Alkaline semen diluent combined with R848 for separation and enrichment of dairy goat X-sperm.

A large number of female goats are needed for the dairy goat industry; therefore, the development of a method to ensure the birth of more females than males in a single pregnancy will lead to economic benefits. Increasing the number of X-sperm would be an effective way to increase the proportion of female offspring. In this study, goat semen was incubated at pH 7.4 in alkaline diluent combined with resiquimod (R848) and the number of X-sperm was enriched by the swim-up method. The percentage of X-sperm was determined using the double TaqMan qPCR method. Sperm total motility, progressive motility, average path velocity, straight-line velocity, and curvilinear velocity were measured using a computer-aided sperm analysis system, and the functional parameters of the sperm plasma membrane, the acrosome, mitochondrial activity, ATP content, and reactive oxygen species levels were also measured. Lastly, the ratio of female embryos was determined by in vitro fertilization, and the number of female kids and the pregnancy rate of does was assessed by artificial insemination. The results showed that dilution of semen in an alkaline buffer containing R848 could enrich the number of X-sperm to 85.57% ± 3.27%. The progressive motility, average path velocity, straight-line velocity, curvilinear velocity, mitochondrial activity, and ATP level of the collected X-sperm-enriched semen were significantly reduced, but its total motility, plasma membrane, and acrosome were not affected. The in vitro fertilization experiments showed that the rate of female embryo production using X-sperm-rich seminal fluid could reach 83.25% (174/209), which was significantly higher than the proportion of female embryos in the control group, 47.71% ± 1.80% (104/218). As determined by artificial insemination, the number of female kids in the test group increased by 62.79% (243/387), which was significantly higher than that in the control group (47.65%, 193/405). There was no significant difference in pregnancy rate between the test group and the control group (71.71% vs. 78.48%). Therefore, this study demonstrated that use of a pH 7.4 diluent containing R848 is a simple and effective method of X-sperm enrichment for dairy goat production. Its application would allow does to produce more female offspring for herd expansion and milk production.

Intratumoral Polymorphism of Peroxisome Proliferator-Activated Receptor Delta-87 T>C in Colorectal Cancer.

Peroxisome proliferator activated receptor delta (PPARD) is a nuclear receptor transcription factor whose single nucleotide polymorphism (SNP), especially PPARD-87 T>C (rs2016520), may play an important role in expression regulation of PPARD. But its expression patterns as well as contribution in colorectal cancer (CRC) are still controversial. In this study, whether the intratumoral heterogeneity of polymorphism of PPARD-87 T>C (rs2016520) existed and its influence in CRC were investigated. Tumor masses from primary CRC patients were collected during the operation of tumorectomy, specimens at the different sites of the same tumor mass were sampled and stored individually. The SNP of PPARD-87 T>C was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and the expression of PPARD in vivo was observed by immunohistochemistry. The correlation of PPARD -87 T>C intratumoral polymorphism and the clinicopathological parameters of patients was analyzed statistically. Tumor samples were collected from 106 CRC patients (70 males and 36 females) with an average age of 61.04±13.67 years. A total number of 808 samples (7.60±1.60 per patient) were mainly harvested at peripheral superficial (n=376), central superficial (n=163), invasive front (n=112) and mesenteric cancer foci (n=42) of tumor tissues as well as cancerous adjacent mucosa (n=104). PCR-RFLP analysis showed that T/T (n=460, 56.9%) and T/C (n=334, 41.3%) were the main genotypes of -87 T>C among these samples. Furthermore, intratumoral genotype of -87 T>C was homogeneous in 90 patients and heterogeneous in other 16 patients. The intratumoral heterogeneity was related to patients' age (P=0.016), tumor location (P=0.011) and the grade of differentiation (P=0.022). For patients with intratumoral heterogeneity, immunochemistry showed the expressions of PPARD were not influenced by T/T or T/C genotypes. Intratumoral heterogeneity of PPARD-87 T>C wildly existed in CRC, and associated with patients' age, tumor location and differentiation. However, the immunochemistry assay revealed that there's no significant link between heterogeneity and expression of PPARD.

[Poorly-differentiated chordoma with INI1 loss: a clinicopathologic study].

