CD30 induces Reed-Sternberg cell-like morphology and chromosomal instability in classic Hodgkin lymphoma cell lines.

Classic Hodgkin lymphoma (cHL) is characterized by multinucleated cells called Reed-Sternberg (RS) cells and genetic complexity. Although CD30 also characterizes cHL cells, its biological roles are not fully understood. In this report, we examined the link between CD30 and these characteristics of cHL cells. CD30 stimulation increased multinucleated cells resembling RS cells. We found chromatin bridges, a cause of mitotic errors, among the nuclei of multinucleated cells. CD30 stimulation induced DNA double-strand breaks (DSBs) and chromosomal imbalances. RNA sequencing showed significant changes in the gene expression by CD30 stimulation. We found that CD30 stimulation increased intracellular reactive oxygen species (ROS), which induced DSBs and multinucleated cells with chromatin bridges. The PI3K pathway was responsible for CD30-mediated generation of multinucleated cells by ROS. These results suggest that CD30 involves generation of RS cell-like multinucleated cells and chromosomal instability through induction of DSBs by ROS, which subsequently induces chromatin bridges and mitotic error. The results link CD30 not only to the morphological features of cHL cells, but also to the genetic complexity, both of which are characteristic of cHL cells.

CD30 stimulation induces multinucleation and chromosomal instability in HTLV-1-infected cell lines.

A recent report indicated involvement of CD30 in progression of human leukemia virus type 1 (HTLV-1) infection, but the exact roles of CD30 in this process remain unclear. This study was conducted to determine the role of CD30 by stimulating CD30 expressed on HTLV-1-infected cell lines with CD30 ligand and observing its effects. CD30 stimulation increased multinucleated cells and inhibited proliferation of HTLV-1-infected cells. This inhibition was recovered by interruption of CD30 stimulation. Chromatin bridges found in multinucleated cells suggested DNA damage. CD30 stimulation triggered DNA double-strand breaks (DSBs) and chromosomal imbalances. CD30 stimulation induced reactive oxygen species (ROS), which induced DSBs. Generation of ROS and multinucleated cells by CD30 was dependent on phosphoinositide 3-kinase. RNA sequencing showed that CD30 stimulation produced significant changes in gene expression profiles, including upregulation of programmed death ligand 1 (PD-L1). Tax, which has also been shown to induce multinucleation and chromosomal instability, failed to induce CD30. These results suggest that induction of CD30, independent of Tax, triggers morphological abnormalities, chromosomal instability, and alteration of gene expression in HTLV-1-infected cells.

PTCH1-null induced pluripotent stem cells exclusively differentiate into immature ectodermal cells with large areas of medulloblastoma-like tissue.

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder with an increased incidence of tumors, such as basal cell carcinomas and medulloblastomas. The PTCH1 gene, responsible for NBCCS, suppresses the hedgehog signaling pathway, which is recognized as one of the important pathways in tumorigenesis and, thus, is a therapeutic target in cancer. In the present study, we generated PTCH1-/- induced pluripotent stem cells (iPSCs) from NBCCS patient-derived iPSCs (PTCH1+/-) by gene editing. The proliferation of PTCH1-/- iPSCs was accelerated due to the activation of the hedgehog signaling pathway. When PTCH1-/- iPSCs were subcutaneously injected into immunodeficient mice, the resulting teratomas almost exclusively contained immature ectodermal lineage cells expressing medulloblastoma markers, and the percentages of the area occupied by medulloblastoma-like tissue were larger in PTCH1-/- teratomas than in PTCH1+/- teratomas. In contrast, in PTCH1+/+ teratomas, medulloblastoma-like tissue positive for all of these medulloblastoma markers was not observed. The present results indicate the importance of PTCH1 in medulloblastoma formation and the suitability of these gene-edited iPSCs and PTCH1-/- teratomas as models for the formation of tumors, such as medulloblastomas and Hh-related tumors.

Dysregulated DNA methylation of GLA gene was associated with dysfunction of autophagy.

