Molecular mechanisms of the preventable causes of cancer in the United States.

Annually, there are 1.6 million new cases of cancer and nearly 600,000 cancer deaths in the United States alone. The public health burden associated with these numbers has motivated enormous research efforts into understanding the root causes of cancer. These efforts have led to the recognition that between 40% and 45% of cancers are associated with preventable risk factors and, importantly, have identified specific molecular mechanisms by which these exposures modify human physiology to induce or promote cancer. The increasingly refined knowledge of these mechanisms, which we summarize here, emphasizes the need for greater efforts toward primary cancer prevention through mitigation of modifiable risk factors. It also suggests exploitable avenues for improved secondary prevention (which includes the development of therapeutics designed for cancer interception and enhanced techniques for noninvasive screening and early detection) based on detailed knowledge of early neoplastic pathobiology. Such efforts would complement the current emphasis on the development of therapeutic approaches to treat established cancers and are likely to result in far greater gains in reducing morbidity and mortality.

A vertebrate gene, ticrr, is an essential checkpoint and replication regulator.

Eukaryotes have numerous checkpoint pathways to protect genome fidelity during normal cell division and in response to DNA damage. Through a screen for G2/M checkpoint regulators in zebrafish, we identified ticrr (for TopBP1-interacting, checkpoint, and replication regulator), a previously uncharacterized gene that is required to prevent mitotic entry after treatment with ionizing radiation. Ticrr deficiency is embryonic-lethal in the absence of exogenous DNA damage because it is essential for normal cell cycle progression. Specifically, the loss of ticrr impairs DNA replication and disrupts the S/M checkpoint, leading to premature mitotic entry and mitotic catastrophe. We show that the human TICRR ortholog associates with TopBP1, a known checkpoint protein and a core component of the DNA replication preinitiation complex (pre-IC), and that the TICRR-TopBP1 interaction is stable without chromatin and requires BRCT motifs essential for TopBP1's replication and checkpoint functions. Most importantly, we find that ticrr deficiency disrupts chromatin binding of pre-IC, but not prereplication complex, components. Taken together, our data show that TICRR acts in association with TopBP1 and plays an essential role in pre-IC formation. It remains to be determined whether Ticrr represents the vertebrate ortholog of the yeast pre-IC component Sld3, or a hitherto unknown metazoan replication and checkpoint regulator.

Comparative oncogenomic analysis of copy number alterations in human and zebrafish tumors enables cancer driver discovery.

The identification of cancer drivers is a major goal of current cancer research. Finding driver genes within large chromosomal events is especially challenging because such alterations encompass many genes. Previously, we demonstrated that zebrafish malignant peripheral nerve sheath tumors (MPNSTs) are highly aneuploid, much like human tumors. In this study, we examined 147 zebrafish MPNSTs by massively parallel sequencing and identified both large and focal copy number alterations (CNAs). Given the low degree of conserved synteny between fish and mammals, we reasoned that comparative analyses of CNAs from fish versus human MPNSTs would enable elimination of a large proportion of passenger mutations, especially on large CNAs. We established a list of orthologous genes between human and zebrafish, which includes approximately two-thirds of human protein-coding genes. For the subset of these genes found in human MPNST CNAs, only one quarter of their orthologues were co-gained or co-lost in zebrafish, dramatically narrowing the list of candidate cancer drivers for both focal and large CNAs. We conclude that zebrafish-human comparative analysis represents a powerful, and broadly applicable, tool to enrich for evolutionarily conserved cancer drivers.

Highly aneuploid zebrafish malignant peripheral nerve sheath tumors have genetic alterations similar to human cancers.

