Chemosensitivity of primary human fibroblasts with defective unhooking of DNA interstrand cross-links

Xeroderma pigmentosum (XP) is characterised by defects in nucleotide excision repair, ultraviolet (UV) radiation sensitivity and increased skin carcinoma. Compared to other complementation groups, XP-F patients show relatively mild cutaneous symptoms. DNA interstrand cross-linking agents are a highly cytotoxic class of DNA damage induced by common cancer chemotherapeutics such as cisplatin and nitrogen mustards. Although the XPF-ERCC1 structure-specific endonuclease is required for the repair of ICLs cellular sensitivity of primary human XP-F cells has not been established. In clonogenic survival assays, primary fibroblasts from XP-F patients were moderately sensitive to both UVC and HN2 compared to normal cells (2- to 3-fold and 3- to 5-fold, respectively). XP-A fibroblasts were considerably more sensitive to UVC (10- to 12-fold) but not sensitive to HN2. The sensitivity of XP-F fibroblasts to HN2 correlated with the defective incision or 'unhooking' step of ICL repair. Using the comet assay, XP-F cells exhibited only 20% residual unhooking activity over 24 h. Over the same time, normal and XP-A cells unhooked greater than 95% and 62% of ICLs, respectively. After HN2 treatment, ICL-associated DNA double-strand breaks (DSBs) are detected by pulse field gel electrophoresis in dividing cells. Induction and repair of DNA DSBs was normal in XP-F fibroblasts. These findings demonstrate that in primary human fibroblasts, XPF is required for the unhooking of ICLs and not for the induction or repair of ICL-associated DNA DSBs induced by HN2. In terms of cancer chemotherapy, people with mild DNA repair defects affecting ICL repair may be more prevalent in the general population than expected. Since cellular sensitivity of primary human fibroblasts usually reflects clinical sensitivity such patients with cancer would be at risk of increased toxicity.     

Homoeologous chromosomes of Xenopus laevis are highly conserved after whole-genome duplication

It has been suggested that whole-genome duplication (WGD) occurred twice during the evolutionary process of vertebrates around 450 and 500 million years ago, which contributed to an increase in the genomic and phenotypic complexities of vertebrates. However, little is still known about the evolutionary process of homoeologous chromosomes after WGD because many duplicate genes have been lost. Therefore, Xenopus laevis (2n=36) and Xenopus (Silurana) tropicalis (2n=20) are good animal models for studying the process of genomic and chromosomal reorganization after WGD because X. laevis is an allotetraploid species that resulted from WGD after the interspecific hybridization of diploid species closely related to X. tropicalis. We constructed a comparative cytogenetic map of X. laevis using 60 complimentary DNA clones that covered the entire chromosomal regions of 10 pairs of X. tropicalis chromosomes. We consequently identified all nine homoeologous chromosome groups of X. laevis. Hybridization signals on two pairs of X. laevis homoeologous chromosomes were detected for 50 of 60 (83%) genes, and the genetic linkage is highly conserved between X. tropicalis and X. laevis chromosomes except for one fusion and one inversion and also between X. laevis homoeologous chromosomes except for two inversions. These results indicate that the loss of duplicated genes and inter- and/or intrachromosomal rearrangements occurred much less frequently in this lineage, suggesting that these events were not essential for diploidization of the allotetraploid genome in X. laevis after WGD.     

A Xenopus tropicalis oligonucleotide microarray works across species using RNA from Xenopus laevis

Microarrays have great potential for the study of developmental biology. As a model system Xenopus is well suited for making the most of this potential. However, Xenopus laevis has undergone a genome wide duplication meaning that most genes are represented by two paralogues. This causes a number of problems. Most importantly the presence of duplicated genes mean that a X. laevis microarray will have less or even half the coverage of a similar sized microarray from the closely related but diploid frog Xenopus tropicalis. However, to date, X. laevis is the most commonly used amphibian system for experimental embryology. Therefore, we have tested if a microarray based on sequences from X. tropicalis will work across species using RNA from X. laevis. We produced a pilot oligonucleotide microarray based on sequences from X. tropicalis. The microarray was used to identify genes whose expression levels changed during early X. tropicalis development. The same assay was then carried out using RNA from X. laevis. The cross species experiments gave similar results to those using X. tropicalis RNA. This was true at the whole microarray level and for individual genes, with most genes giving similar results using RNA from X. laevis and X. tropicalis. Furthermore, the overlap in genes identified between a X. laevis and a X. tropicalis set of experiments was only 12% less than the overlap between two sets of X. tropicalis experiments. Therefore researchers can work with X. laevis and still make use of the advantages offered by X. tropicalis microarrays.     

