must cause the temperature-induced cell cycle block of the mutant. To identify genes involved in the relief of the cell cycle arrest under heat stress, we screened a multicopy yeast genomic library for clones that restore the growth of the rad6-149 mutant. A plasmid was isolated carrying the WSC2 gene, which is closely related to WSC1/SLG1/HCS77, a putative membrane heat sensor. Overexpression of WSC2 reverses the heat-induced cell cycle arrest of rad6-149 but not of rad6 null mutants. Taken together the findings point to the existence of an unidentified heat stress-activated cell cycle checkpoint pathway, which is antagonized by Rad6p by a mechanism also involving Wsc2p.

Cyclins↗
PubMed1996

Molecular Biology of the Cell Cycle: Potential for Therapeutic Applications in Radiation Oncology.

The cell cycle was first described by radiation biologists more than 40 years ago. Since then, radiation oncologists have used information regarding the cell cycle, in particular cell cycle kinetics, to design various treatment protocols; these have resulted in only modest improvements in patient outcome. Over the past 10 to 15 years there has been an explosion of scientific knowledge regarding the cell cycle, in particular the molecular regulation of the cell cycle checkpoints. In this review we will discuss the genetic events involved in regulating the G1 and G2 checkpoints and how this information may lead to future therapeutic breakthroughs. In addition we will discuss the potential clinical impact of the recent cloning of the gene for ataxia telangiectasia.

Journal Article↗
PubMed2001

Intermediates in V(D)J recombination: a stable RAG1/2 complex sequesters cleaved RSS ends.

Rearrangement of gene segments to generate antigen receptor coding regions depends on the RAG1/2 recombinase, which assembles a synaptic complex between two DNA signal sequences and then cleaves the DNA directly adjacent to the paired signals. After coupled cleavage of complementary signal sequences, virtually all of the cleaved signal ends remained associated with RAG1/2 in stable complexes. These signal end complexes were distinct from various precleavage RAG1/2 signal complexes in that they were resistant to treatment with heparin. A mammalian joining apparatus consisting of purified Ku70/86, XRCC4, and DNA ligase IV proteins was sufficient to join deproteinized cleaved ends, but retention of signal sequences within the signal end complex blocked access to the DNA ends and prevented their joining by these proteins. Sequestration of cleaved ends within the signal end complex would account for the persistence of these ends in the cell after cleavage and may explain why they do not normally activate the DNA-damage-dependent cell cycle checkpoint.

Antigens, Nuclear↗
PubMed2001

A genome-wide screen in Saccharomyces cerevisiae for genes affecting UV radiation sensitivity.

The recent completion of the deletion of essentially all of the ORFs in yeast is an important new resource for identifying the phenotypes of unknown genes. Each ORF is replaced with a cassette containing unique tag sequences that allow rapid parallel analysis of strains in a pool by using hybridization to a high-density oligonucleotide array. We examined the utility of this system to identify genes conferring resistance to UV irradiation by using a pool of 4,627 individual homozygous deletion strains (representing deletions of all nonessential genes). We identified most of the nonessential genes previously shown to be involved in nucleotide excision repair, in cell cycle checkpoints, in homologous recombination, and in postreplication repair after UV damage. We also identified and individually confirmed, by replacing the genes, three new genes, to our knowledge not previously reported to confer UV sensitivity when deleted. Two of these newly identified genes have human orthologs associated with cancer, demonstrating the potential of this system to uncover human genes affecting sensitivity to DNA-damaging agents and genes potentially involved in cancer formation.

DNA Damage↗
PubMed1997

Architectural accommodation in the complex of four p53 DNA binding domain peptides with the p21/waf1/cip1 DNA response element.