Objective: To analyze the clinicopathologic characteristics of poorly-differentiated chordoma with INI1 loss in children and to discuss the differential diagnosis. Methods: The clinical, radiological, histopathological profiles and molecular pathologic characteristics of two pediatric poorly differentiated chordoma cases with INI1 loss were reviewed. Results: The patients were a girl and a boy. Both lesions involved the slope. Both patients were presented with progressive muscle weakness or neck pain. Radiological examination showed clivus bone destruction and compression of the brain stem and cervical spinal cord. Histologically, the tumor cells lacked typical organization and were associated with inflammatory cells infiltration. On high power field, the tumor cells were ovoid or fusiform with prominent atypia, vacuolated nuclei and prominent nucleoli. By immunohistochemistry, the tumor cells expressed cytokeratin, epithelial membrane antigen, brachyury and were negative for INI1. In both cases, INI1 gene deletion was detected by FISH. Conclusions: Poorly-differentiated chordoma with INI1 loss mainly occurs in children. The morphology is different from classical chordoma.INI1 gene deletion is detectable by FISH. It can be distinguished from atypical teratoid/rhabdoid tumors and other neoplasms by the identification of nuclear brachyury expression. The loss of INI1 expression in poorly-differentiated chordoma might be associated with a poorly-differentiated morphology and an adverse prognosis.

[Magnetic resonance imaging characteristics of encephalopathy of prematurity].

Objective: To explore the MRI manifestation of encephalopathy of prematurity (EOP), so as to find an access to the early prevention, early diagnosis, effective treatment and prognosis. Methods: A total of 2 718 premature infants were collected, MRI and clinical data were analyzed who were admitted to NICU of Children's Hospital of Fudan University between January 1, 2009 and December 31, 2014. The manifestation and lesions distribution in MRI were analyzed. Results: All the 2 718 preterm infants underwent MRI. 58.8% (1 599/2 718) of which had normal MRI apperance, whereas 24.9% (678/2 718) showed manifestations of EOP.78.8% (534/678) EOP were non-cystic EOP. 21.2% (144/678) EOP were cystic EOP. Periventricular and cerebral lobe white matter were primary distributions of these lesions. Cystic lesions were primarily distributed in the body of periventricular whiter matter (49.3%). However, more non-cystic EOP were found in cerebral parietal lobe whiter matter (25.1%). Non-cystic EOP were also distributed in the body of periventricular whiter matter, frontal lobe and basal ganglia(20.8%, 20.2% and 18.9%, respectively ). Conclusions: The morbidity rate of EOP in preterm infants was 24.9%. 21.2% (144/678) EOP were cystic EOP. 78.8% (534/678) EOP were non-cystic EOP. Cystic lesions were primarily distributed in the body of periventricular whiter matter. Non-cystic EOP were also distributed in the body of periventricular whiter matter, frontal lobe and basal ganglia.

Correlations of TNF-α gene promoter polymorphisms with the risk of thymoma-associated myasthenia gravis in a northern Chinese Han population.

This study was performed with the aim to investigate the correlations of tumor necrosis factor-alpha (TNF-α) gene promoter polymorphisms with the risk of thymoma-associated myasthenia gravis (T-MG) in a northern Chinese Han population. Between June 2005 and June 2015, 305 MG patients (150 males and 155 females, MG group) and 293 healthy volunteers (negative control (NC) group) were enrolled in this study. Among the MG patients, there were 121 patients with thymoma-associated MG (T-MG group) and 184 without T-MG (NT-MG group). Enzyme-linked immunosorbent assay (ELISA) was used for the serum TNF-α level. Polymerase chain reaction-restriction fragment length polymorphism was conducted to determine genotype and allele frequencies of TNF-α gene promoter -1031T/C, -857C/T and -863C/A. The haplotype was analyzed with the SHEsis software. Logistic regression analysis was performed for correlations between TNF-α gene promoter polymorphisms and the risk of T-MG. The T-MG group had higher frequencies of the CT/TT genotype and T allele of -857C/T than the NT-MG and NC groups. The frequencies of the CC genotype and C allele of -1031T/C were higher in the T-MG group than in the NT-MG and NC groups, and higher in male patients in the T-MG group than in male patients in the NC group. TTA and TTC haplotypes exhibited lower frequencies in the T-MG group than in the NT-MG group. The ocular MG patients exhibited lower frequencies of the TT genotype and T allele of -857C/T than the generalized MG patients did. The TNF-α level was elevated in the T-MG group compared with that in the NC and NT-MG groups, indicating that the TC+CC and CT+TT genotypes were increased compared with the TT and CC genotypes in the -1031T/C and -857C/T, respectively. Logistic regression analysis suggested that expressions of anti-acetylcholine receptor antibodies, Osserman's classification, -1031T/C and -857C/T polymorphisms and the TTA haplotype were the independent risk factors for T-MG. These findings reveal that TNF-α -1031T/C and -857C/T polymorphisms and the TTA haplotype may be correlated with the occurrence of T-MG in a Northern Chinese Han population.

