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Abstract Four single-nucleotide polymorphisms have been found in the human BUB1 gene, which encodes a kinase involved in the mitotic spindle checkpoint. A cytosine-to-thymine change in exon 10, corresponding to codon 375 (c.1124C>T), causes an amino acid substitution of serine to phenylalanine. A guanine/cytosine polymorphism in exon 4 (c.279G>C) and a thymine/cytosine polymorphism in exon 12 (c.1293T>C) do not cause amino acid substitution. The other polymorphism, of thymine/cytosine (IVS9-8T>C), is found at 8bp upstream of exon 10. As mutations of the hBUB1 gene were reported in a subset of human cancers, these polymorphisms could provide useful tools for the genetic study of susceptibility to various human cancers. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs) have been positively associated with child neurodevelopment in some cohort studies. However, there is a lack of consistency in the association between HMOs and benefits to infants' brains. Moreover, the quantification methods for HMOs have not yet been standardized. In this study, we developed a quantification method for evaluating eight HMOs (2'-fucosyllactose [2'-FL], 3'-fucosyllactose [3'-FL], 3'-sialyllactose [3'-SL], 6'-sialyllactose [6'-SL], lactosialyltetrasaccharide a [LSTa], lactosialyltetrasaccharide b [LSTb], lactosialyltetrasaccharide c [LSTc], and disialyllacto-N-tetraose [DSLNT]) in breast milk. After validating the method, we applied it to 1-month breast milk samples (n = 150) from the Tohoku Medical Megabank Project Birth and Three-Generation Cohort Study to assess HMO profiles in breast milk and their possible association with changes in head circumference z-score (ΔHCZ) and neurodevelopmental scores of children (as measured by the Ages and Stages Questionnaire, Third Edition). The validation demonstrated that the method had relative standard deviation ≤ 12.7% of precision and 79.5-110.9% of accuracy. Using this method, eight HMO levels (2'-FL, 0-4.74 mg/mL; 3'-FL, 0.02-1.52 mg/mL; 3'-SL, 0.07-0.32 mg/mL; 6'-SL, 0.01-0.70 mg/mL; LSTa, 0.002-0.043 mg/mL; LSTb, 0.02-0.31 mg/mL; LSTc, 0.001-0.47 mg/mL; and DSLNT, 0.09-0.71 mg/mL [min-max, all participants]) and the ratio of low secretors (16.0%) in the Japanese cohort were obtained. The obtained HMO levels in breast milk were subjected to multivariate analysis to screen for HMOs showing a positive association with ΔHCZ and neurodevelopmental scores. The results proposed that ΔHCZ was positively associated with LSTb and 2'-FL levels, whereas neurodevelopmental scores were positively associated with 2'-FL levels (among all participants) and 3'-SL and DSLNT levels (among secretor participants). This study showed that the developed method provides HMO profiles in Japanese breast milk, as well as additional information on the associations between specific HMOs and neurodevelopment, reinforcing the sum of evidence for the role of HMOs in the brain., (© 2024 The Author(s). Journal of Food Science published by Wiley Periodicals LLC on behalf of Institute of Food Technologists.)

Background: The purpose of this study was to report the basic profile of the Miyagi Prefecture part of a repeated center-based survey during the second period of the Tohoku Medical Megabank Community-Based Cohort Study (TMM CommCohort Study), as well as the participants' characteristics based on their participation type in the baseline survey., Methods: The second period survey, conducted from June 2017 to March 2021, included participants of the TMM CommCohort Study (May 2013 to March 2016). In addition to the questionnaire, blood, urine, and physiological function tests were performed during the second period survey. There were three main ways of participation in the baseline survey: Type 1, Type 1 additional, or Type 2 survey. The second period survey was conducted in the same manner as the Type 2 survey, which was based on the community support center (CSC)., Results: In Miyagi Prefecture, 29,383 (57.7%) of 50,967 participants participated in the second period survey. The participation rate among individuals who had visited the CSC was approximately 80%. Although some factors differed depending on the participation type in the baseline survey, the second period survey respondents in the Type 1 and Type 2 survey groups at baseline had similar traits., Conclusion: The second period survey of the TMM CommCohort Study provided detailed follow-up information. Following up on the health conditions of the participants will clarify the long-term effects of disasters and contribute to personalized prevention.