High resolution chemical footprinting and cross-linking experiments have provided a basis for elucidating the overall architecture of the complex between the core DNA binding domain of p53 (p53DBD, amino acids 98-309) and the p21/waf1/cip1 DNA response element implicated in the G1/S phase cell cycle checkpoint. These studies complement both a crystal structure and earlier biophysical studies and provide the first direct experimental evidence that four subunits of p53DBD bind to the response element in a regular staggered array having pseudodyad symmetry. The invariant guanosines in the highly conserved C(A/T)|(T/A)G parts of the consensus half-sites are critical to the p53DBD-DNA binding. Molecular modeling of the complex using the observed peptide-DNA contacts shows that when four subunits of p53DBD bind the response element, the DNA has to bend approximately 50 degrees to relieve steric clashes among different subunits, consistent with recent DNA cyclization studies. The overall lateral arrangement of the four p53 subunits with respect to the DNA loop comprises a novel nucleoprotein assembly that has not been reported previously in other complexes. We suggest that this kind of nucleoprotein superstructure may be important for p53 binding to response elements packed in chromatin and for subsequent transactivation of p53-mediated genes.

Base Sequence↗
PubMed2001

The hCds1 (Chk2)-FHA domain is essential for a chain of phosphorylation events on hCds1 that is induced by ionizing radiation.

hCds1 (Chk2) is an evolutionarily conserved kinase that functions in DNA damage response and cell cycle checkpoint. The Cds1 family of kinases are activated by a family of large phosphatidylinositol 3-kinase-like kinases. In humans, ataxia telangiectasia-mutated (ATM) and ataxia-telangiectasia and Rad3-related kinases activate hCds1 by phosphorylating Thr(68) . hCds1 and Cds1-related kinases contain the FHA (forkhead-associated) domain, which appears to be important for integrating the DNA damage signal. It is not known how ATM phosphorylation activates hCds1 function and whether the phosphorylation is linked to the FHA. Here, we demonstrate that the hCds1-FHA domain is essential for Thr(68) phosphorylation. Thr(68) phosphorylation, in turn, is required for ionizing radiation-induced autophosphorylation of two amino acid residues in hCds1, Thr(383) and Thr(387). These two amino acid residues, located in the activation loop of hCds1, are conserved in hCds1-related kinases and are essential for hCds1 activity. Thus, the hCds1-FHA domain mediates a chain of phosphorylation events on hCds1, which includes phosphorylation by ATM and hCds1 autophosphorylation, in response to DNA damage.

Amino Acid Sequence↗
PubMed2002

Determination of substrate motifs for human Chk1 and hCds1/Chk2 by the oriented peptide library approach.

Mammalian Chk1 and Chk2 are two Ser/Thr effector kinases that play critical roles in DNA damage-activated cell cycle checkpoint signaling pathways downstream of ataxia telangiectasia-mutated and ataxia telangiectasia-related. Endogenous substrates have been identified for human hCds1/Chk2 and Chk1; however, the sequences surrounding the substrate residues appear unrelated, and consensus substrate motifs for the two Ser/Thr kinases remain unknown. We have utilized peptide library analyses to develop specific, highly preferred substrate motifs for hCds1/Chk2 and Chk1. The optimal motifs are similar for both kinases and most closely resemble the previously identified Chk1 and hCds1/Chk2 substrate target sequences in Cdc25C and Cdc25A, the regulation of which plays an important role in S and G(2)M arrest. Essential residues required for the definition of the optimal motifs were also identified. Utilization of the peptides to assay the substrate specificities and catalytic activities of Chk1 and hCds1/Chk2 revealed substantial differences between the two Ser/Thr kinases. Structural modeling analyses of the peptides into the Chk1 catalytic cleft were consistent with Chk1 kinase assays defining substrate suitability. The library-derived substrate preferences were applied in a genome-wide search program, revealing novel targets that might serve as substrates for hCds1/Chk2 or Chk1 kinase activity.

Amino Acid Sequence↗
PubMed2003

Identification of phosphorylation sites on the yeast ribonucleotide reductase inhibitor Sml1.