Lysosomes are an essential organ for cellular metabolism and play an important role in autophagy. We examined the association between methylation and autophagy in a severely affected female patient with Fabry disease, which is caused by mutation of the GLA gene on the X chromosome, and her two sisters, who had few symptoms. We confirmed autophagic flux by LC3 turnover assay using fibroblasts from each sister. In the severe female patient, autophagic flux showed abnormal while her two sisters with few symptoms had normal autophagic flux, revealing the direct relationship between symptoms and autophagic flux. Furthermore, we observed the levels of p62, which is a substrate for autophagy, and lysosome morphology. In the severe patient of this family, lysosomes were enlarged and p62 was accumulated. The methylated allele of the GLA gene in the severe patient had a high proportion of wild alleles; conversely, the sisters' methylated allele had a high proportion of mutant alleles. Therefore, we examined the mRNA expression level of the mutant allele by allele-specific PCR. It was high in the severe patient and low in the siblings with few symptoms. That is, the correlation between the mRNA expression level of the mutant allele and disease severity was confirmed. We showed a correlation between severe symptoms, dysfunction of autophagy and methylation of wild alleles in Fabry disease. It was suggested that allele-specific PCR may lead to a diagnosis and help to determine the prognosis of female patients with Fabry disease.

L-leucine and SPNS1 coordinately ameliorate dysfunction of autophagy in mouse and human Niemann-Pick type C disease.

Lysosomal storage disorders are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. 573C10 is a Schwann cell line derived from a mouse model of Niemann-Pick type C disease-1, NPC (-/-). Under serum-starved conditions, NPC (-/-) cells manifested impaired autophagy accompanied by an increase in the amount of p62 and lysosome enlargement. Addition of L-leucine to serum-starved NPC (-/-) cells ameliorated the enlargement of lysosomes and the p62 accumulation. Similar autophagy defects were observed in NPC (-/-) cells even without serum starvation upon the knockdown of Spinster-like 1 (SPNS1), a putative transporter protein thought to function in lysosomal recycling. Conversely, SPNS1 overexpression impeded the enlargement of lysosomes, p62 accumulation and mislocalization of the phosphorylated form of the mechanistic Target of rapamycin in NPC (-/-) cells. In addition, we found a reduction in endogenous SPNS1 expression in fibroblasts derived from NPC-1 patients compared with normal fibroblasts. We propose that SPNS1-dependent L-leucine export across the lysosomal membrane is a key step for triggering autophagy, and that this mechanism is impaired in NPC-1.

Somatic mosaicism containing double mutations in PTCH1 revealed by generation of induced pluripotent stem cells from nevoid basal cell carcinoma syndrome.

BACKGROUND: Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterised by developmental defects and tumorigenesis, such as medulloblastomas and basal cell carcinomas, caused by mutations of the patched-1 (PTCH1) gene. In this article, we seek to demonstrate a mosaicism containing double mutations in PTCH1 in an individual with NBCCS. METHODS AND RESULTS: A de novo germline mutation of PTCH1 (c.272delG) was detected in a 31-year-old woman with NBCCS. Gene analysis of two out of four induced pluripotent stem cell (iPSC) clones established from the patient unexpectedly revealed an additional mutation, c.274delT. Deep sequencing confirmed a low-prevalence somatic mutation (5.5%-15.6% depending on the tissue) identical to the one found in iPSC clones. CONCLUSIONS: This is the first case of mosaicism unequivocally demonstrated in NBCCS. Furthermore, the mosaicism is unique in that the patient carries one normal and two mutant alleles. Because these mutations are located in close proximity, reversion error is likely to be involved in this event rather than a spontaneous mutation. In addition, this study indicates that gene analysis of iPSC clones can contribute to the detection of mosaicism containing a minor population carrying a second mutation.

Nevoid basal cell carcinoma syndrome caused by splicing mutations in the PTCH1 gene.

