Showing papers by "Fred Hutchinson Cancer Research Center published in 2001"

Predicting Deleterious Amino Acid Substitutions

Abstract: Many missense substitutions are identified in single nucleotide polymorphism (SNP) data and large-scale random mutagenesis projects. Each amino acid substitution potentially affects protein function. We have constructed a tool that uses sequence homology to predict whether a substitution affects protein function. SIFT, which sorts intolerant from tolerant substitutions, classifies substitutions as tolerated or deleterious. A higher proportion of substitutions predicted to be deleterious by SIFT gives an affected phenotype than substitutions predicted to be deleterious by substitution scoring matrices in three test cases. Using SIFT before mutagenesis studies could reduce the number of functional assays required and yield a higher proportion of affected phenotypes. may be used to identify plausible disease candidates among the SNPs that cause missense substitutions.

Phases of Biomarker Development for Early Detection of Cancer

Abstract: Recent developments in such areas of research as geneexpression microarrays, proteomics, and immunology offer new approaches to cancer screening (1). The surge in research to develop cancer-screening biomarkers prompted the establishment of the Early Detection Research Network (EDRN) by the National Cancer Institute (2). The purpose of the EDRN is to coordinate research among biomarker-development laboratories, biomarker-validation laboratories, clinical repositories, and population-screening programs. By coordination of research efforts, the hope is to facilitate collaboration and to promote efficiency and rigor in research. With the goals of the EDRN in mind, the purpose of this commentary is to define a formal structure to guide the process of biomarker development. We categorize the development into five phases that a biomarker needs to pass through to produce a useful population-screening tool. The phases of research are generally ordered according to the strength of evidence that each provides in favor of the biomarker, from weakest to strongest. In addition, the results of earlier phases are generally necessary to design later phases. Therapeutic drug development has had such a structure in place for some time (3). The clinical phases of testing a new cancer drug are as follows: phase 1, determinations of toxicity, pharmacokinetics, and optimal dose levels; phase 2, determinations of biologic efficacy; and phase 3, definitive controlled trials of effects on clinical endpoints. For each phase, guidelines exist for subject selection, outcome measures, relevant comparisons for evaluating study results, and so forth. Although deviations are common, the basic structure facilitates coherent, thorough, and efficient development of new therapies. A phased approach has also been proposed for prevention trials (4,5). In a similar vein, we hope that our proposed guidelines or some related construct will facilitate the development of biomarker-based screening tools for early detection of cancer. Although deviations from these guidelines may be necessary in specific applications, our proposal will, at the minimum, provide a checklist of issues that should be addressed at each phase of development before proceeding to the next.

The centromere paradox: stable inheritance with rapidly evolving DNA.

Abstract: Every eukaryotic chromosome has a centromere, the locus responsible for poleward movement at mitosis and meiosis. Although conventional loci are specified by their DNA sequences, current evidence favors a chromatin-based inheritance mechanism for centromeres. The chromosome segregation machinery is highly conserved across all eukaryotes, but the DNA and protein components specific to centromeric chromatin are evolving rapidly. Incompatibilities between rapidly evolving centromeric components may be responsible for both the organization of centromeric regions and the reproductive isolation of emerging species.

Variation is the spice of life.

Abstract: Assembling a comprehensive catalog of common human sequence polymorphisms is a key goal of the Human Genome Project. The International SNP Map Working Group has compiled a map of 1.4 million single-nucleotide polymorphisms. What fraction of all human sequence variation is captured in this collection, and what further advances are needed before the map becomes a useful tool for genetic studies?

Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on virus-infected cells.

