A model for investigating developmental eye repair in Xenopus laevis.

Vertebrate eye development is complex and requires early interactions between neuroectoderm and surface ectoderm during embryogenesis. In the African clawed frog, Xenopus laevis, individual eye tissues such as the retina and lens can undergo regeneration. However, it has been reported that removal of either the specified eye field at the neurula stage or the eye during tadpole stage does not induce replacement. Here we describe a model for investigating Xenopus developmental eye repair. We found that tailbud embryos can readily regrow eyes after surgical removal of over 83% of the specified eye and lens tissues. The regrown eye reached a comparable size to the contralateral control by 5 days and overall animal development was normal. It contained the expected complement of eye cell types (including the pigmented epithelium, retina and lens), and is connected to the brain. Our data also demonstrate that apoptosis, an early mechanism that regulates appendage regeneration, is also required for eye regrowth. Treatment with apoptosis inhibitors (M50054 or NS3694) blocked eye regrowth by inhibiting caspase activation. Together, our findings indicate that frog embryos can undergo successful eye repair after considerable tissue loss and reveals a required role for apoptosis in this process. Furthermore, this Xenopus model allows for rapid comparisons of productive eye repair and developmental pathways. It can also facilitate the molecular dissection of signaling mechanisms necessary for initiating repair.

Effects of the biocide methylisothiazolinone on Xenopus laevis wound healing and tail regeneration.

The South African clawed frog, Xenopus laevis, has a strong history as a suitable model for environmental studies. Its embryos and transparent tadpoles are highly sensitive to the environment and their developmental processes are well described. It is also amenable for molecular studies. These characteristics enable its use for rapid identification and understanding of exposure-induced defects. To investigate the consequences of chemical exposure on aquatic animals, Xenopus laevis embryos and tadpoles were exposed to the biocide, methylisothiazolinone (MIT). Frog tadpoles exposed to MIT following tail amputation lost their natural regenerative ability. This inhibition of regeneration led to a failure to regrow tissues including the spinal cord, muscle, and notochord. This MIT-dependent regenerative defect is due to a failure to close the amputation wound. A wound healing assay revealed that while untreated embryos close their wounds within one day after injury, MIT-treated animals maintained open wounds that did not reduce in size and caused lethality. Concomitant exposure of MIT with chemicals containing thiol groups such as glutathione and N-acetyl cysteine restored normal wound healing and regeneration responses in tadpoles. Together these results indicate that exposure to MIT impairs developmental wound repair and tissue regeneration in Xenopus laevis. Thus, this study reveals new aspects of MIT activity and demonstrates that Xenopus laevis is a well-suited model for facilitating future research into chemical exposure effects on injury responses.

Diabetes mellitus and cancer in Werner syndrome.

Onishi et al. recently reported here an association of diabetes mellitus (DM) with neoplasm type in newly identified patients with Werner syndrome (WS), an autosomal recessive cancer predisposition syndrome with features of premature aging that include a high risk of DM (Onishi et al. in Acta Diabetol 49(Suppl 1):259-260, 2012; Epstein et al. in Medicine 45:177-121, 1966). In contrast, we did not detect an association between DM and neoplasm type in an independent WS cohort we assembled to determine the histopathologic spectrum and type-specific risk of neoplasia in WS.

Spectrum and risk of neoplasia in Werner syndrome: a systematic review.

BACKGROUND: Werner syndrome (WS) is an autosomal recessive genetic instability and progeroid ('premature aging') syndrome which is associated with an elevated risk of cancer. OBJECTIVES: Our study objectives were to characterize the spectrum of neoplasia in WS using a well-documented study population, and to estimate the type-specific risk of neoplasia in WS relative to the general population. METHODS: We obtained case reports of neoplasms in WS patients through examining previous case series and reviews of WS, as well as through database searching in PubMed, Google Scholar, and J-EAST, a search engine for articles from Japan. We defined the spectrum (types and sites) of neoplasia in WS using all case reports, and were able to determine neoplasm type-specific risk in Japan WS patients by calculating standardized incidence and proportionate incidence ratios (SIR and SPIR, respectively) relative to Osaka Japan prefecture incidence rates. RESULTS: We used a newly assembled study population of 189 WS patients with 248 neoplasms to define the spectrum of neoplasia in WS. The most frequent neoplasms in WS patients, representing 2/3 of all reports, were thyroid neoplasms, malignant melanoma, meningioma, soft tissue sarcomas, leukemia and pre-leukemic conditions of the bone marrow, and primary bone neoplasms. Cancer risk defined by SIRs was significantly elevated in Japan-resident WS patients for the six most frequent neoplasms except leukemia, ranging from 53.5-fold for melanoma of the skin (95% CI: 24.5, 101.6) to 8.9 (95% CI: 4.9, 15.0) for thyroid neoplasms. Cancer risk as defined by SPIR was also significantly elevated for the most common malignancies except leukemia. CONCLUSIONS: WS confers a strong predisposition to several specific types of neoplasia. These results serve as a guide for WS clinical care, and for additional analyses to define the mechanistic basis for cancer in WS and the general population.

