Kazuaki Nagao

Investigation of glucocorticoid-induced apoptotic pathway: Processing of Caspase-6 but not Caspase-3

Glucocorticoids (GCs) are essential therapeutic reagents for the treatment of lymphomas and leukemias. GCs cause cell death in certain types of lymphoid cells mediated by the process known as apoptosis. This cell death is completely inhibited by Bcl-2. Here we report that Bcl-2 and benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), a broad spectrum caspase inhibitor, prevent loss of mitochondrial membrane potential (ΔΨCm) and the production of reactive oxygen species (ROS) caused by GC, while acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), an inhibitor of the caspase-3 family proteases, does not. This suggests that the inhibition by Bcl-2 and activation of some initiator caspases are upstream events of mitochondrial damage, whereas the activation of caspase-3 family proteases occurs downstream of mitochondrial changes. We also demonstrate that caspase-6 but not caspase-3 is cleaved and activated during GC-mediated apoptosis and that poly(ADP-ribose) polymerase (PARP), a substrate of caspases, also undergoes proteolysis. In addition, we provide the evidence that DNA fragmentation is markedly inhibited by Ac-DEVD-CHO, while cell death, assessed by the damage of the plasma membrane, is marginally inhibited or merely delayed.

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

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

AbstractMutations 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.

Entire PTCH1 deletion is a common event in point mutation-negative cases with nevoid basal cell carcinoma syndrome in Japan.

To the Editor : Nevoid basal cell carcinoma syndrome [NBCCS (OMIM 109400)], also called Gorlin syndrome, is an autosomal dominant neurocutaneous disorder characterized by large body size, developmental and skeletal abnormalities, sensitivity to radiation, and an increased incidence of cancers such as basal cell carcinoma (BCC) and medulloblastoma (1). NBCCS is caused by inactivating mutations in the Patched-1 (PTCH1 ) gene (2, 3). Heterozygous loss of PTCH1 found in certain sporadic and familial cases of BCC indicates that PTCH1 is also a tumor suppressor gene (4, 5). Despite extensive efforts to detect mutations, they are still unidentified in 25–60% of patients (6–8). To date, we have analyzed 38 patients with NBCCS from 32 families. Eight of the families did not harbor any PTCH1 mutations detectable by polymerase chain reaction (PCR)based direct sequencing of the exons. To investigate the possibility of large deletions involving the PTCH1 gene, we employed a high-resolution array-based comparative genomic hybridization technology. Consequently, we identified genomic deletions involving PTCH1 in seven individuals from five of the eight point mutation-negative families (Fig. S1). These patients are listed in Table 1. Some of them have been reported previously by us (9) and one patient reported by others (NS6) (10) is also included in the table, all of which are of Japanese origin. To our knowledge, this table includes all the patients with PTCH1 deletions in which the breakpoints have been identified at the nucleotide level. A schematic representation of each deletion’s size together with the deleted genes is shown in Fig. 1a. Unlike in cases of Sotos syndrome and neurofibromatosis type 1, no recurrent breakpoints were observed in these patients (11, 12). Whereas deletions larger than 2.4 Mb were generated by non-homologous end joining, smaller ones (less than 1.2 Mb) were produced by Alu-mediated nonallelic homologous recombination (Fig. S2). G19 and G36 inherited the deletion from their mothers (G27 and G43, respectively), whereas the deletion in NS6 is of paternal origin. The breakpoint sequences in these cases were completely conserved through generations. Other patients (G35, G10 and G5) did not have a family history of NBCCS and, therefore, the deletions seemed to be de novo. Patients harboring deletions of less than 2.4 Mb did not exhibit any phenotypes atypical for NBCCS despite that up to 22 RefSeq genes (four disease genes) were included in the deleted region. This implies that hemizygous loss of these genes, except for PTCH1, might not have an influence on any observable phenotypes. In contrast, deletions larger than 5.3 Mb led to phenotypes unusual for NBCCS including severe mental and motor retardation, epilepsy, and hypotonia (Table 1). Interestingly, each Alu-mediated deletion was mediated by a distinct path of rearrangement (Fig. 1b). G36/43 had a crossing over point within the Alu elements generating a hybrid Alu element. In G19/27, however, the crossing over occurred near the poly-A tail of the proximal Alu element (9). Therefore, the proximal Alu remained intact while the distal Alu was deleted. In the third case, NS6, crossing over occurred at the 5′ end of the Alu elements and removed both Alu sequences leaving two short direct repeats flanking an Alu element on both sides called target-site duplications (10). To date, we have analyzed 32 NBCCS families and identified entire deletions of PTCH1 in 5 families. This implies that 16% of NBCCS families (five of the eight point mutation-negative families) can be explained by the entire loss of PTCH1. Mutations are not observed in the PTCH1 -coding sequences in considerable numbers of NBCCS cases not only in Japanese but also in other ethnicities and, apart from PTCH1, only one PTCH2 and one SUFU mutation in NBCCS have been

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

AbstractA 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.

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.

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.

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.

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.