[Novel compound heterozygous TBC1D24 mutations in a boy with infantile focal myoclonic epilepsy and literature review].

Objective: To investigate the clinical features and genetic characteristics of patients with TBC1D24 gene mutations. Method: The clinical data of a patient with novel TBC1D24 compound heterozygous mutations from Children's Hospital of Fudan University were collected, the related literature was searched from China National Knowledge Infrastructure, Wanfang Data Knowledge Service Platform, National Center from Biotechnology Information and Pubmed (up to April 2016) by using search terms "TBC1D24" "epilepsy" . The clinical features, electroencephalogram (EEG) and prognosis of the patients with TBC1D24 gene mutations were studied. Result: The patient was a boy with non-consanguineous healthy parents.He had an acute episode of focal continuous myoclonus lasting a few hours with consciousness preserved at the age of 3 months.Myoclonic jerks alternatively affected the eyelids, either the right or left limbs, sometimes triggered by fever or fatigue.The frequency was once 3-7 days.At the age of 6 months he was found to have myoclonus seizures with onset from a unilateral eyes lid and limb lasting 10 more minutes and subsequently affected four extremities or the trunk.They occurred once 3-4 months with perserved consciousness and lasted from several hours to up to ten more hours.They mostly disappeared during sleep.He had ataxia and mild mental retarding.Paroxysmal anomalies were not found on ictal traces.A novel compound heterozygous mutation of TBC1D24 gene, c. 730G>A, p.A244T and c. 1571G>C, p.R524P were found in the patient.Further study showed that c. 730G>A mutation was inherited from his father and c. 1571G>C from his mother. These two were not reported in public databases and predicted deleterious by Mutation Taster and polyphen-2.Literature relevant to TBC1D24 published all around the world was reviewed, no Chinese cases with TBC1D24 gene mutations had been reported. The total of 24 cases including the present case with TBC1D24 gene mutation were reported.Among them, 11 cases had compound heterozygous mutations and 13 cases had homozygous mutations.Ten mutations were identified, including 1 termination mutation, 1 frameshift mutation and 8 missense mutations. Conclusion: TBC1D24 gene mutational analysis should be performed on patients with early-onset focal continuous myoclonus, if the etiology was unclear.

Seropositivity and identification of paramyosin for sparganosis in the Kangwon and Incheon provinces of the Republic of Korea.

Sparganosis is one of the top three tissue-dwelling heterologous helminthic diseases, along with cysticercosis and paragonimiasis, in Korea. Due to a lack of effective early diagnosis and treatment methods, this parasitic disease is regarded as a public health threat. This study evaluated reactivity, against sparganum extracts, of sera from inhabitants of Cheorwon-gun, Goseong-gun and Ongjin-gun in Korea. The sera from 836 subjects were subjected to enzyme-linked immunosorbent assay and immunoblot analysis. The sera from 18 (5.8%) and 15 (5.1%) inhabitants in Cheorwon-gun (n = 312) and Goseong-gun (n = 294), respectively, exhibited highly positive reactions to the sparganum antigen, whereas only two (0.9%) inhabitants in Ongjin-gun (n = 230) showed positivity. We sought antigenic proteins for serodiagnosis of positive sera by immunoproteomic approaches. Total sparganum lysates were separated by two-dimensional electrophoresis and then subjected to immunoblot analysis with mixed sparganosis-positive sera. We found seven antigenic spots and identified paramyosin as an antigenic protein by liquid chromatography-mass spectrometry. By two-dimensional (2D)-based mass analysis and immunoblotting against sparganosis-positive sera, paramyosin was identified as a candidate antigen for serodiagnosis of sparganosis.

A small subunit processome protein promotes cancer by altering translation.