Abstract: NKG2D is an activating receptor that stimulates innate immune responses by natural killer cells upon engagement by MIC ligands, which are induced by cellular stress. Because NKG2D is also present on most CD8αβ T cells, it may modulate antigen-specific T cell responses, depending on whether MIC molecules—distant homologs of major histocompatibility complex (MHC) class I with no function in antigen presentation—are induced on the surface of pathogen-infected cells. We found that infection by cytomegalovirus (CMV) resulted in substantial increases in MIC on cultured fibroblast and endothelial cells and was associated with induced MIC expression in interstitial pneumonia. MIC engagement of NKG2D potently augmented T cell antigen receptor (TCR)-dependent cytolytic and cytokine responses by CMV-specific CD28− CD8αβ T cells. This function overcame viral interference with MHC class I antigen presentation. Combined triggering of TCR-CD3 complexes and NKG2D induced interleukin 2 production and T cell proliferation. Thus NKG2D functioned as a costimulatory receptor that can substitute for CD28.

Transplantation of bone marrow as compared with peripheral-blood cells from HLA-identical relatives in patients with hematologic cancers

Abstract: Background In recipients of allogeneic hematopoietic-cell transplants, peripheral-blood cells mobilized with the use of filgrastim (recombinant granulocyte colony-stimulating factor) engraft more rapidly than bone marrow. However, the relative effects of these techniques on the rates of acute and chronic graft-versus-host disease, overall survival, and disease-free survival have not been determined in randomized studies. Methods Between March 1996 and July 1999, 172 patients (12 to 55 years of age) with hematologic cancer were randomly assigned to receive either bone marrow or filgrastim-mobilized peripheral-blood cells from HLA-identical relatives for hematopoietic rescue after the treatment of hematologic cancer with high doses of chemotherapy, with or without radiation. Results The recovery of both neutrophils and platelets was faster with peripheral-blood cells than with marrow (P<0.001 for both comparisons). The cumulative incidence of grade II, III, or IV acute graft-versus-host disease at 100 days ...

Night Shift Work, Light at Night, and Risk of Breast Cancer

Abstract: Background Exposure to light at night may increase the risk of breast cancer by suppressing the normal nocturnal production of melatonin by the pineal gland, which, in turn, could increase the release of estrogen by the ovaries. This study investigated whether such exposure is associated with an increased risk of breast cancer in women. Methods Case patients (n = 813), aged 20-74 years, were diagnosed from November 1992 through March 1995; control subjects (n = 793) were identified by random-digit dialing and were frequency matched according to 5-year age groups. An in-person interview was used to gather information on sleep habits and bedroom lighting environment in the 10 years before diagnosis and lifetime occupational history. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by use of conditional logistic regression, with adjustment for other potential risk factors. Results Breast cancer risk was increased among subjects who frequently did not sleep during the period of the night when melatonin levels are typically at their highest (OR = 1.14 for each night per week; 95% CI = 1.01 to 1.28). Risk did not increase with interrupted sleep accompanied by turning on a light. There was an indication of increased risk among subjects with the brightest bedrooms. Graveyard shiftwork was associated with increased breast cancer risk (OR = 1.6; 95% CI = 1.0 to 2.5), with a trend of increased risk with increasing years and with more hours per week of graveyard shiftwork (P =.02, Wald chi-squared test). Conclusion The results of this study provide evidence that indicators of exposure to light at night may be associated with the risk of developing breast cancer.

A physical map of the human genome.

Abstract: The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.

Efficacy and Safety of Gemtuzumab Ozogamicin in Patients With CD33-Positive Acute Myeloid Leukemia in First Relapse

Abstract: PURPOSE: Three open-label, multicenter trials were conducted to evaluate the efficacy and safety of single-agent Mylotarg (gemtuzumab ozogamicin; CMA-676; Wyeth Laboratories, Philadelphia, PA), an antibody-targeted chemotherapy agent, in patients with CD33-positive acute myeloid leukemia (AML) in untreated first relapse. PATIENTS AND METHODS: The study population comprised 142 patients with AML in first relapse with no history of an antecedent hematologic disorder and a median age of 61 years. All patients received Mylotarg as a 2-hour intravenous infusion, at a dose of 9 mg/m2, at 2-week intervals for two doses. Patients were evaluated for remission, survival, and treatment-emergent adverse events. RESULTS: Thirty percent of patients treated with Mylotarg obtained remission as characterized by 5% or less blasts in the marrow, recovery of neutrophils to at least 1,500/μL, and RBC and platelet transfusion independence. Although patients treated with Mylotarg had relatively high incidences of myelosuppressi...

Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation.

Abstract: Epigenetic silenced alleles of the Arabidopsis SUPERMANlocus (the clark kent alleles) are associated with dense hypermethylation at noncanonical cytosines (CpXpG and asymmetric sites, where X = A, T, C, or G). A genetic screen for suppressors of a hypermethylated clark kent mutant identified nine loss-of-function alleles of CHROMOMETHYLASE3(CMT3), a novel cytosine methyltransferase homolog. These cmt3 mutants display a wild-type morphology but exhibit decreased CpXpG methylation of the SUP gene and of other sequences throughout the genome. They also show reactivated expression of endogenous retrotransposon sequences. These results show that a non-CpG DNA methyltransferase is responsible for maintaining epigenetic gene silencing.

Second Malignant Neoplasms in Five-Year Survivors of Childhood Cancer: Childhood Cancer Survivor Study

Abstract: Background: Because survival rates among childhood cancer patients are increasing, assessing the risk of second and subsequent malignant neoplasms (SMNs) is ever more important. Using the Childhood Cancer Survivor Study cohort, we identified the risk of SMNs. Methods: A retrospective cohort of 13 581 children diagnosed with common cancers before age 21 years and surviving at least 5 years was constructed with the use of data from patients treated at 25 U.S. and Canadian institutions. SMNs were ascertained through selfadministered questionnaires and verified by pathology reports. Information on therapeutic exposures was abstracted from medical records. The risk of SMN was evaluated by standardized incidence ratios (SIRs) and excess absolute risk. Poisson multiple regression models were used to assess the impact of host and therapy factors on the risk of developing SMNs. All statistical tests were two-sided. Results: In 298 individuals, 314 SMNs were identified (SIR = 6.38; 95% confidence interval [CI] = 5.69 to 7.13). The largest observed excess SMNs were bone and breast cancers (SIR = 19.14 [95% CI = 12.72 to 27.67] and SIR = 16.18 [95% CI = 12.35 to 20.83], respectively). A statistically significant excess of SMNs followed all childhood cancers. In multivariate regression models adjusted for therapeutic radiation exposure, SMNs of any type were independently associated with female sex (P<.001), childhood cancer at a younger age (P for trend <.001), childhood Hodgkin’s disease or soft-tissue sarcoma (P<.001 and P = .01, respectively), and exposure to alkylating agents (P for trend = .02). Twenty years after the childhood cancer diagnosis, the cumulative estimated SMN incidence was 3.2%. However, only 1.88 excess malignancies occurred per 1000 years of patient follow-up. Conclusions: Success in treating children with cancer should not be overshadowed by the incidence of SMNs. However, patients and health-care providers must be aware of risk factors for SMNs so that surveillance is focused and early prevention strategies are implemented. [J Natl Cancer Inst 2001;93:618–29]

Chemosensitivity prediction by transcriptional profiling

Abstract: In an effort to develop a genomics-based approach to the prediction of drug response, we have developed an algorithm for classification of cell line chemosensitivity based on gene expression profiles alone. Using oligonucleotide microarrays, the expression levels of 6,817 genes were measured in a panel of 60 human cancer cell lines (the NCI-60) for which the chemosensitivity profiles of thousands of chemical compounds have been determined. We sought to determine whether the gene expression signatures of untreated cells were sufficient for the prediction of chemosensitivity. Gene expression-based classifiers of sensitivity or resistance for 232 compounds were generated and then evaluated on independent sets of data. The classifiers were designed to be independent of the cells’ tissue of origin. The accuracy of chemosensitivity prediction was considerably better than would be expected by chance. Eighty-eight of 232 expression-based classifiers performed accurately (with P < 0.05) on an independent test set, whereas only 12 of the 232 would be expected to do so by chance. These results suggest that at least for a subset of compounds genomic approaches to chemosensitivity prediction are feasible.

Principles for the buffering of genetic variation.