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental defects and tumorigenesis such as medulloblastomas and basal cell carcinomas, caused by mutations of the patched-1 (PTCH1) gene. To date, we have detected 73 mutations in PTCH1 and ten of them (14 %) were suspected splicing mutations. Eight out of the ten mutations were localized near the splice donor site. Five mutations were localized within the invariant GT-AG splice site, whereas the other five mutations occurred outside the invariant GT-AG site including the last exonic nucleotide. When the transcripts were examined, all mutations resulted in aberrant splicing, including exon skipping or the activation of cryptic splice sites. This is the first extensive report of NBCCS focusing on splice site mutations, and it highlights the importance of analyzing transcripts especially for mutations lying outside the GT-AG splicing consensus site. In addition, the splice site score calculated by Splice-Site Analyzer Tool provided by Tel Aviv University helped predict aberrant splice patterns in most of the cases.

Molecular pathogenesis of keratocystic odontogenic tumors developing in nevoid basal cell carcinoma syndrome.

OBJECTIVE: The aim of this study was to investigate the molecular pathogenesis of keratocystic odontogenic tumors (KCOTs) that developed in nevoid basal cell carcinoma syndrome (NBCCS) patients. STUDY DESIGN: We analyzed germline and somatic mutations of the PTCH1 and its related genes, SMO and SUFU in 10 KCOTs that developed in 8 unrelated NBCCS patients. Methylation status of the PTCH1 promoter was also investigated by bisulfite sequencing. RESULTS: Somatic mutations of PTCH1 were detected in 3 KCOTs. Two out of 3 somatic mutations were either identified as a polymorphism or located on the same allele as the germline mutation. Neither abnormal methylation of the PTCH1 promoter, loss of PTCH1, nor somatic mutation of SMO or SUFU was detected in any of the samples. CONCLUSIONS: Our results suggest that the tumorigenesis of most KCOTs associated with NBCCS cannot be explained by the classical 2-hit theory.

RCAN1 is an important mediator of glucocorticoid-induced apoptosis in human leukemic cells.

Glucocorticoid (GC) is a major therapeutic agent for the treatment of leukemia because of its ability to induce apoptosis in lymphoid cells. The mechanism causing apoptosis, however, is still controversial. Since the glucocorticoid receptor is a transcription factor, some of its target genes are expected to be implicated in apoptosis. In this study, using a GC-sensitive human pre-B leukemia cell line, Nalm-6, the FK506 binding protein 51 (FKBP5) and regulator of calcineurin 1 (RCAN1) genes were disrupted by homologous recombination, since the expression of both is up-regulated by GC in GC-sensitive but not in GC-resistant leukemic cell lines. While the disruption of FKBP5 had a marginal effect on GC-induced apoptosis, that of RCAN1 resulted in marked resistance to GC. In addition, overexpression of RCAN1 rendered cells more sensitive to DEX. In RCAN1-disrupted cells, levels of some pro-apoptotic and anti-apoptotic Bcl-2 family proteins were decreased and increased, respectively. Finally, phosphorylation of cAMP-response element binding protein (CREB) and up-regulation of CREB target genes by GC were inhibited by RCAN1 disruption, and treatment with a cAMP-inducing agent, forskolin, restored the sensitivity to GC in RCAN1-disrupted Nalm-6 cells. These findings suggest that up-regulation of RCAN1 expression followed by activation of the CREB pathway is required in GC-induced apoptosis.

Heterozygous tandem duplication within the PTCH1 gene results in nevoid basal cell carcinoma syndrome.

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental defects and tumorigenesis. The gene responsible for NBCCS is PTCH1. Using multiplex ligation-dependent probe amplification, we identified a heterozygous tandem duplication within the PTCH1 gene in a 14-year-old girl with typical NBCCS. We have sequenced the chromosomal breakpoint and determined the duplication as tandem in orientation and 18,814 bp in size. The fusion occurred between non-repetitive elements with an overlap of three nucleotides. The duplicated segment began at exon 10 and ended at intron 17. Subsequent analysis of cDNA from the patient showed the expression of mutant mRNA species containing a duplicated segment spanning exons 11-17, resulting in a frameshift and premature stop codon. This is the first reported case of NBCCS due to a tandem multiexon duplication of PTCH1 representing a novel mechanism leading to the NBCCS phenotype, and highlights the importance of copy number analysis as an adjunct to exon sequencing in identifying infrequent mutational events in PTCH1.