The human WRN and BLM RecQ helicases differentially regulate cell proliferation and survival after chemotherapeutic DNA damage.

Loss-of-function mutations in the human RecQ helicase genes WRN and BLM respectively cause the genetic instability/cancer predisposition syndromes Werner syndrome and Bloom syndrome. To identify common and unique functions of WRN and BLM, we systematically analyzed cell proliferation, cell survival, and genomic damage in isogenic cell lines depleted of WRN, BLM, or both proteins. Cell proliferation and survival were assessed before and after treatment with camptothecin, cis-diamminedichloroplatinum(II), hydroxyurea, or 5-fluorouracil. Genomic damage was assessed, before and after replication arrest, by gamma-H2AX staining, which was quantified at the single-cell level by flow cytometry. Cell proliferation was affected strongly by the extent of WRN and/or BLM depletion, and more strongly by BLM than by WRN depletion (P = 0.005). The proliferation of WRN/BLM-codepleted cells, in contrast, did not differ from BLM-depleted cells (P = 0.34). BLM-depleted and WRN/BLM-codepleted cells had comparably impaired survival after DNA damage, whereas WRN-depleted cells displayed a distinct pattern of sensitivity to DNA damage. BLM-depleted and WRN/BLM-codepleted cells had similar, significantly higher gamma-H2AX induction levels than did WRN-depleted cells. Our results provide new information on the role of WRN and BLM in determining cell proliferation, cell survival, and genomic damage after chemotherapeutic DNA damage or replication arrest. We also provide new information on functional redundancy between WRN and BLM. These results provide a strong rationale for further developing WRN and BLM as biomarkers of tumor chemotherapeutic responsiveness.

Hinge residue I174 is critical for proper dNTP selection by DNA polymerase beta.

DNA polymerase beta (pol beta) is the key gap-filling polymerase in base excision repair, the DNA repair pathway responsible for repairing up to 20000 endogenous lesions per cell per day. Pol beta is also widely used as a model polymerase for structure and function studies, and several structural regions have been identified as being critical for the fidelity of the enzyme. One of these regions is the hydrophobic hinge, a network of hydrophobic residues located between the palm and fingers subdomains. Previous work by our lab has shown that hinge residues Y265, I260, and F272 are critical for polymerase fidelity by functioning in discrimination of the correct from incorrect dNTP during ground state binding. Our work aimed to elucidate the role of hinge residue I174 in polymerase fidelity. To study this residue, we conducted a genetic screen to identify mutants with a substitution at residue I174 that resulted in a mutator polymerase. We then chose the mutator mutant I174S for further study and found that it follows the same general kinetic pathway as and has an overall protein folding similar to that of wild-type (WT) pol beta. Using single-turnover kinetic analysis, we found that I174S exhibits decreased fidelity when inserting a nucleotide opposite a template base G, and this loss of fidelity is due primarily to a loss of discrimination during ground state dNTP binding. Molecular dynamics simulations show that mutation of residue I174 to serine results in an overall tightening of the hinge region, resulting in aberrant protein dynamics and fidelity. These results point to the hinge region as being critical in the maintenance of the proper geometry of the dNTP binding pocket.

DNA polymerases and human diseases.

DNA polymerases function in DNA replication, repair, recombination and translesion synthesis. Currently, 15 DNA polymerase genes have been identified in human cells, belonging to four distinct families. In this review, we briefly describe the biochemical activities and known cellular roles of each DNA polymerase. Our major focus is on the phenotypic consequences of mutation or ablation of individual DNA polymerase genes. We discuss phenotypes of current mouse models and altered polymerase functions and the relationship of DNA polymerase gene mutations to human cell phenotypes. Interestingly, over 120 single nucleotide polymorphisms (SNPs) have been identified in human populations that are predicted to result in nonsynonymous amino acid substitutions of DNA polymerases. We discuss the putative functional consequences of these SNPs in relation to human disease.