Aneuploidy is a hallmark of human cancers, but most mouse cancer models lack the extensive aneuploidy seen in many human tumors. The zebrafish is becoming an increasingly popular model for studying cancer. Here we report that malignant peripheral nerve sheath tumors (MPNSTs) that arise in zebrafish as a result of mutations in either ribosomal protein (rp) genes or in p53 are highly aneuploid. Karyotyping reveals that these tumors frequently harbor near-triploid numbers of chromosomes, and they vary in chromosome number from cell to cell within a single tumor. Using array comparative genomic hybridization, we found that, as in human cancers, certain fish chromosomes are preferentially overrepresented, whereas others are underrepresented in many MPNSTs. In addition, we obtained evidence for recurrent subchromosomal amplifications and deletions that may contain genes involved in cancer initiation or progression. These focal amplifications encompassed several genes whose amplification is observed in human tumors, including met, cyclinD2, slc45a3, and cdk6. One focal amplification included fgf6a. Increasing fgf signaling via a mutation that overexpresses fgf8 accelerated the onset of MPNSTs in fish bearing a mutation in p53, suggesting that fgf6a itself may be a driver of MPNSTs. Our results suggest that the zebrafish is a useful model in which to study aneuploidy in human cancer and in which to identify candidate genes that may act as drivers in fish and potentially also in human tumors.

Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development.

To rapidly identify genes required for early vertebrate development, we are carrying out a large-scale, insertional mutagenesis screen in zebrafish, using mouse retroviral vectors as the mutagen. We will obtain mutations in 450 to 500 different genes--roughly 20% of the genes that can be mutated to produce a visible embryonic phenotype in this species--and will clone the majority of the mutated alleles. So far, we have isolated more than 500 insertional mutants. Here we describe the first 75 insertional mutants for which the disrupted genes have been identified. In agreement with chemical mutagenesis screens, approximately one-third of the mutants have developmental defects that affect primarily one or a small number of organs, body shape or swimming behavior; the rest of the mutants show more widespread or pleiotropic abnormalities. Many of the genes we identified have not been previously assigned a biological role in vivo. Roughly 20% of the mutants result from lesions in genes for which the biochemical and cellular function of the proteins they encode cannot be deduced with confidence, if at all, from their predicted amino-acid sequences. All of the genes have either orthologs or clearly related genes in human. These results provide an unbiased view of the genetic construction kit for a vertebrate embryo, reveal the diversity of genes required for vertebrate development and suggest that hundreds of genes of unknown biochemical function essential for vertebrate development have yet to be identified.

The first board examination in pediatric surgery.

The inaugural certifying examination for special competence in pediatric surgery in North America was given by the American Board of Surgery (ABS) in April 1975, the day before the sixth meeting of the American Pediatric Surgical Association at a resort near San Juan, PR. The event came after failed applications before the ABS and the Advisory Board for Medical Specialties in 1957, 1961, and 1967. The specialty had matured with a scholarly publication devoted to the field (Journal of Pediatric Surgery, 1965), the establishment of standards for training and training programs (1966), and a society independent of pediatrics and devoted solely to pediatric surgery (American Pediatric Surgical Association, first meeting 1970). Harvey Beardmore had guided the successful campaign for a certificate for pediatric surgery under the aegis of the ABS that was approved in June 1972. Pediatric surgery had thus gained full recognition as a specialty of surgery. A group photograph of its participants became one of the iconic images in our specialty. Thanks to Jim and Nancy Hopkins of Windsor Heights, IA, and to their many friends and colleagues, nearly half (71 of 151) of the pediatric surgeons in the photo were identified, marking their places in the history of pediatric surgery.

Loss of p53 synthesis in zebrafish tumors with ribosomal protein gene mutations.

Zebrafish carrying heterozygous mutations for 17 different ribosomal protein (rp) genes are prone to developing malignant peripheral nerve sheath tumors (MPNSTs), a tumor type that is seldom seen in laboratory strains of zebrafish. Interestingly, the same rare tumor type arises in zebrafish that are homozygous for a loss-of-function point mutation in the tumor suppressor gene p53. For these reasons, and because p53 is widely known to be mutated in the majority of human cancers, we investigated the status of p53 in the rp(+/-) MPNSTs. Using monoclonal antibodies that we raised to zebrafish p53, we found that cells derived from rp(+/-) MPNSTs are significantly impaired in their ability to produce p53 protein even in the presence of a proteasome inhibitor and gamma-irradiation. Although the coding regions of the p53 gene remain wild type, the gene is transcribed, and overall protein production rates appear normal in rp(+/-) MPNST cells, p53 protein does not get synthesized. This defect is observed in all MPNSTs we examined that were derived from our 17 zebrafish lines with rp gene mutations. To date, studies of p53 in malignancies have focused predominantly on either p53 gene mutations or the aberrant posttranslational regulation of the p53 protein. Our results show that the appropriate amount of numerous ribosomal proteins is required for p53 protein production in vivo and that disruption of this regulation most likely contributes to tumorigenesis.