Selective haploinsufficiency of longer isoforms of PTCH1 protein can cause nevoid basal cell carcinoma syndrome.

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental defects and tumorigenesis. The gene responsible for NBCCS is PTCH1. The PTCH1 gene has five alternatively used first exons resulting in the translation of three isoforms of the PTCH1 protein; that is, PTCHL, PTCHM and PTCHS. However, the biological significance of each isoform is unclear. Here we show an individual with NBCCS carrying a nonsense mutation in PTCH1 exon2, c.387G>A (p.W129X). As the mutation lay upstream of the ATG codon used for PTCHS translation, the mutant allele still expressed RNA isoforms that encode PTCHS. These results clearly demonstrate that a selective haploinsufficiency of longer isoforms of the PTCH1 protein, PTCHL and PTCHM, but not PTCHS is sufficient to cause NBCCS. Although mice selectively deficient in PTCHS isoforms are currently unavailable, this study sheds light on the complex in vivo roles of PTCH1 isoforms.

Thermal [2+2] cycloaddition of morpholinoenamines with C60 via a single electron transfer.

The thermal reaction of C(60) with five- and six-membered morpholinocycloalkenes in refluxing toluene exclusively gave the [2+2] cycloadducts in high yields. However, a seven-membered homologue sluggishly reacted with C(60) because of the increasing steric hindrance. This cycloaddition reaction is likely to proceed via a single electron transfer (SET), a radical-coupling, and subsequent ion cyclization rather than the prior proton transfer between the radical ions.

EYA4, deleted in a case with middle interhemispheric variant of holoprosencephaly, interacts with SIX3 both physically and functionally.

Holoprosencephaly (HPE) is the most common congenital malformation of the developing human forebrain, in which the two cerebral hemispheres fail to separate to various degrees. Although pathological mutations have been identified in up to nine genes, a number of other genes as well as environmental factors are likely to be involved in HPE. Here, we describe a case with the middle interhemispheric variant, a milder variant of HPE, carrying a deletion of approximately 10.4 Mb at 6q22.31-q23.2, which includes the EYA4 gene. EYA4 is one of four vertebrate orthologs of the Drosophila melanogaster gene, eyes absent. EYA4 was co-immunoprecipitated with SIX3, the product of one of the known HPE genes. Moreover, the EYA4 protein was observed to be recruited to the nucleus by the nuclear protein SIX3 under a confocal microscope. EYA4 is a transcriptional coactivator, and was shown to cooperate with transcription factor SIX3 by reporter gene assays. These results demonstrate physical and functional association between EYA4 and SIX3, suggesting that EYA4 is a novel candidate gene of HPE, whose haploinsufficiency leads to HPE through the compromised function of SIX3.

U7 snRNA-mediated correction of aberrant splicing caused by activation of cryptic splice sites.

A considerable fraction of mutations associated with hereditary disorders and cancers affect splicing. Some of them cause exon skipping or the inclusion of an additional exon, whereas others lead to the inclusion of intronic sequences or deletion of exonic sequences through the activation of cryptic splice sites. We focused on the latter cases and have designed a series of vectors that express modified U7 small nuclear RNAs (snRNAs) containing a sequence antisense to the cryptic splice site. Three cases of such mutation were investigated in this study. In two of them, which occurred in the PTCH1 and BRCA1 genes, canonical splice donor sites had been partially impaired by mutations that activated nearby intronic cryptic splice donor sites. Another mutation found in exonic region in CYP11A created a novel splice donor site. Transient expression of the engineered U7 snRNAs in HeLa cells restored correct splicing in a sequence-specific and dose-dependent manner in the former two cases. In contrast, the third case, in which the cryptic splice donor site in the exonic sequence was activated, the expression of modified U7 snRNA resulted in exon skipping. The correction of aberrant splicing by suppressing intronic cryptic splice sites with modified U7 is expected be a promising alternative to gene replacement therapy.

Brain- and heart-specific Patched-1 containing exon 12b is a dominant negative isoform and is expressed in medulloblastomas.