Dysregulation of ribosome biogenesis or translation can promote cancer, but the underlying mechanisms remain unclear. UTP18 is a component of the small subunit processome, a nucleolar multi-protein complex whose only known function is to cleave pre-ribosomal RNA to yield the 18S ribosomal RNA component of 40S ribosomal subunits. Here, we show that UTP18 also alters translation to promote stress resistance and growth, and that UTP18 is frequently gained and overexpressed in cancer. We observed that UTP18 localizes to the cytoplasm in a subset of cells, and that serum withdrawal increases cytoplasmic UTP18 localization. Cytoplasmic UTP18 associates with the translation complex and Hsp90 to upregulate the translation of IRES-containing transcripts such as HIF1a, Myc and VEGF, thereby inducing stress resistance. Hsp90 inhibition decreases cytoplasmic UTP18 and UTP18-induced increases in translation. Importantly, elevated UTP18 expression correlates with increased aggressiveness and decreased survival in numerous cancers. Enforced UTP18 overexpression promotes transformation and tumorigenesis, whereas UTP18 knockdown inhibits these processes. This stress adaptation mechanism is thus co-opted for growth by cancers, and its inhibition may represent a promising new therapeutic target.

KAP regulates ROCK2 and Cdk2 in an RNA-activated glioblastoma invasion pathway.

Aberrant splicing of the cyclin-dependent kinase-associated phosphatase, KAP, promotes glioblastoma invasion in a Cdc2-dependent manner. However, the mechanism by which this occurs is unknown. Here we show that miR-26a, which is often amplified in glioblastoma, promotes invasion in phosphatase and tensin homolog (PTEN)-competent and PTEN-deficient glioblastoma cells by directly downregulating KAP expression. Mechanistically, we find that KAP binds and activates ROCK2. Thus, RNA-mediated downregulation of KAP leads to decreased ROCK2 activity and this, in turn, increases Rac1-mediated invasion. In addition, the decrease in KAP expression activates the cyclin-dependent kinase, Cdk2, and this directly promotes invasion by increasing retinoblastoma phosphorylation, E2F-dependent Cdc2 expression and Cdc2-mediated inactivation of the actomyosin inhibitor, caldesmon. Importantly, glioblastoma cell invasion mediated by this pathway can be antagonized by Cdk2/Cdc2 inhibitors in vitro and in vivo. Thus, two distinct RNA-based mechanisms activate this novel KAP/ROCK2/Cdk2-dependent invasion pathway in glioblastoma.

SKLB-287, a novel oral multikinase inhibitor of EGFR and VEGFR2, exhibits potent antitumor activity in LoVo colorectal tumor model.

Colorectal cancer (CRC) is the third common cancer and most of the chemotherapies of CRC currently used often suffer limited efficacy and large side effects. Targeted small-molecule by anti-tumor drugs are thought a promising strategy for improving the efficacy and reducing the side effects. In this investigation, we report a novel multikinase inhibitor, termed SKLB-287, which was discovered by us recently. SKLB-287 could efficiently inhibit the activation of endothelial growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2). It displayed very good anti-proliferative activity against LoVo CRC cells and considerable antiangiogenic potency in transgenic zebrafish embryos. Oral administration of SKLB-287 resulted in dose-dependent suppression of tumor growth in LoVo xenograft mouse model. Immunohistochemistry was adopted to examine the in vivo anti-tumor mechanism of action of SKLB-287.

A novel allergen Tab y 1 with inhibitory activity of platelet aggregation from salivary glands of horseflies.

BACKGROUND: Horsefly sting causes allergic reactions in human body. However, our knowledge on horsefly allergens remains poor. OBJECTIVES: To identify the novel horsefly allergens and characterize their properties. METHODS: A native allergen protein Tab y 1 (apyrase) was purified from the salivary glands of the horsefly Tabanus yao Macquart by gel filtration and ion exchange chromatography. Its sequence was determined by Edman degradation and cDNA cloning. Its allergenicity was assessed by immunoblotting for specific IgE, basophil activation test, skin prick test (SPT), and competitive enzyme-linked immunosorbent assay (ELISA). RESULTS: Tab y 1 showed a single diffusion band of 70 kDa on SDS-PAGE. Seventy percent (7/10) of patients with horsefly allergy tested positive to Tab y 1 in SPT; sera from 81% (30/37) of patients reacted to Tab y 1 on western blots. Purified Tab y 1 reduced approximately 42% sera IgE reactivity to horsefly salivary gland extract on a competitive ELISA. Tab y 1 upregulated the expression of CD63 and CCR3 on passively sensitized basophils by up to approximately 4.9-fold. Tab y 1 also showed enzymatic activity to hydrolyze ATP and ADP, and potent antiplatelet aggregation and antithrombotic activities. CONCLUSION: The current work identified a novel major allergen of horsefly, Tab y 1, with antiplatelet aggregation and antithrombotic activities, which implicates Tab y 1 in helping horseflies suck host blood, meanwhile causing allergy in their human hosts.