With the number of samples increasing in many biobanks, one of the most pressing tasks is recording the correct relationships between information and the specimens. Genomic information is useful in determining the identity of these specimens. The Tohoku Medical Megabank Organization is running one of the largest biobanks in Japan. Here, we introduce a management system, which includes the development of a new probe set for the MassARRAY system for use during the production of proliferating T cells (T cells) and lymphoblastoid cell lines (LCLs). We selected single nucleotide variants that could be detected by next-generation sequencing and showed high resolution with ∼0.5 minor allele frequencies. After checking the set of probes against 96 samples from 48 people, we obtained no contradictory results in comparison with our genome sequence information. When we applied the set to our 3035 LCLs and 2256 T cells, the result showed 98.93% consistency with the corresponding genomic information. We surveyed the handling records of the 1.07% of samples that showed inconsistencies, and found that most had resulted from human errors (ID swapping between samples) during manual operations. After improving a few error-prone protocols, the error rate dropped to 0.47% for LCLs and 0% for T cells. Overall, the system that we developed shows high accuracy with easy and fast operability, and provides a good opportunity to improve the validation procedure to facilitate high-quality banking, especially in cases involving genomic information.

(1) Background: Breast milk is the only source of nutrition for breastfed infants, but few studies have examined the relationship between breast milk micronutrients and infant neurodevelopmental outcome in exclusively breastfed infants. The aim of this study was to characterize the association between nicotinamide adenine dinucleotide (NAD)-related compounds in the breast milk of Japanese subjects and infant neurodevelopmental outcome. (2) Methods: A total of 150 mother-child pairs were randomly selected from the three-generation cohort of the Tohoku Medical Megabank in Japan. Infants were exclusively breastfed for up to 6 months. Breast milk was collected at 1 month postpartum, and the quantity of NAD-related substances in the breast milk was quantified. The mothers also completed developmental questionnaires at 6, 12, and 24 months. The relationship between the concentration of NAD-related substances in breast milk and developmental indicators was evaluated via ordinal logistic regression analysis. (3) Results: Nicotinamide mononucleotide (NMN) was quantified as the major NAD precursor in breast milk. The median amount of NMN in the breast milk was 9.2 μM. The NMN concentration in breast milk was the only NAD-related substance in breast milk that showed a significant positive correlation with neurodevelopmental outcome in infants at 24 months. (4) Conclusions: The results suggest that NMN in human milk may be an important nutrient for early childhood development.

To reveal gene-environment interactions underlying common diseases and estimate the risk for common diseases, the Tohoku Medical Megabank (TMM) project has conducted prospective cohort studies and genomic and multiomics analyses. To establish an integrated biobank, we developed an integrated database called "dbTMM" that incorporates both the individual cohort/clinical data and the genome/multiomics data of 157,191 participants in the Tohoku Medical Megabank project. To our knowledge, dbTMM is the first database to store individual whole-genome data on a variant-by-variant basis as well as cohort/clinical data for over one hundred thousand participants in a prospective cohort study. dbTMM enables us to stratify our cohort by both genome-wide genetic factors and environmental factors, and it provides a research and development platform that enables prospective analysis of large-scale data from genome cohorts., (© 2021. The Author(s).)

Human lymphoblastoid cell lines (LCLs) are valuable for the functional analyses of diseases. We have established more than 4200 LCLs as one of the resources of an integrated biobank. While oxidative and inflammatory stresses play critical roles in the onset and progression of various diseases, the responsiveness of LCLs, especially that of biobank-made LCLs, to these stresses has not been established. To address how LCLs respond to these stresses, in this study, we performed RNA sequencing of eleven human LCLs that were treated with an electrophile, diethyl maleate (DEM) and/or an inflammatory mediator, lipopolysaccharide (LPS). We found that over two thousand genes, including those regulated by a master regulator of the electrophilic/oxidative stress response, NRF2, were upregulated in LCLs treated with DEM, while approximately three hundred genes, including inflammation-related genes, were upregulated in LPS-treated LCLs. Of the LPS-induced genes, a subset of proinflammatory genes was repressed by DEM, supporting the notion that DEM suppresses the expression of proinflammatory genes through NRF2 activation. Conversely, a part of DEM-induced gene was repressed by LPS, suggesting reciprocal interference between electrophilic and inflammatory stress-mediated pathways. These data clearly demonstrate that LCLs maintain, by and large, responsive pathways against oxidative and inflammatory stresses and further endorse the usefulness of the LCL supply from the biobank., (Copyright © 2020. Published by Elsevier Inc.)

