Establishment of an oral squamous cell carcinoma cell line expressing vascular endothelial growth factor a and its two receptors.

Background/purpose: Vascular endothelial growth factor receptor (VEGFR) expression in oral squamous cell carcinoma (OSCC) promotes tumor growth through both autocrine and paracrine signaling. VEGF-positive OSCC cases are associated with a high depth of invasion, increased metastasis, and poor prognosis. In this study we established and then molecularly and functionally analyzed an OSCC cell line that co-expresses VEGF-A, VEGFR-1, and VEGFR-2, termed HCM-SqCC010 cells. Materials and methods: VEGF-A, VEGFR-1, and VEGFR-2 expression in HCM-SqCC010 cells were examined by immunohistochemistry and immunoblotting. Expression and inhibition of VEGF-A, VEGFR-1, and VEGFR-2 in HCM-SqCC010 cells were verified by quantitative real-time PCR. Results: Our analysis of HCM-SqCC010 cells revealed that their proliferation depended on VEGF-A, and selective inhibition of VEGFR-1 or VEGFR-2 resulted in decreased cell growth. Conclusion: We established an OSCC cell line, HCM-SqCC010, that expresses VEGF-A, VEGFR-1, and VEGFR-2. This triple-positive cell line showed no effect from a molecular targeted drug toward VEGF-A, but it did show strong cell growth inhibition in response to a VEGFR inhibitor. Thus, new therapeutic strategies against OSCC should include a VEGFR inhibitor.

Establishment of a patient-derived mucoepidermoid carcinoma cell line with the CRTC1-MAML2 fusion gene.

Mucoepidermoid carcinoma (MEC) is the most common malignant tumor of the major and minor salivary glands. Surgical resection is the only curative treatment and there is no effective post-operative therapy for MEC. The present study reports an Institutional Review Board-approved case of a 45-year-old Japanese female diagnosed with low-grade MEC in the hard palate. Radical resection, supraomohyoid neck dissection and antero-lateral thigh flap reconstruction was performed. A MEC cell line was then established from the resected tumor tissue. Short tandem repeat profiling confirmed the origin and authenticity of the cell line, that harbors a CRTC1-MAML2 translocation, which is frequently observed in MEC. Amphiregulin (AREG), identified as one of the targets of the CRTC1-MAML2 fusion gene, was expressed in the cell line. The AREG receptor, epidermal growth factor receptor (EGFR) was also highly phosphorylated. The results predicted that AREG-EGFR signaling, which is required for tumor growth and survival, might be activated in the cell line in a cell-autonomous manner. As AREG expression is associated with EGFR-targeted drug resistance, this cell line might assist with the identification of novel strategies for MEC treatment.

In vitro investigation of fracture load and aging resistance of high-speed sintered monolithic tooth-borne zirconia crowns.

OBJECTIVES: The purpose of this study was to evaluate the fit, fracture load and aging resistance of the monolithic zirconia tooth-borne crowns with conventional and high-speed sintering. METHODS: The Y-TZP block was machined and sintered with conventional and high-speed sintering furnace. The marginal and internal gap between the crown and abutment was measured using a microscope and a fit checking material. A total of 28 crowns were further divided into an undegraded and a degraded group. An accelerated aging test was carried out on the degraded group. The crown was cemented and a fracture resistance was tested. X-ray diffraction was used to evaluate the crystalline structure. The data were analyzed with Student's t-test, and a one-way ANOVA and Tukey's multiple comparison test. RESULTS: There was no significant difference in mean marginal gap between the two groups. The mean internal gap was significantly greater in the speed sintering than in the conventional sintering (P <0.001). The mean fracture load of the conventional sintering crowns was not significantly different from that of speed sintering crowns after aging. The occurrence of monoclinic crystals of degraded crown was significantly higher than that of undegraded crown both in the conventional (P <0.001) and speed-sintering group (P <0.001). CONCLUSIONS: It was concluded that the monolithic zirconia crowns produced by high-speed sintering showed no significant difference in the marginal gap and the fracture load after aging compared to conventional sintering. Therefore, the high-speed sintering seems a valid method of producing tooth-borne monolithic zirconia crowns.

Stories of spinster with various faces: from courtship rejection to tumor metastasis rejection.