Mutations in the human tumor suppressor gene, Patched-1, are associated with nevoid basal cell carcinoma syndrome characterized by developmental abnormalities and tumorigenesis, such as basal cell carcinoma and medulloblastoma. During the investigation of complex alternative splicing in Patched-1, we identified an alternative exon, exon 12b, located between exon 12 and 13, both in humans and in mice. Since exon 12b has an in-frame stop codon, the mRNA isoform containing this exon (Patched12b) encodes a truncated patched-1 protein. RT-PCR and whole mount in situ hybridization revealed that mouse exon 12b was expressed in the brain and heart, particularly in the cerebellum, in both adults and embryos. We next performed a functional analysis of Patched12b using a GLI-responsive luciferase reporter. Luciferase activity was suppressed when transfected with a plasmid encoding Patched-1, but not with a plasmid for Patched12b. The suppressive activity of Patched-1 was relieved when cotransfected with a plasmid for Patched12b. This implies that the Patched12b protein has a dominant negative effect on Patched-1. Interestingly, Patched12b was found to be expressed in some of the medulloblastoma tissues and cell lines, indicating an important role in the pathogenesis of medulloblastoma as well as brain development.

Detecting tissue-specific alternative splicing and disease-associated aberrant splicing of the PTCH gene with exon junction microarrays.

Mutations in the human ortholog of Drosophila patched (PTCH) have been identified in patients with autosomal dominant nevoid basal cell carcinoma syndrome (NBCCS), characterized by minor developmental anomalies and an increased incidence of cancers such as medulloblastoma and basal cell carcinoma. We identified many isoforms of PTCH mRNA involving exons 1-5, exon 10 and a novel exon, 12b, generated by alternative splicing (AS), most of which have not been deposited in GenBank nor discussed earlier. To monitor splicing events of the PTCH gene, we designed oligonucleotide arrays on which exon probes and exon-exon junction probes as well as a couple of intron probes for the PTCH gene were placed in duplicate. Probe intensities were normalized on the basis of the total expression of PTCH and probe sensitivity. Tissue-specific regulation of AS identified with the microarrays closely correlated with the results obtained by RT-PCR. Of note, the novel exon, exon 12b, was specifically expressed in the brain and heart, especially in the cerebellum. Additionally, using these microarrays, we were able to detect disease-associated aberrant splicings of the PTCH gene in two patients with NBCCS. In both cases, cryptic splice donor sites located either in an exon or in an intron were activated because of the partial disruption of the consensus sequence for the authentic splice donor sites due to point mutations. Taken together, oligonucleotide microarrays containing exon junction probes are demonstrated to be a powerful tool to investigate tissue-specific regulation of AS and aberrant splicing taking place in genetic disorders.

Frequent occurrence of protein isoforms with or without a single amino acid residue by subtle alternative splicing: the case of Gln in DRPLA affects subcellular localization of the products.

Protein isoforms with or without a single amino acid residue make a subtle difference. It has been documented on a few genes that alternative splicing generated such isoforms; however, the fact has attracted little attention. We became aware of a subtle sequence difference in DRPLA, a polyglutamine disease gene for dentatorubral pallidoluysian atrophy. Some reported cDNA sequences lacked 3 nucleotides (nt) (CAG), which were positioned apart from the expandable and polymorphic CAG repeats and also coded for glutamine. We experimentally confirmed that the difference was indeed generated by alternative splicing utilizing two acceptors separated by 3 nt. In DRPLA, the expression ratio of two mRNA isoforms was almost constant among tissues, with the CAG-included form being major. The glutamine-included protein isoform was more predominantly localized in the nucleus. Database searching revealed that alternative splice acceptors, as well as donors, are frequently situated very close to each other. We experimentally confirmed two mRNA isoforms of 3 nt difference in more than 200 cases by RT-PCR and found interesting features associated with this phenomena. Inclusion of 3 nt tends to result in single amino acid inclusion despite the phase of translational frame. The expression ratio sometimes varied extensively among tissues.

Identification and characterization of multiple isoforms of a murine and human tumor suppressor, patched, having distinct first exons.