Isolation and characterization of multipotent stem cells from human cruciate ligaments.

OBJECTIVES: Mesenchymal stem cells have great potential for tissue regeneration, and these cells can be harvested from a variety of tissues; however, up to now it has not been clear whether stem cells could be isolated from cruciate ligaments of the knee joint. The aim of our study was to isolate and characterize stem cells from both anterior and posterior cruciate ligaments (ACL and PCL) of humans. MATERIALS AND METHODS: Cruciate ligaments were obtained from patients receiving total knee arthroplasty for advanced osteoarthritis and plastic-adherent cells were serially passaged. In vitro chondrogenic, osteogenic and adipogenic abilities of the cells were evaluated by reverse transcriptase-polymerase chain reaction and histological study. Karyotyping and surface immunophenotyping of the cells were performed. RESULTS: It was found that a population of ligament-derived cells could be expanded and subcultured extensively. These cells were able to differentiate into osteoblasts, chondrocytes and adipocytes under appropriate inductions. Their phenotypic characteristics were similar to those of bone marrow mesenchymal stem cells. Karyotyping was normal after serial passage. CONCLUSIONS: In summary, our study demonstrates that human multipotent stem cells can be isolated and expanded from human ACL and PCL, which are easily obtained from patients following total knee or cruciate ligament reconstructive surgery. Self-renewal and mesodermal differentiation potential of these cells make them a viable alternative source for use in regenerative medicine.

Relationship between HPV16/18 E6 and 53, 21WAF1, MDM2, Ki67 and cyclin D1 expression in esophageal squamous cell carcinoma: comparative study by using tissue microarray technology.

AIM: To investigate the role of human papillomavirus (HPV) HPV16/18 E6 oncogene in the carcinogenesis of esophageal cell carcinoma (ESCC). MATERIALS AND METHODS: Tissue microarray (TMA) block was constructed from 60 cases of paraffin-embedded ESCC tissues and pair-matched controls (adjacent normal epithelium). Immunohistochemistry (IHC) methods were applied to detect the expression of HPV16/18 E6, p53, p21(WAF1), MDM2, Ki67 and cyclin D1 proteins on TMA slides. In situ hybridization (ISH) targeting HPV gene was also used. RESULTS: In ESCC samples, 18.3% (11/60) were revealed HPV16/18 E6 positive by IHC, while 40.0% (24/60) HPV positive by ISH; HPV16/18 E6 expression was significantly higher than that of control samples. In ESCC samples, the expressions of p53, p21(WAF1), Cyclin D1, MDM2 and Ki67 were recorded in 60.0% (36/60), 40.0% (24/60), 51.7% (31/60), 65.0% (39/60) and 88.3% (53/60) cases respectively, In ESCC samples, p53, MDM2 and Ki67 expression correlated with the HPV16/18 E6 expression (p less, similar 0.01), p21(WAF1) expression - with these of MDM2 and cyclin D1 (p less, similar 0.01) whilst expression of Ki67 - with ESCC grade (p less, similar 0.01). CONCLUSION: HPV might be one of etiological factor of esophageal carcinoma in Shantou, China. p53, MDM2 proteins may play important roles in the pathogenesis of HPV-associated ESCC.

Adenocarcinoma arising from an ectopic pancreas in the stomach.

Ectopic pancreas is rare, being found in between 0.6 % and 15 % at autopsy. Heterotopic pancreas is usually an asymptomatic condition which is found incidentally at surgery or at autopsy. Occasionally, significant symptoms arise from complications, such as recurrent upper gastrointestinal bleeding, biliary or intestinal obstruction, or malignant degeneration. Malignant change is very rare. We report a case of malignant change (adenocarcinoma) in an ectopic pancreas in the stomach. In the literature, there are eight reported cases of malignant change in ectopic gastric pancreas. The prognosis in the other reported cases is unknown, but in our patient, the tumor was confined to the muscle of the stomach and there was no lymph node invasion.