The Drosophila spinster (spin) mutant was isolated as a mutant that showed abnormal morphology and function in the nervous system. The spin defect induces neural degeneration similar to human lysosomal storage diseases. Various studies have shown that Spin proteins are localized in lysosomes and participate in the late stages of the autophagic process. Vertebrates have three spinster orthologs, Spns1, Spns2, and Spns3. A defect in Spns1 caused a short lifespan with aberrant lysosomal function in zebrafish. Spns2 was originally isolated as the gene responsible for abnormal heart development and was identified as a sphingosine 1-phosphate transporter in zebrafish. An endothelial cell-specific defect in Spns2 resulted in impaired egress of lymphocytes and the prevention of tumor metastasis in mice. Herein, I reviewed the history of spin/Spns research and discussed the conserved and newly diverged spin/Spns function and possible implications for human diseases.

Fluorescence of thulium-doped translucent zirconia.

The fluorescence and physical properties of thulium-doped zirconia were investigated. A standard grade of zirconia (TZ-3Y-E) and two translucent dental zirconia materials (Zpex and Zpex Smile) were examined. The specimens were prepared by addition of 0-1.5 wt% Tm2O3 and then sintering. When exposed to UV light, the Tm2O3-doped zirconia exhibited blue fluorescence with a peak wavelength of 460 nm. The fluorescence intensity of Zpex and Zpex Smile was higher than that of TZ-3Y-E, with Zpex being more intense than Zpex Smile. Zpex exhibited maximum fluorescence intensity when doped with 0.8 wt% Tm2O3. XRD analysis revealed that TZ-3Y-E and Zpex contained primarily tetragonal zirconia, while Zpex Smile contained largely cubic phase zirconia. There were no changes observed in the microstructure or physical properties of the zirconia specimens when doped with Tm2O3.

Molecular analysis of keratocystic odontogenic tumor cell lines derived from sporadic and basal cell nevus syndrome patients.

Keratocystic odontogenic tumor (KCOT) is a benign tumor often associated with basal cell nevus syndrome (BCNS). Mutations in Patched 1 (PTCH1), the Hedgehog (Hh) receptor, are responsible for BCNS. BCNS is distinguished by morphological anomalies and predisposition to benign and malignant tumors, including medulloblastoma, basal cell carcinoma, KCOT and ovarian fibromas. Among these tumors, KCOT is the least well studied because a suitable model system is not available for its investigation. To enable KCOT to be studied, we established two KCOT cell lines, one from a BCNS case (designated as iKCOT1) and one from a sporadic KCOT case (designated as sKCOT1). The BCNS­derived KCOT cell line, iKCOT1, retained a germline-mutated PTCH1 allele and a wild-type PTCH1 allele. The sporadic KCOT-derived KCOT cell line, sKCOT1, had different loss-of-function PTCH1 mutations on both alleles. Both cell lines expressed stem cell markers (CD44, SOX2 and BMI1), mesenchymal cell markers (CDH2, VIM and SNAI2) and a neurogenic marker (NEFL). Culture of the cell lines in high calcium concentration media induced expression of epithelial cell and keratinocyte marker proteins (CDH1, CLDN1, KRT10 and IVL). Parakeratosis, which is characteristic for KCOTs, was observed in 2-D cultures. The similarities in protein expression patterns between the two cell lines suggested that common mechanisms underlie the development of both types of KCOT and a probable common origin of KCOT cells.

[Corrigendum] Functional analysis of Zyxin in cell migration and invasive potential of oral squamous cell carcinoma cells.

Following the publication of this article, an interested reader drew to our attention an anomaly associated with the presentation of Figs. 2 and 3. Essentially, there was a direct duplication of certain of the western blotting data between Fig. 2 (the E-cadherin and Actin data) and Fig. 3C (the Zyxin and Actin data). After having re-examined our original data, we realize that we inadvertently duplicated the data from Fig. 2 in Fig. 3C (the Zyxin and Actin data). A corrected version of Fig. 3C (containing the true Zyxin and Actin data), and, by natural process, also of Fig. 6, are presented below, in which the Zyxin data in Figs. 3C and 6 are now correctly shown. Since the Zyxin data was a control for the siZyxin knockout of HOC313, this error did not affect the results of the Rho family analysis in this study. We sincerely apologize for this mistake, and thank the reader of our article who drew this matter to our attention. Furthermore, we regret any inconvenience this mistake has caused. [the original article was published in the International Journal of Oncology 42: 873-880, 2013; DOI: 10.3892/ijo.2013.1761].

Translucency and low-temperature degradation of silica-doped zirconia: A pilot study.