The Tohoku Medical Megabank biobank (TMM biobank) is the first major population-based biobank established in Japan. The TMM biobank was established based on two population cohorts and is a reconstruction program from the Great East Japan Earthquake and Tsunami of 2011. The biobank stores more than 3.4 million tubes of biospecimens and associated health and analytic data obtained from approximately 150,000 TMM cohort participants between May 2013 and December 2018, and the TMM biobank currently shares high-quality specimens and data. Various biospecimens, including peripheral and cord blood mononuclear cells, buffy coat, plasma, serum, urine, breast milk and saliva have been collected in the TMM biobank. To minimize human error and maintain the quality of data and specimens, we have been utilizing laboratory information management system into various biobank procedures from registration to storage with various automation systems, such as liquid dispensing, DNA extraction and their storage. The biobank procedures for the quality management system (ISO 9001:2015) and information security management system (ISO 27001:2013) are certified by the International Organization for Standardization. The quality of our biobank samples fulfills the pre-analytical requirements for researchers conducting next-generation whole genome sequencing, DNA array analyses, proteomics, metabolomics, etc. We established analytical centers to conduct standard genomic and multiomic analyses in-house and share the generated data. Additionally, we generate thousands of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines and proliferating T cells for functional studies. The TMM biobank serves as an indispensable infrastructure for academic, clinical and industrial research to actualize next-generation medicine in Japan.

Malignant peripheral nerve sheath tumors (MPNST) are chemoresistant sarcomas with poor 5-year survival that arise in patients with neurofibromatosis type 1 (NF1) or sporadically. We tested three drugs for single and combinatorial effects on collected MPNST cell lines and in MPNST xenografts. The mammalian target of rapamycin complex 1 inhibitor RAD001 (Everolimus) decreased growth 19% to 60% after 4 days of treatment in NF1 and sporadic-derived MPNST cell lines. Treatment of subcutaneous sporadic MPNST cell xenografts with RAD001 significantly, but transiently, delayed tumor growth, and decreased vessel permeability within xenografts. RAD001 combined with the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib caused additional inhibitory effects on growth and apoptosis in vitro, and a small but significant additional inhibitory effect on MPNST growth in vivo that were larger than the effects of RAD001 with doxorubicin. RAD001 plus erlotinib, in vitro and in vivo, reduced phosphorylation of AKT and total AKT levels, possibly accounting for their additive effect. The results support the consideration of RAD001 therapy in NF1 patient and sporadic MPNST. The preclinical tests described allow rapid screening strata for drugs that block MPNST growth, prior to tests in more complex models, and should be useful to identify drugs that synergize with RAD001.

Excess levels of circulating amino acids (AAs) play a causal role in specific human pathologies, including obesity and type 2 diabetes. Moreover, obesity and diabetes are contributing factors in the development of cancer, with recent studies suggesting that this link is mediated in part by AA activation of mammalian target of rapamycin (mTOR) Complex 1. AAs appear to mediate this response through class III phosphatidylinositol 3-kinase (PI3K), or human vacuolar protein sorting 34 (hVps34), rather than through the canonical class I PI3K pathway used by growth factors and hormones. Here we show that AAs induce a rise in intracellular Ca(2+) ([Ca(2+)](i)), which triggers mTOR Complex 1 and hVps34 activation. We demonstrate that the rise in [Ca(2+)](i) increases the direct binding of Ca(2+)/calmodulin (CaM) to an evolutionarily conserved motif in hVps34 that is required for lipid kinase activity and increased mTOR Complex 1 signaling. These findings have important implications regarding the basic signaling mechanisms linking metabolic disorders with cancer progression.