Mutations in mouse and human patched (PTCH) genes are associated with birth defects and cancer. PTCH, a 12-pass transmembrane protein, is a receptor for Sonic hedgehog (Shh) signaling proteins. Shh proteins activate transcription of target genes, including PTCH, via GLI transcription factors. Here we identified seven and five isoforms of human and mouse PTCH mRNA, respectively, which are generated by the complex alternative use of five exons as the first exon (exons 1a to 1e in the 5'-to-3' order). Although expression profiles of these isoforms were highly variable among human tissues, three of them, PTCHa, PTCHb, and PTCHd, were predominantly expressed in most tissues, PTCHd being most ubiquitous. In contrast, PTCHb was always predominant and reached a maximum at E10.5 during mouse development. These three mRNA isoforms encode three PTCH proteins with distinct N-termini, PTCH(L), PTCH(M), and PTCH(S). The expression of these three isoforms was regulated by GLI transcription factors, and at least two functional GLI-binding sequences were identified, one in exon 1a and the other between exon 1a and exon 1b. PTCH(L) and PTCH(M) were equally active in terms of suppressing GLI-mediated transcription and inducing apoptosis. PTCH(S) protein (encoded by PTCHd), lacking the first transmembrane domain, was more unstable than the other two, resulting in a reduced activity. This study may shed light on the mechanism whereby a single PTCH gene plays a role in both tumor cell growth and embryonic development.

Identification of a novel polymorphism involving a CGG repeat in the PTCH gene and a genome-wide screening of CGG-containing genes.

Mutations in the human homologue of the Drosophila patched gene (PTCH) are responsible for the hereditary disorder called nevoid basal cell carcinoma syndrome (NBCCS). PTCH has a CGG triplet repeat located 4 bp upstream of the first methionine codon. Here we report a novel polymorphism involving the number of the CGG-repeat. The major allele (86.3%) contained a repeat size of seven, whereas the minor allele contained eight. No significant difference in the distributions of genotypes was observed between normal and NBCCS individuals. However, when the repeat was inserted between a heterologous promoter and the luciferase gene, the longer repeats tended to induce higher luciferase activities, suggesting that the repeat length potentially affects the levels of gene expression. A genome-wide screening revealed that 68 and 146 genes contained a CGG/CCG repeat in the coding region and in the 5'-untranslated region (5'-UTR), respectively. None of the genes had this repeat in 3'-UTR. Interestingly, the number of genes with a CGG repeat in the 5'-UTR was significantly higher than that with a CCG repeat in the 5'-UTR. The localization of a CGG/CCG repeat in PTCH is quite unique in that only four other genes have been found in which the repeat is localized up to 4 bp upstream of the first methionine.

Dentatorubral-pallidoluysian atrophy protein is phosphorylated by c-Jun NH2-terminal kinase.

Dentatorubral-pallidoluysian atrophy (DRPLA) is a dominant-inherited neurodegenerative disease characterized by selective cell loss in particular neuronal pathways. This is caused by expansion of CAG repeats in the coding region of the DRPLA gene, and the extended polyglutamine tract (polyQ) confers a toxic activity. It is valuable to characterize disease gene products for elucidation of the mechanism underlying neuron death at specific anatomical areas of the brain. Here, we define the DRPLA protein as a phosphoprotein, and c-Jun NH(2)-terminal kinase (JNK) is one of the major factors involved in its phosphorylation. Endogenous DRPLA protein was serine-phosphorylated. Phosphorylation was demonstrated in a recombinant JNK activation system in vitro and also in overexpressing cells by transfection after the JNK activation with osmotic pressure. One of the phospho-acceptor sites for JNK appearing in the DRPLA sequence was indeed phosphorylated, which was confirmed by a specific antibody raised against the phosphopeptide. Kinetic studies in the JNK recombinant system showed that expanded polyQ slightly reduced the affinity of JNK to the protein. Thus, the abnormal DRPLA protein seems to be slowly phosphorylated in a certain condition of JNK activation in patients. It may delay a process that is essential in keeping neurons alive.