The purpose of this study was to examine the translucency and low-temperature degradation of silica-doped experimental Y-TZP (Yttria-stabilized tetragonal zirconia polycrystal) containing almost no alumina. The experimental Y-TZP samples were sintered at either 1,450 or 1,500°C. The samples of commercially available translucent Y-TZP and conventional Y-TZP were used as controls. The contrast ratio (CR) and translucency parameter (TP) were obtained to compare the translucencies. In addition, the specimens were also subjected to an accelerated aging test. The results showed that the experimental Y-TZP sintered at 1,500°C and translucent Y-TZP exhibited almost the same level of translucency. During the accelerated aging test, the translucent Y-TZP underwent a substantial increase in monoclinic content, an index of degradation after the aging test. However, neither the experimental Y-TZP nor the conventional Y-TZP exhibited any appreciable change. It was concluded that the silica-doped Y-TZP will develop translucency and resistance to degradation when sintered at 1,500°C.

Fabrication of a radiopaque fit-testing material to evaluate the three-dimensional accuracy of dental prostheses.

OBJECTIVES: The objective of this study was to fabricate a radiopaque prosthetic fit-testing material, and to develop methodology to evaluate the fitting accuracy of prostheses three-dimensionally (3D) using a combination of the silicone replica technique and micro-computed tomography (µCT). METHODS: Eight types of prototype specimens of fit-testing materials were prepared by adding contrast agents (zirconia, alumina, and barium-glass) to a commercially available fit-testing material. These specimens were evaluated on their mechanical properties, X-ray absorption coefficients, reproducibility of cement space, and suitability for 3D analysis by µCT. Then, silicone replicas made from prototype specimens were assessed for accurate 3D morphology. Subsequently, color-mapping analyses of the silicone replicas were performed according to replica thickness, and the results were compared with stereomicroscopic images. RESULTS: The mechanical properties, X-ray absorption coefficients, and reproducibility of the cement space demonstrated that prototypes containing 20wt% zirconia (Zr-20) or barium glass (diameter 2µm; Ba2-20) were useful as fit-testing materials. However, the morphology of the Ba2-20 silicone replica was unable to be accurately described using µCT because of its low X-ray absorption threshold. Zr-20, however, could be clearly observed on µCT imaging. Furthermore, color-mapping analysis of the µCT images demonstrated that Zr-20 was the most suitable for 3D observation of prosthetic fit. SIGNIFICANCE: This method could allow any professional to evaluate the fit of any type of dental prosthesis, such as inlays, crowns, and fixed and removable dentures. This study demonstrated that the technique presented in the current study is able to accurately describe the abutment-crown prosthetic discrepancy based on silicone replicas.

Genomic profiling of the genes on chromosome 3p in sporadic clear cell renal cell carcinoma.

Somatic mutations of the BRCA1 associated protein-1 (BAP1) gene, which maps to 3p21, have been found in several tumors including malignant mesothelioma, uveal melanoma, and renal cell carcinoma (RCC). The role of BAP1 inactivation in tumor development remains unclear. It has been reported that Vhl knock-out mice did not develop RCC, but Vhl knock-out mice with single allele loss of Bap1 in nephron progenitor cells developed RCC, indicating that Bap1 inactivation may be essential in murine renal tumorigenesis. To clarify the role of BAP1 in human RCC development, we performed mutation analyses, including copy number detection of BAP1 and assessment of allelic imbalance using microsatellite polymorphisms on 3p, in 45 RCC samples derived from 45 patients without VHL or BAP1 germline mutation. Additionally, we analyzed the sequences of the VHL, PBRM1, and SETD2 genes, and examined promoter methylation of VHL. Using immunostaining, we also checked for expression of BAP1 protein, which is normally located in the nuclei. None of the RCCs had biallelic deletion of BAP1, but five (11.1%) showed a biallelic mutation (four with a sequence-level mutation with monoallelic loss and one with a biallelic sequence-level mutation); these cells were negative for nuclear BAP1 staining. These patients had worse recurrence-free survival than the patients without a biallelic mutation (p=0.046). However, there were no significant differences in worse outcome by multivariate analysis combined with age, T stage, histological subtype, infiltration and vascular invasion. In 35 RCCs (77.8%), monoallelic loss of BAP1 was accompanied by VHL biallelic mutation or VHL promoter hypermethylation. In five RCCs (11.1%), we detected 3p loss-of-heterozygosity, but the copy number of BAP1 was normal. Surprisingly, nuclear staining of BAP1 was negative in 10 out of 31 tumors (32.3%) with hemizygous normal BAP1, suggesting that haploinsufficiency may relate to RCC development.