Essential genes for astroglial development and axon pathfinding during zebrafish embryogenesis.

The formation of the central nervous system depends on the coordinated development of neural and glial cell types that arise from a common precursor. Using an existing group of zebrafish mutants generated by viral insertion, we performed a "shelf-screen" to identify genes necessary for astroglial development and axon scaffold formation. We screened 274 of 315 viral insertion lines using antibodies that label axons (anti-Acetylated Tubulin) and astroglia (anti-Gfap) and identified 25 mutants with defects in gliogenesis, glial patterning, neurogenesis, and axon guidance. We also identified a novel class of mutants affecting radial glial cell numbers. Defects in astroglial patterning were always associated with axon defects, supporting an important role for axon-glial interactions during axon scaffold development. The genes disrupted in these viral lines have all been identified, providing a powerful new resource for the study of axon guidance, glio- and neurogenesis, and neuron-glial interactions during development of the vertebrate CNS.

Mutagenesis strategies in zebrafish for identifying genes involved in development and disease.

It has been nearly a decade since the completion of two large-scale chemical mutagenesis screens in zebrafish, and two years since the completion of a large-scale insertional mutagenesis. In this article, we use the accumulated data from these screens to compare the efficiency of each mutagen to isolate mutants and to identify mutated genes, and argue that the two mutagens target the same set of genes. We then review how both forward genetic screens and reverse genetic techniques, such as morpholinos and TILLING, and transgenics are being used to develop models of human disease.

Many ribosomal protein genes are cancer genes in zebrafish.

We have generated several hundred lines of zebrafish (Danio rerio), each heterozygous for a recessive embryonic lethal mutation. Since many tumor suppressor genes are recessive lethals, we screened our colony for lines that display early mortality and/or gross evidence of tumors. We identified 12 lines with elevated cancer incidence. Fish from these lines develop malignant peripheral nerve sheath tumors, and in some cases also other tumor types, with moderate to very high frequencies. Surprisingly, 11 of the 12 lines were each heterozygous for a mutation in a different ribosomal protein (RP) gene, while one line was heterozygous for a mutation in a zebrafish paralog of the human and mouse tumor suppressor gene, neurofibromatosis type 2. Our findings suggest that many RP genes may act as haploinsufficient tumor suppressors in fish. Many RP genes might also be cancer genes in humans, where their role in tumorigenesis could easily have escaped detection up to now.

A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression.

BACKGROUND: Craniofacial birth defects result from defects in cranial neural crest (NC) patterning and morphogenesis. The vertebrate craniofacial skeleton is derived from cranial NC cells and the patterning of these cells occurs within the pharyngeal arches. Substantial efforts have led to the identification of several genes required for craniofacial skeletal development such as the endothelin-1 (edn1) signaling pathway that is required for lower jaw formation. However, many essential genes required for craniofacial development remain to be identified. RESULTS: Through screening a collection of insertional zebrafish mutants containing approximately 25% of the genes essential for embryonic development, we present the identification of 15 essential genes that are required for craniofacial development. We identified 3 genes required for hyomandibular development. We also identified zebrafish models for Campomelic Dysplasia and Ehlers-Danlos syndrome. To further demonstrate the utility of this method, we include a characterization of the wdr68 gene. We show that wdr68 acts upstream of the edn1 pathway and is also required for formation of the upper jaw equivalent, the palatoquadrate. We also present evidence that the level of wdr68 activity required for edn1 pathway function differs between the 1st and 2nd arches. Wdr68 interacts with two minibrain-related kinases, Dyrk1a and Dyrk1b, required for embryonic growth and myotube differentiation, respectively. We show that a GFP-Wdr68 fusion protein localizes to the nucleus with Dyrk1a in contrast to an engineered loss of function mutation Wdr68-T284F that no longer accumulated in the cell nucleus and failed to rescue wdr68 mutant animals. Wdr68 homologs appear to exist in all eukaryotic genomes. Notably, we found that the Drosophila wdr68 homolog CG14614 could substitute for the vertebrate wdr68 gene even though insects lack the NC cell lineage. CONCLUSION: This work represents a systematic identification of approximately 25% of the essential genes required for craniofacial development. The identification of zebrafish models for two human disease syndromes indicates that homologs to the other genes are likely to also be relevant for human craniofacial development. The initial characterization of wdr68 suggests an important role in craniofacial development for the highly conserved Wdr68-Dyrk1 protein complexes.