Genomic structure and mutational analysis of the human KIF1B gene which is homozygously deleted in neuroblastoma at chromosome 1p36.2.

In order to clone candidate tumor suppressor genes whose loss contributes to the pathogenesis of neuroblastoma (NB), we performed polymerase chain reaction (PCR) screening using a high-density sequence tagged site-content map within a commonly deleted region (chromosome band 1p36) in 24 NB cell lines. We found a approximately 480 kb homozygously deleted region at chromosome band 1p36.2 in one of the 24 NB cell lines, NB-1, and cloned the human homologue (KIF1B-beta) of the mouseKif1B-beta gene in this region. The KIF1B-beta gene had at least 47 exons, all of which had a classic exon-intron boundary structure. Mouse Kif1B is a microtubule-based putative anterograde motor protein for the transport of mitochondria in neural cells. We performed mutational analysis of the KIF1B-beta gene in 23 cell lines using 46 sets of primers and also an allelic imbalance (AI) analysis of KIF1B-beta in 50 fresh NB samples. A missense mutation at codon 1554, GTG (Gly) to ATG (Met), silent mutations at codon 409 (ACG to ACA) and codon 1721 (ACC to ACT), and polymorphisms at codon 170, GAT (Asp) to GAA (Glu), and at codon 1087, TAT (Tyr), to TGT (Cys), were all identified, although their functional significances remain to be determined. The AI for KIF1B-beta was slightly higher (38%) than those for the other two markers (D1S244, D1S1350) (35 and 32%) within the commonly deleted region (1p36). Reverse transcriptase-PCR analysis of the KIF1B-beta gene revealed obvious expression in all NB cell lines except NB-1, although decreased expression of the KIF1B-beta gene was found in a subset of early- and advanced-stage NBs. These results suggest that the KIF1B-beta gene may not be a candidate for tumor suppressor gene of NB.

Homozygous deletion in a neuroblastoma cell line defined by a high-density STS map spanning human chromosome band 1p36.

Recent molecular studies have shown a relatively high rate of loss of heterozygosity (LOH) in neuroblastoma (NB) as well as other types of tumors in human chromosome band 1p36. To identify candidate tumor suppressor genes in NB, we searched for homozygous deletions in NB cell lines with PCR according to a high-density sequence tagged site (STS)-content map spanning 1p35-36. Among 25 NB cell lines examined, only one cell line, NB-1, showed no signal with 27 STSs in a 480 kb region in 1p36.2. The sequence analysis has revealed that the defective region included seven known genes (E4, KIF1B, SCYA5, PGD, Cortistatin, DFF45, and PEX14), nine expressed sequence tags (ESTs), and two microsatellite markers. These genes are related to apoptosis, an ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule, and components of a common translocation machinery. The region between the DFF45 and KIF1B genes was defined as homozygous deletion by Southern blotting. The search in LOH regions with high-density STSs may be useful for the isolation and identification of tumor suppressor genes in other tumors as well as NBs.

Allelic imbalance on chromosome 2q and alterations of the caspase 8 gene in neuroblastoma.

We previously reported a high incidence of loss of heterozygosity (LOH) on chromosome 2q33 in neuroblastoma (NB), observed in various types of human cancers including lung cancer, head and neck cancer and follicular thyroid carcinoma. To better elucidate the role of chromosome 2q aberrations in NB, we examined common allelic imbalance (AI) regions on chromosome 2q in 82 NB patients using 10 polymorphic microsatellite markers. AI on 2q was detected in 26 (32%) of 82 NB cases. There was a distinct common AI region between the D2S115 and D2S307 markers on 2q33. The distance between these markers was about 2.0 cM. Recently, the caspase 8 and caspase 10 genes, both of which encode cystein protease, were mapped to chromosome 2q33. Since the common AI region on 2q33 includes the caspase 8 and caspase 10 genes, the alterations of these genes were examined further. Absent or reduced expression of caspase 8 and caspase 10 were found in 19 (70%) of 27 and two (7%) of 27 NB cell lines by reverse transcription-polymerase chain reaction, respectively. A missense mutation was detected at codon 96, GCT (Alanine) to GTT (Valine), of the caspase 8 gene in one of the NB cell lines lacking caspase 8 expression. Thirteen (68%) of 19 cell lines lacking caspase 8 expression displayed methylation of the CpG island of the caspase 8 gene, whereas only one (13%) of eight cell lines with caspase 8 expression showed caspase 8 methylation (P=0.031). Furthermore, there was a significant association between AI at 2q33 and loss of caspase 8 expression (P=0.026). These results indicated that there was a tumor suppressor gene in the common AI region on chromosome 2q33 involved in the pathogenesis of a subset of NB. It is possible that the caspase 8 gene is one of the candidate tumor suppressor genes for NB and inactivation of this gene plays an important role in the tumorigenesis of NB through mainly its methylation.