Spatially and temporally regulated alpha6 integrin cleavage during Xenopus laevis development

The α6 integrin is essential for early nervous system development in Xenopus laevis. We have previously reported a uPA cleaved form of integrin α6 (α6p), in invasive human prostate cancer tissue, whose presence correlates with increased migration and invasive capacity. We now report that α6 is cleaved during the normal development of Xenopus in a spatially and temporally controlled manner. In addition, unlike normal mammalian tissues, which lack α6p, the major form of the α6 integrin present in adult Xenopus is α6p. The protease responsible for the cleavage in mammals, uPA, is not involved in the cleavage of Xenopus α6. Finally, overexpression of a mammalian α6 mutant which cannot be cleaved leads to developmental abnormalities suggesting a potential role for the cleavage in development.     

Differences in regulation of the first two M-phases in Xenopus laevis embryo cell-free extracts

The first embryonic M-phase is special, being the time when paternal and maternal chromosomes mix together for the first time. Reports from a variety of species suggest that the regulation of first M-phase has many particularities; however, no systematic comparative study of the biochemical aspects of first and the following M-phases has been previously undertaken. Here, we ask whether the regulation of the first embryonic M-phase is modified, using Xenopus cell-free extracts. We developed new types of extract specific for the first and the second M-phase obtained either from parthenogenetic or from in vitro fertilized embryos. Analyses of these extracts confirmed that the amplitude of histone H1 kinase activity reflecting CDK1/cyclin B (or MPF for M-phase Promoting Factor) activity is higher and persists longer than during the second M-phase, and that levels of cyclins B1 and B2 are correspondingly higher during the first than the second embryonic M-phase. Inhibition of protein synthesis shortly before M-phase entry reduced mitotic histone H1 kinase amplitude, shortened the period of mitotic phosphorylation of chosen marker proteins, and reduced cyclin B1 and B2 levels, suggesting a role of B-type cyclins in regulating the duration of mitotic events. Moreover, addition of exogenous cyclin B to the extract prior the second mitosis brought forward the activation of mitotic histone H1 kinase but prolonged the duration of this activity. We also confirmed that the inhibitory phosphorylation of CDK1 on tyrosine 15 oscillates between the first two embryonic M-phases, but is clearly more pronounced before the first than the second mitosis, while the MAP kinase ERK2 tended to show greater activation during the first embryonic M-phase but with a similar duration of activation. We conclude that discrete differences exist between the first two M-phases in Xenopus embryo and that higher CDK1/cyclin B activity and B-type cyclin levels could account for the different characteristics of these M-phases.     

Repair of 8-methoxypsoralen photoinduced cross-links in yeast

Summary The repair of interstrand cross-links induced by 8-methoxypsoralen plus UVA (365 nm) radiation DNA was analyzed in diploid strains of the yeast Saccharomyces cerevisiae. The strains employed were the wild-type D₇ and derivatives homozygous for the rad18-1 or the rad3-12 mutation. Alkaline step-elution and electron microscopy were performed to follow the process of induction and removal of photoinduced crosslinks. In accordance with previous reports, the D₇ rad3-12 strain failed to remove the induced lesions and could not incise cross-links. The strain D₇ rad18-1 was nearly as efficient in the removal of 8-MOP photoadducts after 2 h of post-treatment incubation as the D₇ RAD⁺ wild-type strain. However, as demonstrated by alkaline step-elution and electron microscopic analysis, the first incision step at DNA cross-links was three times more effective in D₇ rad18-1 than in D₇ RAD⁺. This is consistent with the hypothesis that the RAD18 gene product is involved in the filling of gaps resulting from persistent non-informational DNA lesions generated by the endonucleolytic processing of DNA cross-links. Absence of this gene product may lead to extensive strand breakage and decreased recognition of such lesions by structural repair systems.     