The Hippo pathway transcriptional co-activator, YAP, confers resistance to cisplatin in human oral squamous cell carcinoma.

Cisplatin (CDDP) is widely used to treat oral squamous cell carcinoma (OSCC), however, many patients exhibit acquired drug resistance. Yes-associated protein (YAP) is a transcriptional co-activator of the Hippo pathway that regulates organ size and promotes cell proliferation. YAP overexpression correlates with epithelial-mesenchymal transition and nodal metastasis, resulting in anti-tubulin drug resistance. Whether YAP overexpression is the cause of CDDP resistance in cancer cells is unclear, therefore, we investigated the correlation between YAP expression and CDDP sensitivity. We established three CDDP-resistant cell lines (OSC-19-R, SCCKN-R and HSC-3-R) from the OSCC parental cell lines. We also examined the expression levels of ATP7B, GST-π and ERCC1, which are strongly associated with CDDP resistance, and Hippo pathway-related proteins by western blotting. Using immunocytochemistry, we examined the cellular localization of YAP. Additionally, following knockdown of YAP using short interfering RNAs (siRNAs), we analyzed changes in sensitivity to CDDP. Compared with parental OSC-19 cells, OSC-19-R cells were obviously larger. Expression levels of YAP were not significantly different between OSC-19 and OSC-19-R. However, expression levels of phosphorylated YAP in OSC-19-R were decreased. We observed translocation of YAP from the cytoplasm to the nucleus in OSC-19-R cells. Knockdown of YAP using siRNAs revealed that sensitivity to CDDP was significantly increased. Translocation of YAP correlated with the acquisition of CDDP resistance. YAP could be a new therapeutic target for the treatment of patients with cancer that are resistant to CDDP.

Nevoid basal cell carcinoma syndrome with a unilateral giant ovarian fibroma in a Japanese 6-year-old girl.

Nevoid basal cell carcinoma syndrome (NBCCS) is characterized by basal cell carcinoma, skeletal abnormalities, benign tumors including ovarian fibroma, and various other phenotypic expressions. Ovarian fibromas in NBCCS before puberty are very rare. We report a 6-year-old prepubescent girl with NBCCS showing skeletal abnormalities, medulloblastoma, and ovarian fibromas. The patient was referred to our hospital owing to abdominal distension. On admission, a huge elastic hard tumor was palpable and computed tomography showed a huge tumor of the left ovary. We performed a left salpingo-oophorectomy and diagnosed the tumor as a benign fibroma. Further examination of the computed tomography images showed skeletal abnormalities. In addition, the patient had a history of medulloblastoma at the age of 4 years. Therefore, we diagnosed NBCCS. A genetic examination indicated a novel 1 bp deletion in exon 18 (c.3055delG). Sequence analysis of exon 18 using DNA from the ovarian tumor revealed a mutant allele (c.3055delG) dominant to the wild-type allele, thus suggesting loss of heterozygosity in the PTCH1 gene, which is known to be associated with NBCCS. Conclusion On the basis of our experience, physicians treating pediatric ovarian tumors should be aware that such huge benign ovarian tumors may be a phenotype of NBCCS, as shown in our patient. In addition, genetic examination focusing on the PTCH1 gene might be important for diagnosis of NBCCS in pediatric patients.

Functional analysis of Zyxin in cell migration and invasive potential of oral squamous cell carcinoma cells.