DNA fragmentation factor 45 (DFF45) gene at 1p36.2 is homozygously deleted and encodes variant transcripts in neuroblastoma cell line.

Recently, loss of heterozygosity (LOH) studies suggest that more than two tumor suppressor genes lie on the short arm of chromosome 1 (1p) in neuroblastoma (NB). To identify candidate tumor suppressor genes in NB, we searched for homozygous deletions in 20 NB cell lines using a high-density STS map spanning chromosome 1p36, a common LOH region in NB. We found that the 45-kDa subunit of the DNA fragmentation factor (DFF45) gene was homozygously deleted in an NB cell line, NB-1. DFF45 is the chaperon of DFF40, and both molecules are necessary for caspase 3 to induce apoptosis. DFF35, a splicing variant of DFF45, is an inhibitor of DFF40. We examined 20 NB cell lines for expression and mutation of DFF45 gene by reverse transcription (RT)-polymerase chain reaction (PCR) and RT-PCR-single-strand conformation polymorphism. Some novel variant transcripts of the DFF45 gene were found in NB cell lines, but not in normal adrenal gland and peripheral blood. These variants may not serve as chaperons of DFF40, but as inhibitors like DFF35, thus disrupting the balance between DFF45 and DFF40. No mutations of the DFF45 gene were found in any NB cell line, suggesting that the DFF45 is not a tumor suppressor gene for NB. However, homozygous deletion of the DFF45 gene in the NB-1 cell line may imply the presence of unknown tumor suppressor genes in this region.

Loss of p73 gene expression in lymphoid leukemia cell lines is associated with hypermethylation.

The expression of the p73 gene and the methylation status was examined in 61 acute lymphoblastic leukemia (ALL) cell lines and lymphocytes from seven healthy individuals. p73 mRNA was not expressed in 19 (31.1%) of 61 ALL cell lines, including 11 (31.4%) of 35 B-precursor ALL cell lines, 2 (16.7%) of 12 B-ALL/Burkitt lymphoma (BL) cell lines (totally 27.7% of B-lineage cell lines), 6 (42.9%) of 14 T-ALL cell lines, and expressed in all of normal lymphocytes, by reverse transcriptase-polymerase chain reaction (RT-PCR). Restriction-enzyme related PCR (REP) and methylation-specific PCR (MSP) revealed that the cell lines lacking p73 mRNA expression were hypermethylated. In contrast, normal lymphocytes and most cell lines that expressed detectable p73 mRNA were not hypermethylated with the exception of five cell lines. Furthermore, bisulfite genomic sequencing confirmed the results obtained by REP and MSP. Our results suggest that p73 inactivation may be involved in the pathogenesis of both T- and B-ALLs, and that hypermethylation is the predominant mechanism of inactivation of the p73 gene in ALL.

Absent or reduced expression of the caspase 8 gene occurs frequently in neuroblastoma, but not commonly in Ewing sarcoma or rhabdomyosarcoma.

PROCEDURE: To clarify whether the caspase 8 gene is involved in the pathogenesis of neuroblastoma (NB), we examined alterations of the caspase 8 gene in 15 NB, seven Ewing sarcoma (ES), and eight rhabdomyosarcoma (RMS) cell lines, using reverse transcription-polymerase chain reaction (RT-PCR) and RT-PCR single-strand conformation polymorphism (SSCP) analyses. RESULTS: The caspase 8 gene was not expressed in 11 (73%) of 15 NB cell lines, it was absent in only one of seven ES cell lines, but was present in all eight RMS cell lines examined. No mutations were detected in any cell lines examined. CONCLUSIONS: Inactivation of the caspase 8 gene is considered to be involved in the pathogenesis of NB, but not ES or RMS.