Endocannabinoid system in Xenopus laevis development: CB1 receptor dynamics

This study investigates for the first time the dynamics of endocannabinoid system appearance during low vertebrate Xenopus laevis development. We observed that the CB1 gene started to be expressed during the organogenesis period (+/- 1 dpf, st. 28) and expression persisted throughout the three further stages analyzed. Attention was focused on the localization of the CB1 messenger that was found both at the central level (in romboencephalon and in olfactory placods) and at the peripheral level (in the gastrointestinal tract) at +/- 3 dpf (st. 41), +/- 4 dpf (st. 46) and +/- 12 dpf (st. 49). We also considered the synthesis of CB1 protein that occurred from st. 41 onwards and, from this stage, we tested the receptor functionality in response to anandamide using cytosensor microphysiometry. CB1 functionality increased with development at both central and peripheral level. These data provide sufficient evidence to encourage further analysis on endocannabinoid physiological roles during embryonic and larval X. laevis growth.     

Engineered telomeres in transgenic Xenopus laevis

The expanding roles of telomeres in epigenetic gene regulation, nuclear organization, and human disease have necessitated the establishment of model organisms in which to study telomere function under normal developmental conditions. We present an efficient system for generating numerous vertebrate animals containing engineered telomeres using a Xenopus laevis transgenesis technique. Our results indicate Xenopus zygotes efficiently recognize telomeric repeats at chromosome break points and form telomeric complexes thus generating a new telomere. The resulting transgenic animals progress through normal development and successfully metamorphose into froglets despite the chromosome breakage. Overall, this presents an efficient mechanism for generating engineered telomeres in a vertebrate system and provides an opportunity to investigate epigenetic aspects of telomere function during normal vertebrate development.     

Hermes is a localized factor regulating cleavage of vegetal blastomeres in Xenopus laevis

We have identified the RNA-binding protein Hermes in a screen for vegetally localized RNAs in Xenopus oocytes. The RNA localizes to the vegetal cortex through both the message transport organizer (METRO) and late pathways. Hermes mRNA and protein are both detected at the vegetal cortex of the oocyte; however, the protein is degraded within a several hour period during oocyte maturation. Injection of antisense morpholino oligonucleotides (HE-MO) against Hermes caused a precocious reduction in Hermes protein present during maturation and resulted in a phenotype characterized by cleavage defects in vegetal blastomeres. The phenotype can be partially rescued by injecting Hermes mRNA. These results demonstrate that the localized RNA-binding protein Hermes functions during oocyte maturation to regulate the cleavage of specific vegetally derived cell lineages. Hermes most likely performs its function by regulating the translation or processing of one or more target RNAs. This is an important mechanism by which the embryo can generate unique cell lineages. The regulation of region-specific cell division is a novel function for a localized mRNA.     

Calcium signalling during neural induction in Xenopus laevis embryos

In Xenopus, experiments performed with isolated ectoderm suggest that neural determination is a 'by default' mechanism, which occurs when bone morphogenetic proteins (BMPs) are antagonized by extracellular antagonists, BMP being responsible for the determination of epidermis. However, Ca(2+) imaging of intact Xenopus embryos reveals patterns of Ca(2+) transients which are generated via the activation of dihydropyridine-sensitive Ca(2+) channels in the dorsal ectoderm but not in the ventral ectoderm. These increases in the concentration of intracellular Ca(2+)([Ca(2+)]i) appear to be necessary and sufficient to orient the ectodermal cells towards a neural fate as increasing the [Ca(2+)]i artificially results in neuralization of the ectoderm. We constructed a subtractive cDNA library between untreated and caffeine-treated ectoderms (to increase [Ca(2+)]i) and then identified early Ca(2+)-sensitive target genes expressed in the neural territories. One of these genes, an arginine methyltransferase, controls the expression of the early proneural gene, Zic3. Here, we discuss the evidence for the existence of an alternative model to the 'by default' mechanism, where Ca(2+) plays a central regulatory role in the expression of Zic3, an early proneural gene, and in epidermal determination which only occurs when the Ca(2+)-dependent signalling pathways are inactive.     

Assembly of correct kinetochore architecture in Xenopus egg extract requires transition of sperm DNA through interphase

Xenopus egg extracts provide a powerful tool for studying the formation and function of chromosomes. Two alternative protocols are generally used to obtain mitotic chromosomes. The first one uses a direct chromatin assembly from sperm nuclei in cytostatic factor (CSF)-arrested meiotic extracts, while the second is based on transition of sperm DNA through a replication step with subsequent reestablishment of CSF arrest. In this study we show that general kinetochore structure is disrupted in chromosomes assembled directly in CSF egg extracts: The amounts of outer kinetochore proteins such as Bub1, BubR1, and Dynactin subunit p150^glued are reduced and the components of the inner centromeric region (Aurora B kinase and Survivin) show compromised recruitment to centromeres. On the contrary, kinetochores on chromosomes assembled according to the second protocol closely resemble those in somatic cells. Our results indicate that the transition of sperm nuclei through interphase is an essential step for proper kinetochore assembly.     