Sml1 is a small protein in Saccharomyces cerevisiae which inhibits the activity of ribonucleotide reductase (RNR). RNR catalyzes the rate-limiting step of de novo dNTP synthesis. Sml1 is a downstream effector of the Mec1/Rad53 cell cycle checkpoint pathway. The phosphorylation by Dun1 kinase during S phase or in response to DNA damage leads to diminished levels of Sml1. Removal of Sml1 increases the population of active RNR, which raises cellular dNTP levels. In this study using mass spectrometry and site-directed mutagenesis, we have identified the region of Sml1 phosphorylation to be between residues 52 and 64 containing the sequence GSSASASASSLEM. This is the first identification of a phosphorylation sequence of a Dun1 biological substrate. This sequence is quite different from the consensus Dun1 phosphorylation sequence reported previously from peptide library studies. The specific phosphoserines were identified to be Ser(56), Ser(58), and Ser(60) by chemical modification of these residues to S-ethylcysteines followed by collision activated dissociation. To investigate further Sml1 phosphorylation, we constructed the single mutants S56A, S58A, S60A, and the triple mutant S56A/S58A/S60A and compared their degrees of phosphorylation with that of wild type Sml1. We observed a 90% decrease in the relative phosphorylation of S60A compared with that of wild type, a 25% decrease in S58A, and little or no decrease in the S56A mutant. There was no observed phosphate incorporation in the triple mutant, suggesting that Ser(56), Ser(58), and Ser(60) in Sml1 are the sites of phosphorylation. Further mutagenesis studies reveal that Dun1 kinase requires an acidic residue at the +3 position, and there is cooperativity between the phosphorylation sites. These results show that Dun1 has a unique phosphorylation motif.

Amino Acid Sequence↗
PubMed1998

Fine localization of the Nijmegen breakage syndrome gene to 8q21: evidence for a common founder haplotype.

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, a birdlike face, growth retardation, immunodeficiency, lack of secondary sex characteristics in females, and increased incidence of lymphoid cancers. NBS cells display a phenotype similar to that of cells from ataxia-telangiectasia patients, including chromosomal instability, radiation sensitivity, and aberrant cell-cycle-checkpoint control following exposure to ionizing radiation. A recent study reported genetic linkage of NBS to human chromosome 8q21, with strong linkage disequilibrium detected at marker D8S1811 in eastern European NBS families. We collected a geographically diverse group of NBS families and tested them for linkage, using an expanded panel of markers at 8q21. In this article, we report linkage of NBS to 8q21 in 6/7 of these families, with a maximum LOD score of 3.58. Significant linkage disequilibrium was detected for 8/13 markers tested in the 8q21 region, including D8S1811. In order to further localize the gene for NBS, we generated a radiation-hybrid map of markers at 8q21 and constructed haplotypes based on this map. Examination of disease haplotypes segregating in 11 NBS pedigrees revealed recombination events that place the NBS gene between D8S1757 and D8S270. A common founder haplotype was present on 15/18 disease chromosomes from 9/11 NBS families. Inferred (ancestral) recombination events involving this common haplotype suggest that NBS can be localized further, to an interval flanked by markers D8S273 and D8S88.

Chromosome Breakage↗
PubMed2002

A CHEK2 genetic variant contributing to a substantial fraction of familial breast cancer.