Zyxin is an evolutionarily conserved protein that has been implicated in the regulation of actin assembly and is mainly located at focal adhesions. However, the biological roles of Zyxin in cancer cells are incompletely understood. We analyzed the functions of Zyxin in cell migration and the invasive potential of OSCC. Zyxin expression was examined using eight OSCC cell lines with two different cell morphologies (6 epithelial type and 2 fibroblastic type). To knockdown Zyxin expression, OSCC cells were transfected with Zyxin siRNA and control siRNA. The cell lines were studied by western blot analysis, immunocytochemical analysis and cell migration and invasion assay. Epithelial type OSCC cells showed a high level of E-cadherin expression and a low level of Zyxin expression. N-cadherin as well as Zyxin were strongly expressed in fibroblastic type OSCC cells. Expression levels of LPP and TRIP6, members of the human Zyxin family, did not differ between epithelial type and fibroblastic type. Knockdown of Zyxin expression by siRNA in fibroblastic type OSCC cells was associated with cell morphological changes from spindle (fibroblastic) to polygonal (epithelial) shape and significantly inhibited cell growth as well as cell migration and invasion. Expression levels of Rac1 and Cdc42 were weaker in Zyxin siRNA-treated fibroblastic type OSCC cells than in control siRNA-treated cells, but the expression of RhoA did not differ significantly. Treatment of fibroblastic type OSCC cells with Rac1 inhibitor decreased the expression of Zyxin mRNA and protein. Zyxin is suggested to promote growth, migration and invasiveness of fibroblastic type OSCC cells by upregulating Rac1 and Cdc42.

Mechanisms and functions of Hedgehog signalling across the metazoa.

Hedgehog proteins constitute one of a small number of families of secreted signals that have a central role in the development of metazoans. Genetic analyses in flies, fish and mice have uncovered the major components of the pathway that transduces Hedgehog signals, and recent genome sequence projects have provided clues about its evolutionary origins. In this Review we provide an updated overview of the mechanisms and functions of this signalling pathway, highlighting the conserved and divergent features of the pathway, as well as some of the common themes in its deployment that have emerged from recent studies.

Novel PTCH1 mutations in Japanese Nevoid basal cell carcinoma syndrome patients: two familial and three sporadic cases including the first Japanese patient with medulloblastoma.

Nevoid basal cell carcinoma syndrome (NBCCS), also known as Gorlin syndrome, is inherited in an autosomal dominant mode, and is characterized by a combination of developmental abnormalities and predisposition to form a variety of tumors. The hedgehog receptor Patched1 (PTCH1) has been identified as the gene mutated in NBCCS. We analyzed PTCH1 in two familial and three sporadic Japanese NBCCS cases, and identified five germline mutations in PTCH1. Two cases have a nonsense mutation (c.3058C>T and c.2760C>A), one a splice site mutation (c.584+2T>G), one a 1 bp insertion (c.2712_2713insA) and one a 1 bp deletion (c.980Gdel). All mutations induce truncation of the PTCH1 protein or could induce nonsense-mediated mRNA decay. The 11-year-old male patient with splice-site mutation (c.584+2T>G) had medulloblastoma (MB) at the age of 1 year. This is the first NBCCS patient with molecularly defined MB in Japan.

Evolution of cuticular hydrocarbons of Hawaiian Drosophilidae.

Hawaiian Drosophila offer an excellent model for adaptive evolution. More than 500 species are reported in Hawaiian islands, and there is considerable diversity in behavior and morphology. Such diversity is mainly driven by sexual selection. In this study qualitative and quantitative chemical compositions of cuticular hydrocarbons (CHCs) in 138 flies belonging to 27 Hawaiian Drosophila species, picture-winged and non picture-winged, were analyzed regarding sexual dimorphism, differences in saturation, branching position, and lengths of CHCs. We found significant variation in the CHC patterns. In several subgroups, new species show decreases in unsaturated hydrocarbons, and gradual increases in branched compounds, monomethylalkanes and dimethylalkanes, not commonly found in Drosophila. Moreover, branching positions gradually shifted towards internal carbons, and chain lengths increased in the new species. The long-term evolution of CHCs in the light of the recent evolutionary migration and adaptation history of Hawaiian Drosophila species along the developing archipelago was discussed.

Phenotypic interactions of spinster with the genes encoding proteins for cell death control in Drosophila melanogaster.

The spin gene was first identified by its mutant phenotype, which is characterized by extremely strong mate refusal by females in response to male courtship in Drosophila. Spin mutants are also known to be accompanied by a remarkable reduction in programmed cell death in the reproductive and nervous systems. To better understand the molecular functions of spin, we searched for its genetic modifiers. Forced expression of spin(+) in somatic cells as driven by ptc-Gal4 in the testis resulted in the invasion of mature sperm into the anterior testes tip, which is otherwise occupied only by immature germ cells. To obtain genes that modulate spin's effect, the gain-of-function spin phenotype was observed in the presence of a chromosome harboring an EP or GS P-element insertion, which initiates transcription of the genomic sequence neighboring the insertion site. We isolated th and emc as suppressors of spin and atg8a as a gene that reproduces the spin phenotype on its own. th encodes Inhibitor of apoptosis-1, and mammalian Id genes homologous to emc are known to inhibit apoptosis. atg8a encodes a protein essential for autophagy. These results suggest that spin promotes cell death mechanisms that are regulated negatively by th and emc and positively by atg8a.