Glucose transporter GLUT12-functional characterization in Xenopus laevis oocytes

We have recently identified and cloned the cDNA of a new member of the glucose transporter family that has been designated GLUT12. GLUT12 possesses the structural features critical to facilitative transport of glucose but the key to understanding the possible physiological roles of this novel protein requires analysis of functional glucose transport. In the current study, we have utilized the Xenopus laevis oocyte expression system to assay transport of the glucose analog 2-deoxy-D-glucose and characterize the glucose transport properties and hexose affinities of GLUT12. Our results demonstrate that GLUT12 facilitates transport of glucose with an apparent preferential substrate affinity for glucose over other hexoses assayed. The results are significant to understanding the potential role and importance of GLUT12 in insulin-sensitive tissues and also cells with high glucose utilization such as cancer cells.     

Reprogramming events of mammalian somatic cells induced by Xenopus laevis egg extracts

It is known that differentiated cells can be reprogrammed to an undifferentiated state in oocyte cytoplasm after nuclear transfer. Recently, some reports suggested that Xenopus egg extracts have the ability to reprogram mammalian somatic cells. Reprogramming events of mammalian cells after Xenopus egg extract treatment and after cell culture of extract-treated cells have not been elucidated. In this experiment, we examined reprogramming events in reversibly permeabilized or nonpermeabilized porcine fibroblast cells after Xenopus egg extract treatment. The Xenopus egg-specific histone B4 was assembled on porcine chromatin and nuclear lamin LIII was incorporated into nuclei. Deacetylation of histone H3 at lysine 9 in extract-treated cells was detected in nonpermeabilized cells, suggesting that a part of reprogramming may be induced even in nonpermeabilized cells. Following culture of extract-treated cells, the cells began to express the pluripotent marker genes such as POU5F1 (OCT4) and SOX2 and to form colonies. Reactivation of the OCT4 gene in extract-treated cells was also confirmed in bovine fibroblasts transformed with an OCT4-EGFP construct. These results suggest that nuclei of mammalian cells can be partially reprogrammed to an embryonic state by Xenopus egg extracts and the remodeled cells partly dedifferentiate after cell culture. A system using egg extracts may be useful for understanding the mechanisms and processes of dedifferentiation and reprogramming of mammalian somatic cells after nuclear transfer.     

Attachment of DNA Loops to an Artificial Matrix Does Not Affect the Replication Origin Specificity in Early Development of Xenopus laevis

Replication initiation proceeds in a random fashion in early development of Xenopus laevis. The replication origins become fixed only at later stages of development after the mid-blastula transition. Specification of replication origins occurs at the same time with the specification of the DNA attachment to the nuclear matrix. Replication origins of many species coincide or are located in the vicinity of sites of DNA attachment to the nuclear matrix. The present work was dedicated to development of an experimental system where DNA loops were specifically attached to an artificial matrix and a study of an effect of this attachment on specificity of DNA replication initiation in extracts of Xenopus laevis oocytes. We have found that DNA attachment to the artificial matrix increases the efficacy of DNA replication as compared to the control, but does not affect the replication specificity. It is likely that the transition from non-specific to specific replication is determined by a combination of several factors, and specificity of DNA attachment to a matrix alone is not sufficient for specification of a replication origin.     

Mismatch repair-dependent metabolism of O6-methylguanine-containing DNA in Xenopus laevis egg extracts

The cytotoxicity of S_N1-type alkylating agents such as N-methyl-N′-nitrosourea (MNU), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), or the cancer chemotherapeutics temozolomide, dacarbazine and streptozotocin has been ascribed to the persistence of O⁶-methylguanine (^meG) in genomic DNA. One hypothesis posits that ^meG toxicity is caused by futile attempts of the mismatch repair (MMR) system to process ^meG/C or ^meG/T mispairs arising during replication, while an alternative proposal suggests that the latter lesions activate DNA damage signaling, cell cycle arrest and apoptosis directly. Attempts to elucidate the molecular mechanism of ^meG-induced cell killing in vivo have been hampered by the fact that the above reagents induce several types of modifications in genomic DNA, which are processed by different repair pathways. In contrast, defined substrates studied in vitro did not undergo replication. We set out to re-examine this phenomenon in replication-competent Xenopus laevis egg extracts, using either phagemid substrates containing a single ^meG residue, or methylated sperm chromatin. Our findings provide further support for the futile cycling hypothesis.     

