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.

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.

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.

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.

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.

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.

Zebrafish Hagoromo mutants up-regulate fgf8 postembryonically and develop neuroblastoma.

We screened an existing collection of zebrafish insertional mutants for cancer susceptibility by histologic examination of heterozygotes at 2 years of age. As most mutants had no altered cancer predisposition, this provided the first comprehensive description of spontaneous tumor spectrum and frequency in adult zebrafish. Moreover, the screen identified four lines, each carrying a different dominant mutant allele of Hagoromo previously linked to adult pigmentation defects, which develop tumors with high penetrance and that histologically resemble neuroblastoma. These tumors are clearly neural in origin, although they do not express catecholaminergic neuronal markers characteristic of human neuroblastoma. The zebrafish tumors result from inappropriate maintenance of a cell population within the cranial ganglia that are likely neural precursors. These neoplasias typically remain small but they can become highly aggressive, initially traveling along cranial nerves, and ultimately filling the head. The developmental origin of these tumors is highly reminiscent of human neuroblastoma. The four mutant Hagoromo alleles all contain viral insertions in the fbxw4 gene, which encodes an F-box WD40 domain-containing protein. However, although one allele clearly reduced the levels of fbxw4 mRNA, the other three insertions had no detectable effect on fbw4 expression. Instead, we showed that all four mutations result in the postembryonic up-regulation of the neighboring gene, fibroblast growth factor 8 (fgf8). Moreover, fgf8 is highly expressed in the tumorigenic lesions. Although fgf8 overexpression is known to be associated with breast and prostate cancer in mammals, this study provides the first evidence that fgf8 misregulation can lead to neural tumors.

Many ribosomal protein mutations are associated with growth impairment and tumor predisposition in zebrafish.

We have characterized 28 zebrafish lines with heterozygous mutations in ribosomal protein (rp) genes, and found that 17 of these are prone to develop zebrafish malignant peripheral nerve sheath tumors (zMPNST). Heterozygotes from the vast majority of tumor-prone rp lines were found to be growth-impaired, though not all growth-impaired rp lines were tumor-prone. Significantly, however, the rp lines with the greatest incidence of zMPNSTs all displayed a growth impairment. Furthermore, heterozygous cells from one tumor-prone rp line were out-competed by wild-type cells in chimeric embryos. The growth impairment resulting from heterozygosity for many rp genes suggests that a global defect in protein translation exists in these lines, raising the possibility that a translation defect that precedes tumor development is predictive of tumorigenesis.

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.

Interaction of polycyclic aromatic hydrocarbons with human cytochrome P450 1B1 in inhibiting catalytic activity.

Eleven polycyclic aromatic hydrocarbons (PAHs) and 14 acetylenic PAHs and biphenyls were used to analyze interactions with cytochrome P450 (P450) 1B1 in inhibiting catalytic activity, using 7-ethoxyresorufin O-deethylation (EROD) as a model reaction. Most of the chemicals examined were direct inhibitors of P450 1B1 except for 4-(1-propynyl)biphenyl, a mechanism-based inhibitor. In the case of direct inhibition of EROD activity {15 of 24 chemicals, e.g., benzo[a]pyrene, 1-(1-propynyl)pyrene, and 3-(1-propynyl)phenanthrene}, restoration of the EROD activity occurred with increasing incubation time, and kinetic analysis showed that EROD K(m) values were higher with these inhibitors at initial stages of incubation but became lower with increasing incubation time. With the other nine chemicals, the K(m) values for P450 1B1-mediated EROD increased during the incubations. Acetylenic inhibitors, but not the 11 PAHs, induced reverse type I spectral changes with P450 1B1, and the low dissociation constants (K(s)) suggested a role for such interaction in the inhibition of catalytic activity. Studies of quenching of P450 1B1-derived fluorescence with inhibitors demonstrated that acetylenic inhibitors and PAHs interacted rapidly with P450 1B1, with K(d) values < 10 microM. However, studies of quenching of inhibitor-derived fluorescence with P450 1B1 showed these interactions to be different, that is, B[a]P interacted with P450 1B1 more slowly. Molecular docking of P450 1B1, based on P450 1A2 crystal structure, suggested that there are clear differences in the interaction of PAH inhibitors with P450 1B1 and 1A2 and that these differences may explain why PAH inhibitors inhibit P450 1 enzymes by different mechanisms. The results suggest that P450 1B1 interacts with synthetic polycyclic aromatic acetylenes and PAHs in different ways, depending on the chemicals, and that these differences in interactions may explain how these chemicals inhibit P450 activities by different mechanisms.

Inactivation of serine protease Matriptase1a by its inhibitor Hai1 is required for epithelial integrity of the zebrafish epidermis.

Epithelial integrity requires the adhesion of cells to each other as well as to an underlying basement membrane. The modulation of adherence properties is crucial to morphogenesis and wound healing, and deregulated adhesion has been implicated in skin diseases and cancer metastasis. Here, we describe zebrafish that are mutant in the serine protease inhibitor Hai1a (Spint1la), which display disrupted epidermal integrity. These defects are further enhanced upon combined loss of hai1a and its paralog hai1b. By applying in vivo imaging, we demonstrate that Hai1-deficient keratinocytes acquire mesenchymal-like characteristics, lose contact with each other, and become mobile and more susceptible to apoptosis. In addition, inflammation of the mutant skin is evident, although not causative of the epidermal defects. Only later, the epidermis exhibits enhanced cell proliferation. The defects of hai1 mutants can be phenocopied by overexpression and can be fully rescued by simultaneous inactivation of the serine protease Matriptase1a (St14a), indicating that Hai1 promotes epithelial integrity by inhibiting Matriptase1a. By contrast, Hepatocyte growth factor (Hgf), a well-known promoter of epithelial-mesenchymal transitions and a prime target of Matriptase1 activity, plays no major role. Our work provides direct genetic evidence for antagonistic in vivo roles of Hai1 and Matriptase1a to regulate skin homeostasis and remodeling.