Identification of zebrafish insertional mutants with defects in visual system development and function.

Genetic analysis in zebrafish has been instrumental in identifying genes necessary for visual system development and function. Recently, a large-scale retroviral insertional mutagenesis screen, in which 315 different genes were mutated, that resulted in obvious phenotypic defects by 5 days postfertilization was completed. That the disrupted gene has been identified in each of these mutants provides unique resource through which the formation, function, or physiology of individual organ systems can be studied. To that end, a screen for visual system mutants was performed on 250 of the mutants in this collection, examining each of them histologically for morphological defects in the eye and behaviorally for overall visual system function. Forty loci whose disruption resulted in defects in eye development and/or visual function were identified. The mutants have been divided into the following phenotypic classes that show defects in: (1) morphogenesis, (2) growth and central retinal development, (3) the peripheral marginal zone, (4) retinal lamination, (5) the photoreceptor cell layer, (6) the retinal pigment epithelium, (7) the lens, (8) retinal containment, and (9) behavior. The affected genes in these mutants highlight a diverse set of proteins necessary for the development, maintenance, and function of the vertebrate visual system.

Impact of ontogeny on linezolid disposition in neonates and infants.

The impact of age on linezolid disposition during the first few months of life has not been previously investigated. We characterized linezolid pharmacokinetics after a single, 10.0-mg/kg intravenous dose in 42 infants stratified as follows: group 1 (n = 9), gestational age <34 weeks and postnatal age <8 days; group 2 (n = 7), gestational age <34 weeks and postnatal age 8 days to 12 weeks; group 3 (n = 11), gestational age >or=34 weeks and postnatal age <8 days; and group 4 (n = 15), gestational age >or=34 weeks and postnatal age 8 days to 12 weeks. Linezolid was quantitated by a validated HPLC-triple-quadrupole mass spectrometer method from repeated blood samples (n = 7, 0.3 mL each) obtained over a 12-hour period. Pharmacokinetic parameters were determined by standard model-dependent techniques. The values (mean +/- SD) for total body clearance (CL) (0.25 +/- 0.12 L x h(-1) x kg(-1)), apparent volume of distribution (VD(ss)) (0.75 +/- 0.19 L/kg), and elimination half-life (t(1/2)) (2.8 +/- 2.1 hours) from the entire study cohort were similar to values reported previously for children and adolescents. Examination of the linezolid pharmacokinetics as a function of age revealed that CL increased rapidly during the first week of life and as a function of postnatal age. Age stratification revealed lower values for CL in those infants aged less than 8 days (group 1, 0.12 +/- 0.06 L x h(-1) x kg(-1); group 3, 0.23 +/- 0.12 L x h(-1) x kg(-1)) as compared with those aged 8 days to 12 weeks (group 2, 0.31 +/- 0.07 L x h(-1) x kg(-1); group 4, 0.31 +/- 0.10 L x h(-1) x kg(-1)). In contrast to the results for CL, gestational age served to be the most useful predictor of VD(ss). Evaluation of the pharmacokinetic data would appear to support the use of linezolid dosing regimens currently approved for infants and young children in neonates with postnatal age greater than 7 days.

Linezolid pharmacokinetics in pediatric patients: an overview.