Abstract: Most genetic research has used inbred organisms and has not explored the complexity of natural genetic variation present in outbred populations. The translation of genotype to phenotype is complicated by gene interactions observed as epistasis, canalization, robustness, or buffering. Analysis of double mutations in inbred experimental organisms suggests some principles for gene interaction that may apply to natural variation as well. The buffering of variation in one gene is most often due to a small number of other genes that function in the same biochemical process. However, buffering can also result from genes functioning in processes extrinsic to that of the primary gene.

High-Throughput Screening for Induced Point Mutations

Abstract: With the completion of genome sequencing projects, emphasis in genomics has shifted from analyzing sequences to understanding gene function, and effective reverse genetic strategies are increasingly in demand. Here we report adaptations of the targeting induced local lesions in genomes (TILLING)

Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility

Abstract: Homing endonucleases confer mobility to their host intervening sequence, either an intron or intein, by catalyzing a highly specific double-strand break in a cognate allele lacking the intervening sequence. These proteins are characterized by their ability to bind long DNA target sites (14–40 bp) and their tolerance of minor sequence changes in these sites. A wealth of biochemical and structural data has been generated for these enzymes over the past few years. Herein we review our current understanding of homing endonucleases, including their diversity and evolution, DNA-binding and catalytic mechanisms, and attempts to engineer them to bind novel DNA substrates.

Ca2+/CaM-Dependent Kinases: From Activation to Function

Abstract: Calmodulin (CaM) is an essential protein that serves as a ubiquitous intracellular receptor for Ca2+. The Ca2+/CaM complex initiates a plethora of signaling cascades that culminate in alteration of...

Intake of Fruits and Vegetables and Risk of Breast Cancer A Pooled Analysis of Cohort Studies

Abstract: Context: Some epidemiologic studies suggest that elevated fruit and vegetable consumption is associated with a reduced risk of breast cancer. However, most have been case-control studies in which recall and selection bias may influence the results. Additionally, publication bias may have influenced the literature on associations for specific fruit and vegetable subgroups. Objective: To examine the association between breast cancer and total and specific fruit and vegetable group intakes using standardized exposure definitions. Data Sources/Study Selection: Eight prospective studies that had at least 200 incident breast cancer cases, assessed usual dietary intake, and completed a validation study of the diet assessment method or a closely related instrument were included in these analyses. Data Extraction: Using the primary data from each of the studies, we calculated study-specific relative risks (RRs) that were combined using a random-effects model. Data Synthesis: The studies included 7377 incident invasive breast cancer cases occurring among 351825 women whose diet was analyzed at baseline. For comparisons of the highest vs lowest quartiles of intake, weak, nonsignificant associations were observed for total fruits (pooled multivariate RR, 0.93; 95% confidence interval [CI], 0.86-1.00; P for trend = .08), total vegetables (RR, 0.96; 95% CI, 0.89-1.04; P for trend = .54), and total fruits and vegetables (RR, 0.93; 95% CI, 0.86-1.00; P for trend = .12). No additional benefit was apparent in comparisons of the highest and lowest deciles of intake. No associations were observed for green leafy vegetables, 8 botanical groups, and 17 specific fruits and vegetables. Conclusion: These results suggest that fruit and vegetable consumption during adulthood is not significantly associated with reduced breast cancer risk.

Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family

Abstract: NKG2D is an activating receptor that is expressed on most natural killer (NK) cells, CD8 alphabeta T cells, and gammadelta T cells. Among its ligands is the distant major histocompatibility complex class I homolog MICA, which has no function in antigen presentation but is induced by cellular stress. To extend previous functional evidence, the NKG2D-MICA interaction was studied in isolation. NKG2D homodimers formed stable complexes with monomeric MICA in solution, demonstrating that no other components were required to facilitate this interaction. MICA glycosylation was not essential but enhanced complex formation. Soluble NKG2D also bound to cell surface MICB, which has structural and functional properties similar to those of MICA. Moreover, NKG2D stably interacted with surface molecules encoded by three newly identified cDNA sequences (N2DL-1, -2, and -3), which are identical to the human ULBP proteins and may represent homologs of the mouse retinoic acid-early inducible family of NKG2D ligands. Because of the substantial sequence divergence among these molecules, these results indicated promiscuous modes of receptor binding. Comparison of allelic variants of MICA revealed large differences in NKG2D binding that were associated with a single amino acid substitution at position 129 in the alpha2 domain. Varying affinities of MICA alleles for NKG2D may affect thresholds of NK-cell triggering and T-cell modulation.