Nerve-independent DNA synthesis and mitosis in regenerating hindlimbs of larval Xenopus laevis

Summary Xenopus laevis larvae at stage 52–53 (according to Nieuwkoop and Faber 1956) were subjected to amputation of both limbs at the thigh level as well as to repeated denervations of the right limb. Results obtained in larvae sacrificed during wound healing (1 after amputation), blastema formation (3 days) and blastema growth (5 and 7 days) showed that denervated right limbs have undergone the same histological modifications observed in innervated left limbs and have formed a regeneration blastema consisting of mesenchymal cells with a pattern of DNA synthesis and mitosis very similar to that in presence of nerves. Also, the patterns of cellular density in regenerating right and left limbs were very similar. On the whole, the data here reported show a highly remarkable degree of nerve-independence for regeneration in hindlimbs of larval Xenopus laevis at stage 52–53 and lend some substance to the hypothesis that, in early limbs, there would exist trophic factors capable of replacing those released by nerves, promoting DNA synthesis and mitosis in blastemal cells. Offprint requests to: S. Filoni     

A novel gene, BENI is required for the convergent extension during Xenopus laevis gastrulation

Activin-like signaling plays an important role in early embryogenesis. Activin A, a TGF-beta family protein, induces mesodermal/endodermal tissues in animal cap assays. In a screen for genes expressed early after treatment with activin A, we isolated a novel gene, denoted as BENI (Brachyury Expression Nuclear Inhibitor). The BENI protein has a conserved domain at the N-terminus that contains a nuclear localization signal (NLS), and two other NLSs in the C-terminal domain. BENI mRNA was localized to the animal hemisphere at the gastrula stages and to ectoderm except for neural regions at stage 17; expression persisted until the tadpole stage. The overexpression of BENI caused gastrulation defects and inhibition of elongation of activin-treated animal caps with reduction of Xbra expression. Moreover, whole-mount in situ hybridization revealed reduced expression of Xbra in BENI mRNA-injected regions of gastrula embryos. Functional knockdown of BENI using an antisense morpholino oligonucleotide also resulted in an abnormal phenotype of embryos curling to the dorsal side, and excessive elongation of activin-treated animal caps without altered expression of mesodermal markers. These results suggested that BENI expression is regulated by activin-like signaling, and that this regulation is crucial for Xbra expression.     

A role of unliganded thyroid hormone receptor in postembryonic development in Xenopus laevis

A fascinating feature of thyroid hormone (T3) receptors (TR) is that they constitutively bind to promoter regions of T3-response genes, providing dual functions. In the presence of T3, TR activates T3-inducible genes, while unliganded TR represses these same genes. Although this dual function model is well demonstrated at the molecular level, few studies have addressed the presence or the role of unliganded TR-induced repression in physiological settings. Here, we analyze the role of unliganded TR in Xenopus laevis development. The total dependence of amphibian metamorphosis upon T3 provides us a valuable opportunity for studying TR function in vivo. First, we designed a dominant negative form of TR-binding corepressor N-CoR (dnN-CoR) consisting of its receptor interacting domain. We confirmed its dominant negative activity by showing that dnN-CoR competes away the binding of endogenous N-CoR to unliganded TR and relieves unliganded TR-induced gene repression in frog oocytes. Next, we overexpressed dnN-CoR in tadpoles through transgenesis and analyzed its effect on gene expression and development. Quantitative RT-PCR revealed significant derepression of T3-response genes in transgenic animals. In addition, transgenic tadpoles developed faster than wild type siblings, with an acceleration of as much as 7 days out of the 30-day experiment. These data thus provide in vivo evidence for the presence and a role of unliganded TR-induced gene repression in physiological settings and strongly support our earlier model that unliganded TR represses T3-response genes in premetamorphic tadpoles to regulate the progress of development.     

Nuclear reconstitution of demembranatedOrychophragmus violaceus sperm inXenopus laevis egg extracts

The cell-free extracts from animalXenopus laevis egg could induce chromatin decondensation and pronuclear formation from demembranated plant (Orychophragmus violaceu) sperm. The demembranatedOrychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.