BACKGROUND: There are a number of physiologic and developmental differences between children and adults that can influence the absorption, distribution, metabolism and elimination of a drug. Therefore it is important to determine the specific pharmacokinetic characteristics for individual drugs in pediatric patients so that appropriate age-specific dosage regimens can be developed and evaluated in clinical trials. This review summarizes the pharmacokinetic parameters of linezolid in pediatric patients and the rationale for the approved dosing recommendations for this population. METHODS: The pharmacokinetics of linezolid in pediatric patients has been evaluated in 4 clinical trials, including >180 patients ranging in age from preterm newborn infants up to 18 years of age. In all of these studies, patients received a single intravenous dose of linezolid. Plasma linezolid concentrations have been determined by validated high performance liquid chromatography (adult studies) or liquid chromatography/mass spectrometry/mass spectrometry (pediatric studies) methods. RESULTS: The pharmacokinetics of linezolid, especially elimination clearance, is age-dependent. Children younger than 12 years of age have a smaller area under the drug concentration-time curve, a faster clearance and a shorter elimination half-life than adults. Although clearance rates in newborn infants are similar to those in adults, clearance increases rapidly during the first week of life, becoming 2- to 3-fold higher than in adults by the seventh day of life. The clearance of linezolid decreases gradually among young children, becoming similar to adult values by adolescence. The pharmacokinetics of linezolid in children age 12 years and older is not significantly different from that of adults. CONCLUSIONS: Because of the higher clearance and lower area under the drug concentration-time curve, a shorter dosing interval for linezolid is required for children younger than 12 years of age to produce adequate drug exposure against target Gram-positive pathogens.

The pharmacokinetics of linezolid are not affected by concomitant intake of the antioxidant vitamins C and E.

In vitro metabolism experiments have suggested a possible role for endogenous reactive oxygen species (ROS) in the in vivo clearance of linezolid, a synthetic antibiotic of the oxazolidinone class. This observation has resulted in the hypothesis that dietary antioxidant supplements might disturb the balance of ROS in vivo and thereby lower the clearance of linezolid. The purpose of this open-label, two-group parallel design study was to investigate whether continuous intake of widely used vitamin C or vitamin E will affect the pharmacokinetics of linezolid. A total of 28 healthy volunteers (27 male and 1 female), including 22 of Chinese origin, were administered a single oral dose of 600 mg linezolid on days 1 and 8. Half of the subjects received daily oral doses of 1000 mg vitamin C on days 2 through 9, whereas the other half were administered daily oral doses of 800 IU vitamin E during the same time period. Serial blood samples for assessment of the pharmacokinetic parameters of linezolid and its two inactive metabolites were collected on days 1 and 8, whereas vitamin concentrations were measured prior to and after the vitamin intake on these days. Urine was collected on days 1 and 8 to assess the fraction of dose excreted as linezolid and its major metabolites. All linezolid samples were analyzed according to validated HPLC/MS/MS methods. Linezolid was well tolerated in both groups with no reported clinically significant adverse events. No significant changes were found between the day 1 and day 8 AUC0- infinity and Cmax values of linezolid in either the vitamin C treatment group (p = 0.55 and p = 0.64, respectively) or the vitamin E treatment group (p = 0.06 and p = 0.49, respectively). Assessment of other pharmacokinetic parameters did not imply any change across the study groups. In conclusion, linezolid pharmacokinetics are not affected by concomitant administration with vitamins C and E. Therefore, no dose adjustment is necessary in patients taking vitamin C or vitamin E. These no-effect drug interaction data are in accord with current literature indicating that antioxidant vitamins have only subtle effects on overall ROS balance in vivo.

Satellite DNA hypomethylation in karyotyped Wilms tumors.

Previously, a high percentage of Wilms tumors was found to be hypomethylated in the unusually long region of pericentromeric satellite DNA on chromosome 1. We now show that these pediatric cancers are also frequently hypomethylated in centromeric satellite DNA throughout the genome and compare satellite DNA hypomethylation with chromosome rearrangements. Relative to normal somatic tissues, 83% of the tumors were hypomethylated in centromeric satellite alpha DNA. This was assessed by blot hybridization under low-stringency conditions after digestion with CpG methylation-sensitive restriction endonucleases. Similar results were obtained with different enzymes, indicating generalized hypomethylation of centromeric DNA. Hypomethylation of another heterochromatic sequence, juxtacentromeric satellite 2 DNA of chromosome 1, was observed in 51% of the tumors. By cytogenetic analysis, rearrangements in the centromeric or juxtacentromeric heterochromatin of chromosome 1 were the most frequent structural aberration and were seen in 14% of the tumors. Tumors with such rearrangements had hypomethylation of satellite DNA in the pericentromeric region. These results show a high degree of targeting of DNA hypomethylation to centromeric and juxtacentromeric satellite DNA sequences in cancer and are consistent with satellite DNA hypomethylation contributing to, but not sufficing for, karyotypic instability in cancer and possibly playing other roles in carcinogenesis.