Selection and fading of resistance mutations in women and infants receiving nevirapine to prevent HIV-1 vertical transmission (HIVNET 012).

Abstract: Objective To examine the emergence and fading of NVP resistance (NVP(R)) mutations in HIV-1-infected Ugandan women and infants who received single dose NVP to prevent HIV-1 vertical transmission. Design We examined NVP(R) in women and infants who received NVP in the HIVNET 012 clinical trial, including 41 out of 48 women with infected infants, 70 randomly-selected women with uninfected infants, and 33 out of 49 infected infants. Methods Plasma HIV-1 was analyzed using the Applied Biosystems ViroSeq HIV-1 Genotyping System. Results NVP(R) mutations were detected in 21 out of 111 (19%) women tested 6-8 weeks after delivery. The rate of NVP(R) was similar among women whose infants were or were not HIV-1 infected. K103N was the most common mutation detected. NVP(R) mutations faded from detection within 12-24 months in all 11 evaluable women. High baseline viral load and low baseline CD4 cell count were associated with development of NVP(R). NVP(R) mutations were detected in 11 out of 24 (46%) evaluable infants who were infected by 6-8 weeks of age. The most common NVP(R) mutation detected in infants was Y181C. Those mutations faded from detection by 12 months of age in all seven evaluable infants. Of nine evaluable infants with late HIV-1 infection, only one had evidence of NVP(R). Conclusions NVP(R) was detected more frequently in infants than women following NVP prophylaxis, and different patterns of NVP(R) mutations were detected in women versus infants. NVP(R) was detected infrequently in infants with late HIV-1 infection. NVP-resistant HIV-1 faded from detection in women and infants over time.

Nutrient Intake and Risk of Subtypes of Esophageal and Gastric Cancer

Abstract: Incidence rates for adenocarcinoma of the esophagus and gastric cardia have been rising rapidly. We examined nutrient intake as a risk factor for esophageal and gastric cancers in a population-based case-control study in Connecticut, New Jersey, and western Washington state. Interviews were completed for cases with histologically confirmed esophageal adenocarcinoma (n = 282), adenocarcinoma of the gastric cardia (n = 255), esophageal squamous cell carcinoma (n = 206), and noncardia gastric adenocarcinoma (n = 352), along with population controls (n = 687). Associations between nutrient intake and risk of cancer were estimated by adjusted odds ratios (ORs), comparing the 75th versus the 25th percentile of intake. The following nutrients were significantly inversely associated with risk of all four tumor types: fiber, beta-carotene, folate, and vitamins C and B6. In contrast, dietary cholesterol, animal protein, and vitamin B12 were significantly positively associated with risk of all four tumor types. Dietary fat [OR, 2.18; 95% confidence interval (CI), 1.27-3.76] was significantly associated with risk of esophageal adenocarcinoma only. Dietary nitrite (OR, 1.65; 95% CI, 1.26-2.16) was associated with noncardia gastric cancer only. Vitamin C supplement use was associated with a significantly lower risk for noncardia gastric cancer (OR, 0.60; 95% CI, 0.41-0.88). Higher intake of nutrients found primarily in plant-based foods was associated with a reduced risk of adenocarcinomas of the esophagus and gastric cardia, whereas higher intake of nutrients found primarily in foods of animal origin was associated with an increased risk.

Antimycin A mimics a cell-death-inducing Bcl-2 homology domain 3.