Recent studies have shown that the nutrient input to the mTOR Complex1/S6K1 signaling pathway is mediated by class 3 PI3K or hVps34, the oldest member of the PI3K family. Moreover, studies to date would suggest that during the evolution of multicellular organisms this ancient branch of the pathway was merged with the growth-factor-hormone-controlled class 1 PI3K pathway at the level of mTOR Complex1 to control the development and growth of the organism. However, hVps34 also plays a role in the regulation of macroautophagy - the mechanism by which cells generate nutrients, such as amino acids, through the degradation of intracellular complexes, including mitochondria and ribosomes. These functions of hVps34 initially appear contradictory, since increased mTOR Complex1 activation is triggered by increased amino acid levels, while autophagy is triggered when cells are faced with amino acid deprivation.

During the evolution of metazoans and the rise of systemic hormonal regulation, the insulin-controlled class 1 phosphatidylinositol 3OH-kinase (PI3K) pathway was merged with the primordial amino acid-driven mammalian target of rapamycin (mTOR) pathway to control the growth and development of the organism. Insulin regulates mTOR function through a recently described canonical signaling pathway, which is initiated by the activation of class 1 PI3K. However, how the amino acid input is integrated with that of the insulin signaling pathway is unclear. Here we used a number of molecular, biochemical, and pharmacological approaches to address this issue. Unexpectedly, we found that a major pathway by which amino acids control mTOR signaling is distinct from that of insulin and that, instead of signaling through components of the insulin/class 1 PI3K pathway, amino acids mediate mTOR activation by signaling through class 3 PI3K, hVps34.

Tumor suppressor genes evolved as negative effectors of mitogen and nutrient signaling pathways, such that mutations in these genes can lead to pathological states of growth. Tuberous sclerosis (TSC) is a potentially devastating disease associated with mutations in two tumor suppressor genes, TSC1 and 2, that function as a complex to suppress signaling in the mTOR/S6K/4E-BP pathway. However, the inhibitory target of TSC1/2 and the mechanism by which it acts are unknown. Here we provide evidence that TSC1/2 is a GAP for the small GTPase Rheb and that insulin-mediated Rheb activation is PI3K dependent. Moreover, Rheb overexpression induces S6K1 phosphorylation and inhibits PKB phosphorylation, as do loss-of-function mutations in TSC1/2, but contrary to earlier reports Rheb has no effect on MAPK phosphorylation. Finally, coexpression of a human TSC2 cDNA harboring a disease-associated point mutation in the GAP domain, failed to stimulate Rheb GTPase activity or block Rheb activation of S6K1.

The evolution of mitogenic pathways has led to the parallel requirement for negative control mechanisms, which prevent aberrant growth and the development of cancer. Principally, such negative control mechanisms are represented by tumor suppressor genes, which normally act to constrain cell proliferation (Macleod, K. 2000. Curr. Opin. Genet. Dev. 10:81-93). Tuberous sclerosis complex (TSC) is an autosomal-dominant genetic disorder, characterized by mutations in either TSC1 or TSC2, whose gene products hamartin (TSC1) and tuberin (TSC2) constitute a putative tumor suppressor complex (TSC1-2; van Slegtenhorst, M., M. Nellist, B. Nagelkerken, J. Cheadle, R. Snell, A. van den Ouweland, A. Reuser, J. Sampson, D. Halley, and P. van der Sluijs. 1998. Hum. Mol. Genet. 7:1053-1057). Little is known with regard to the oncogenic target of TSC1-2, however recent genetic studies in Drosophila have shown that S6 kinase (S6K) is epistatically dominant to TSC1-2 (Tapon, N., N. Ito, B.J. Dickson, J.E. Treisman, and I.K. Hariharan. 2001. Cell. 105:345-355; Potter, C.J., H. Huang, and T. Xu. 2001. Cell. 105:357-368). Here we show that loss of TSC2 function in mammalian cells leads to constitutive S6K1 activation, whereas ectopic expression of TSC1-2 blocks this response. Although activation of wild-type S6K1 and cell proliferation in TSC2-deficient cells is dependent on the mammalian target of rapamycin (mTOR), by using an S6K1 variant (GST-DeltaC-S6K1), which is uncoupled from mTOR signaling, we demonstrate that TSC1-2 does not inhibit S6K1 via mTOR. Instead, we show by using wortmannin and dominant interfering alleles of phosphatidylinositide-3-OH kinase (PI3K) that increased S6K1 activation is contingent upon the suppression of TSC2 function by PI3K in normal cells and is PI3K independent in TSC2-deficient cells.