Germline PTCH1 mutations in Japanese basal cell nevus syndrome patients.

Basal cell nevus syndrome (BCNS or Gorlin syndrome, OMIM: 109400) is a rare autosomal dominant disorder with high penetrance. It is characterized by developmental anomalies and predisposition to tumors (for example, basal cell carcinoma (BCC) and medulloblastoma). PTCH1, the human homolog of the Drosophila patched gene, was identified as a gene responsible for BCNS. The PTCH1 protein is a Hedgehog (Hh) protein receptor and is pivotal for early development, stem cell maintenance and/or differentiation. We analyzed the six Japanese families with BCNS and identified six germline mutations in the PTCH1 gene. One family had a nonsense mutation (c.1196G>A), one had a 1-bp deletion (c.2029delA), two had 2-bp deletions (c.239_240delGA and c.1670_1671delCA) and one had a 58-bp duplication (c.1138_1195dup). They caused premature termination, resulting in the truncation of the PTCH1 protein. Analysis of a high-density single nucleotide polymorphism (SNP) mapping array showed a large approximately 1.2-Mb deletion, including the PTCH1 gene in one allele, in a family in which PTCH1 mutations were not identified at the sequence level. These data indicated that all the six families who were diagnosed with BCNS had mutations in the PTCH1 gene and that a single copy of a PTCH1 mutation causes BCNS.

Human keratinocytes derived from the bulge region of hair follicles are refractory to differentiation.

Human keratinocyte strains derived from the bulge region of plucked human follicles were successfully established from all 43 donors (age 24-76) regardless of the age and gender. The total cell number, number of population doublings and population doubling time were similar among the strains. These bulge-derived keratinocytes, BDKs, expressed keratin family genes specific to basal cell layers of the epidermis. They also expressed CD34, one of the bulge stem cell marker genes. The growth behavior and positivity of CD34 indicate that BDKs contain stem cells. BDKs were cultured until confluency or treated with CaCl2 to induce differentiation. Morphology and expression of keratin family genes in BDKs before and after differentiation induction with CaCl2 were similar to those of epidermal keratinocytes obtained from skin biopsies (NHEKs). However, expression levels of keratin-10, a prickle cell layer marker, in CaCl2-treated BDKs were lower than those in CaCl2-treated NHEKs. Higher expression of integrin-alpha6, a basal cell layer marker, was also noted in BDKs than in NHEKs after differentiation induction. Expression of stem cell marker genes other than CD34, including CD200, Sox2 and NANOG, was about the same at confluency in both cells, but significantly higher in BDKs than NHEKs after differentiation. These results indicate that BDKs were more refractory to differentiation than NHEKs. We then examined Wnt signaling inhibitor genes, DKK-3 and WIF-1 that function as tumor suppressors. DKK-3 expression decreased in both BDKs and NHEKs after CaCl2-induced differentiation. Expression of WIF-1 decreased 50% in BDKs one day after CaCl2 treatment and remained low, but was induced 1.7 times in NHEKs one day after CaCl2 treatment and further induced thereafter (>2.5 times), suggesting that WIF-1 may be involved in maintaining the differentiation-refractory status of BDKs. Since cancer stem cells in the skin have been reported to be similar to bulge-derived stem cells, our BDK strains may be of use in studying characteristics of cancer stem cells of the epidermis.

Changes in temperature preferences and energy homeostasis in dystroglycan mutants.

Temperature affects the physiology, behavior, and evolution of organisms. We conducted mutagenesis and screens for mutants with altered temperature preference in Drosophila melanogaster and identified a cryophilic (cold-seeking) mutant, named atsugari (atu). Reduced expression of the Drosophila ortholog of dystroglycan (DmDG) induced tolerance to cold as well as preference for the low temperature. A sustained increase in mitochondrial oxidative metabolism caused by the reduced expression of DmDG accounted for the cryophilic phenotype of the atu mutant. Although most ectothermic animals do not use metabolically produced heat to regulate body temperature, our results indicate that their thermoregulatory behavior is closely linked to rates of mitochondrial oxidative metabolism and that a mutation in a single gene can induce a sustained change in energy homeostasis and the thermal responses.