Abstract: The Bcl-2-related survival proteins confer cellular resistance to a wide range of agents. Bcl-xL-expressing hepatocyte cell lines are resistant to tumour necrosis factor and anti-cancer drugs, but are more sensitive than isogenic control cells to antimycin A, an inhibitor of mitochondrial electron transfer. Computational molecular docking analysis predicted that antimycin A interacts with the Bcl-2 homology domain 3 (BH3)-binding hydrophobic groove of Bcl-xL. We demonstrate that antimycin A and a Bak BH3 peptide bind competitively to recombinant Bcl-2. Antimycin A and BH3 peptide both induce mitochondrial swelling and loss of DeltaPsim on addition to mitochondria expressing Bcl-xL. The 2-methoxy derivative of antimycin A3 is inactive as an inhibitor of cellular respiration but still retains toxicity for Bcl-xL+ cells and mitochondria. Finally, antimycin A inhibits the pore-forming activity of Bcl-x L in synthetic liposomes, demonstrating that a small non-peptide ligand can directly inhibit the function of Bcl-2-related proteins.

The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint

Abstract: The spindle checkpoint prevents cell cycle progression in cells that have mitotic spindle defects. Although several spindle defects activate the spindle checkpoint, the exact nature of the primary signal is unknown. We have found that the budding yeast member of the Aurora protein kinase family, Ipl1p, is required to maintain a subset of spindle checkpoint arrests. Ipl1p is required to maintain the spindle checkpoint that is induced by overexpression of the protein kinase Mps1. Inactivating Ipl1p allows cells overexpressing Mps1p to escape from mitosis and segregate their chromosomes normally. Therefore, the requirement for Ipl1p in the spindle checkpoint is not a consequence of kinetochore and/or spindle defects. The requirement for Ipl1p distinguishes two different activators of the spindle checkpoint: Ipl1p function is required for the delay triggered by chromosomes whose kinetochores are not under tension, but is not required for arrest induced by spindle depolymerization. Ipl1p localizes at or near kinetochores during mitosis, and we propose that Ipl1p is required to monitor tension at the kinetochore.

Crystal structure of a hairpin ribozyme–inhibitor complex with implications for catalysis

Abstract: The hairpin ribozyme catalyses sequence-specific cleavage of RNA The active site of this natural RNA results from the docking of two irregular helices: stems A and B One strand of stem A harbours the scissile bond The 24 A resolution structure of a hairpin ribozyme-inhibitor complex reveals that the ribozyme aligns the 2'-OH nucleophile and the 5'-oxo leaving group by twisting apart the nucleotides that flank the scissile phosphate The base of the nucleotide preceding the cleavage site is stacked within stem A; the next nucleotide, a conserved guanine, is extruded from stem A and accommodated by a highly complementary pocket in the minor groove of stem B Metal ions are absent from the active site The bases of four conserved purines are positioned potentially to serve as acid-base catalysts This is the first structure determination of a fully assembled ribozyme active site that catalyses a phosphodiester cleavage without recourse to metal ions

Haematopoietic cell transplantation as immunotherapy

Abstract: The graft-versus-tumour effect seen after allogeneic (genetically different) haematopoietic cell transplantation for human malignancies represents the clearest example of the power of the human immune system to eradicate cancer Recent advances in our understanding of the immunobiology of stem-cell engraftment, tolerance and tumour eradication are allowing clinicians to better harness this powerful effect

Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA.

Abstract: The major histocompatibility complex (MHC) class I homolog, MICA, is a stress-inducible ligand for NKG2D, a C-type lectin−like activating immunoreceptor. The crystal structure of this ligand-receptor complex that we report here reveals an NKG2D homodimer bound to a MICA monomer in an interaction that is analogous to that seen in T cell receptor−MHC class I protein complexes. Similar surfaces on each NKG2D monomer interact with different surfaces on either the α1 or α2 domains of MICA. The binding interactions are large in area and highly complementary. The central section of the α2-domain helix, disordered in the structure of MICA alone, is ordered in the complex and forms part of the NKG2D interface. The extensive flexibility of the interdomain linker of MICA is shown by its altered conformation when crystallized alone or in complex with NKG2D.