Osteoblasts and adipocytes are derived from a common precursor in bone marrow, the mesenchymal stem cell (MSC). Factors driving human MSCs (hMSCs) to differentiate down the two lineages play important roles in determining bone density because it has been shown that bone volume loss associated with osteoporosis and aging is accompanied by reduced osteoblastic bone formation and increased marrow adipose tissue. The genes upregulated in hMSCs during osteogenic differentiation were screened using cDNA microarrays and were semi-quantitated by real-time RT-PCR. One of the genes identified was sortilin, which was upregulated one day after osteogenic induction and remained upregulated for a week. The overexpression of sortilin in hMSCs using an adenovirus vector resulted in the acceleration of mineralization during osteogenic differentiation without affecting alkaline phosphatase activity. Lipoprotein lipase (LPL), produced by adipocytes, is bound by sortilin, which may mediate its endocytosis. By adding LPL to osteogenic induction medium, osteoblastic mineralization was inhibited in a dose-dependent manner. Interestingly, sortilin overexpression abolished the LPL-mediated suppression of osteogenic differentiation. hMSCs exist in marrow where LPL-producing adipose cells are abundant and where osteogenesis is negatively regulated by LPL. Sortilin has a counter effect of promoting osteogenesis by acting as a scavenger of LPL., (Copyright 2002 Wiley-Liss, Inc.)

We recently identified TSLC1, a tumor suppressor gene in human lung cancer. Gene silencing by promoter methylation has been observed frequently in adenocarcinoma of the lung, liver, and pancreas. Here, we demonstrate that TSLC1 expression is also absent or markedly reduced in 3 of 4 prostate cancer cell lines. Promoter sequences of TSLC1 were heavily methylated in PPC-1 cells that lacked TSLC1 expression, supporting the idea that promoter methylation is strongly correlated with complete loss of gene expression. Promoter sequences of TSLC1 were also methylated significantly in 7 of 22 (32%) primary prostate cancers. Hypermethylation of the promoter occurred not only in advanced tumors, but also in relatively early-stage tumors. Restoration of TSLC1 expression substantially suppressed tumor formation of PPC-1 cells in nude mice. These findings indicate that alteration of TSLC1 is involved in prostate cancer.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is the predominant myelopathy among Japanese, and is usually diagnosed by ectopic bone formation in the paravertebral ligament in Japanese and other Asians. To detect genetic determinants associated with OPLL, we performed an extensive nonparametric linkage study with 126 affected sib-pairs using markers for various candidate genes by distinct analyses, SIBPAL and GENEHUNTER. Eighty-eight candidate genes were selected by comparing the genes identified by complementary DNA (cDNA) microarray analysis of systematic gene expression profiles during osteoblastic differentiation of human mesenchymal stem cells with the genes known to be involved in bone metabolism. Of the 24 genes regulated during osteoblastic differentiation, only one, the alpha B crystalline gene, showed evidence of linkage (p = 0.016, nonparametric linkage [NPL] score = 1.83). Of 64 genes known to be associated with bone metabolism, 7 showed weak evidence of linkage by SIBPAL analysis (p < 0.05): cadherin 13 (CDH13), bone morphogenetic protein 4 (BMP4), proteoglycan 1 (PRG1), transforming growth factor beta 3 (TGFb3), osteopontin (OPN), parathyroid hormone receptor 1 (PTHR1), and insulin-like growth factor 1 (IGF1). Among these genes, BMP4 (NPL = 2.23), CDH13 (NPL = 2.00), TGFb3 (NPL = 1.30), OPN (NPL = 1.15), and PTHR1 (NPL = 1.00) showed evidence of linkage by GENEHUNTER. Only BMP4 reached criteria of suggestive evidence of linkage. Because this gene is a well-known factor in osteogenetic function, BMP4 should be screened in further study for the polymorphism responsible.