CHEK2 (previously known as "CHK2") is a cell-cycle-checkpoint kinase that phosphorylates p53 and BRCA1 in response to DNA damage. A protein-truncating mutation, 1100delC in exon 10, which abolishes the kinase function of CHEK2, has been found in families with Li-Fraumeni syndrome (LFS) and in those with a cancer phenotype that is suggestive of LFS, including breast cancer. In the present study, we found that the frequency of 1100delC was 2.0% among an unselected population-based cohort of 1,035 patients with breast cancer. This was slightly, but not significantly (P=.182), higher than the 1.4% frequency found among 1,885 population control subjects. However, a significantly elevated frequency was found among those 358 patients with a positive family history (11/358 [3.1%]; odds ratio [OR] 2.27; 95% confidence interval [CI] 1.11-4.63; P=.021, compared with population controls). Furthermore, patients with bilateral breast cancer were sixfold more likely to be 1100delC carriers than were patients with unilateral cancer (95% CI 1.87-20.32; P=.007). Analysis of the 1100delC variant in an independent set of 507 patients with familial breast cancer with no BRCA1 and BRCA2 mutations confirmed a significantly elevated frequency of 1100delC (28/507 [5.5%]; OR 4.2; 95% CI 2.4-7.2; P=.0002), compared with controls, with a high frequency also seen in patients with only a single affected first-degree relative (18/291 [6.2%]). Finally, tissue microarray analysis indicated that breast tumors from patients with 1100delC mutations show reduced CHEK2 immunostaining. The results suggest that CHEK2 acts as a low-penetrance tumor-suppressor gene in breast cancer and that it makes a significant contribution to familial clustering of breast cancer-including families with only two affected relatives, which are more common than families that include larger numbers of affected women.

Breast Neoplasms↗
PubMed2003

Variants in CHEK2 other than 1100delC do not make a major contribution to breast cancer susceptibility.

We recently reported that a sequence variant in the cell-cycle-checkpoint kinase CHEK2 (CHEK2 1100delC) is a low-penetrance breast cancer-susceptibility allele in noncarriers of BRCA1 or BRCA2 mutations. To investigate whether other CHEK2 variants confer susceptibility to breast cancer, we screened the full CHEK2 coding sequence in BRCA1/2-negative breast cancer cases from 89 pedigrees with three or more cases of breast cancer. We identified one novel germline variant, R117G, in two separate families. To evaluate the possible association of R117G and two germline variants reported elsewhere, R145W and I157T with breast cancer, we screened 737 BRCA1/2-negative familial breast cancer cases from 605 families, 459 BRCA1/2-positive cases from 335 families, and 723 controls from the United Kingdom, the Netherlands, and North America. All three variants were rare in all groups, and none occurred at significantly elevated frequency in familial breast cancer cases compared with controls. These results indicate that 1100delC may be the only CHEK2 allele that makes an appreciable contribution to breast cancer susceptibility.

Base Sequence↗
PubMed2003

The CHEK2 1100delC mutation identifies families with a hereditary breast and colorectal cancer phenotype.

Because of genetic heterogeneity, the identification of breast cancer-susceptibility genes has proven to be exceedingly difficult. Here, we define a new subset of families with breast cancer characterized by the presence of colorectal cancer cases. The 1100delC variant of the cell cycle checkpoint kinase CHEK2 gene was present in 18% of 55 families with hereditary breast and colorectal cancer (HBCC) as compared with 4% of 380 families with non-HBCC (P<.001), thus providing genetic evidence for the HBCC phenotype. The CHEK2 1100delC mutation was, however, not the major predisposing factor for the HBCC phenotype but appeared to act in synergy with another, as-yet-unknown susceptibility gene(s). The unequivocal definition of the HBCC phenotype opens new avenues to search for this putative HBCC-susceptibility gene.

Adult↗
PubMed2012

The nontoxic cell cycle modulator indirubin augments transduction of adeno-associated viral vectors and zinc-finger nuclease-mediated gene targeting.

Parameters that regulate or affect the cell cycle or the DNA repair choice between non-homologous end-joining and homology-directed repair (HDR) are excellent targets to enhance therapeutic gene targeting. Here, we have evaluated the impact of five cell-cycle modulating drugs on targeted genome engineering mediated by DNA double-strand break (DSB)-inducing nucleases, such as zinc-finger nucleases (ZFNs). For a side-by-side comparison, we have established four reporter cell lines by integrating a mutated EGFP gene into either three transformed human cell lines or primary umbilical cord-derived mesenchymal stromal cells (UC-MSCs). After treatment with different cytostatic drugs, cells were transduced with adeno-associated virus (AAV) vectors that encode a nuclease or a repair donor to rescue EGFP expression through DSB-induced HDR. We show that transient cell-cycle arrest increased AAV transduction and AAV-mediated HDR up to six-fold in human cell lines and ten-fold in UC-MSCs, respectively. Targeted gene correction was observed in up to 34% of transduced cells. Both the absolute and the relative gene-targeting frequencies were dependent on the cell type, the cytostatic drug, the vector dose, and the nuclease. Treatment of cells with the cyclin-dependent kinase inhibitor indirubin-3'-monoxime was especially promising as this compound combined high stimulatory effects with minimal cytotoxicity. In conclusion, indirubin-3'-monoxime significantly improved AAV transduction and the efficiency of AAV/ZFN-mediated gene targeting and may thus represent a promising compound to enhance DSB-mediated genome engineering in human stem cells, such as UC-MSCs, which hold great promise for future clinical applications.

Blotting, Western↗
PubMed1996

Radiosensitizing nucleosides.

Chemotherapeutic drugs that perturb nucleotide metabolism have the potential to produce substantial sensitization of tumor cells to radiation treatment. The process is called radiosensitization, and the agents that induce it are called radiosensitizers. The clinical effectiveness of fluoropyrimidines as radiosensitizers has been proven in multiple randomized trials. Thymidine analogues and hydroxyurea also appear to produce clinically relevant increases in radiation sensitivity. Recent laboratory investigations have identified difluorodeoxycytidine (gemcitabine) and fludarabine as promising agents to use in combination with radiation. Until recently, little was known about how the biochemical changes caused by these drugs produced radiosensitization. However, advances in related fields, such as cell cycle checkpoint control, have permitted the development of a hypothesis that may explain the relative tumor selectivity of fluoropyrimidine-mediated radiosensitization. In addition, recent findings suggest that the rational manipulation of drug administration schedules and the use of combinations of radiosensitizers have the potential to improve the efficacy of the currently used agents and to establish the benefit of new ones.

Animals↗
PubMed2005

Saccharomyces cerevisiae Mre11 is a high-affinity G4 DNA-binding protein and a G-rich DNA-specific endonuclease: implications for replication of telomeric DNA.

In Saccharomyces cerevisiae, Mre11p/Rad50p/Xrs2p (MRX) complex plays a vital role in several nuclear processes including cellular response to DNA damage, telomere length maintenance, cell cycle checkpoint control and meiotic recombination. Telomeres are comprised of tandem repeats of G-rich DNA and are incorporated into non-nucleosomal chromatin. Although the structure of the yeast telomeric DNA is poorly understood, it has been suggested that the G-rich sequences can fold into G4 DNA, which has been shown to inhibit DNA synthesis by telomerase. However, little is known about the factors and mechanistic aspects of the generation of appropriate termini for DNA synthesis by telomerase. Here, we show that S.cerevisiae Mre11 protein (ScMre11p) possesses substantially higher binding affinity for G4 DNA, over single- or double-stranded DNA, and binding was inhibited by poly(dG) or porphyrin. Binding of ScMre11p to G4 DNA was most robust, compared with G2' DNA and the resulting protein-DNA complexes were strikingly very resistant to dissociation by NaCl. Remarkably, binding of ScMre11p to G4 DNA and G-rich single-stranded DNA was accompanied by the endonucleolytic cleavage at sites flanking the array of G residues and G-quartets in Mn2+-dependent manner. Collectively, these results suggest that ScMre11p is likely to play a major role in generating appropriate substrates for DNA synthesis by telomerase and telomere-binding proteins. We discuss the implications of these findings with regard to telomere length maintenance by telomerase-dependent and independent mechanisms.

DNA↗