We have recently identified the TSLC1 gene as a novel tumor suppressor in human non-small cell lung cancers. TSLC1 encodes a membrane glycoprotein with an extracellular domain homologous to those of immunoglobulin superfamily proteins. Truncation of TSLC1 in the cytoplasmic domain in a primary human tumor suggests that this domain is important for tumor suppressor activity. Here, we report the isolation of two TSLC1-like genes, TSLL1 and TSLL2, based on their structural homology with the sequences corresponding to the cytoplasmic domain of TSLC1. Significant similarity was also observed in the extracellular domain as well as in the overall gene structure, indicating that these three genes form a unique subfamily (the TSLC1-gene family) in the immunoglobulin superfamily genes. In contrast to the ubiquitous expression of TSLC1, TSLL1 is expressed exclusively in adult and fetal human brain, while TSLL2 is expressed in several specific tissues including prostate, brain, kidney and some other organs. Expression of TSLL1 and TSLL2 was lost or markedly reduced in many human glioma cell lines or some prostate cancer cell lines, suggesting that loss of expression of these genes might be involved in some human cancers.

BCNT (a protein named after Bucentaur or craniofacial development protein 1) has a unique structure in Ruminantia. Bovine BCNT contains a region of the endonuclease domain derived from a truncated RTE-1 (previously called Bov-B LINE), a non-LTR retrotransposable repetitive element, and two repeat units (intramolecular repeat, IR) each with 40 amino acids in the C-terminal region. In contrast the human and mouse BCNT proteins contain one repeat unit and lack the RTE-1-derived portion. The 3' UTR of bovine bcnt cDNA also contains an approximately 300-bp portion homologous to the 3'-part of RTE-1. We examined the bovine bcnt genomic DNA sequence to understand how the bovine bcnt gene has been organized. The sequence of 3' UTR homologous portion was found to more closely resemble the Art2 element than the bovine RTE-1. By PCR screening a bovine/hamster hybrid somatic cell panel, the bovine bcnt gene was mapped to chromosome 18, syntenic human chromosome 16q on which human BCNT is located. The bcnt genomic DNA sequence corresponding to the cDNA downstream of a RTE-1 derived portion reveals that each IR unit is flanked by both 5'-side and 3'-side introns and that 3'-UTR consists of one exon. The alignment of the above sequence with a bovine RTE-1 did not show any significant homology downstream of the endonuclease domain. On the other hand, the alignment of the intron sequences with each other revealed that the six sequential homologous segments ranging in size from 40 to 453 bp existed over a 1 kb long sequence between both the 5'- and 3'-side introns flanking each bovine IR unit. In addition, both the 174-bp of 5'-side intron and 80-bp of 3'-side intron neighboring each 120-bp IR exon are significantly homologous among the two bovine IRs, human IR and mouse IR. These results suggest that a truncated bovine RTE-1 was inserted into the intron upstream of an IR unit of an ancestor bcnt gene and that a duplication of a relatively long region that includes IR occurred in the bovine genome.

The existence of tumor-suppressor genes was originally demonstrated by functional complementation through whole-cell and microcell fusion. Transfer of chromosome 11 into a human non-small-cell lung cancer (NSCLC) cell line, A549, suppresses tumorigenicity. Loss of heterozygosity (LOH) on the long arm of chromosome 11 has been reported in NSCLC and other cancers. Several independent studies indicate that multiple tumor-suppressor genes are found in this region, including the gene PPP2R1B at 11q23-24 (ref. 7). Linkage studies of NSCLC are precluded because no hereditary forms are known. We previously identified a region of 700 kb on 11q23.2 that completely suppresses tumorigenicity of A549 human NSCLC cells. Most of this tumor-suppressor activity localizes to a 100-kb segment by functional complementation. Here we report that this region contains a single confirmed gene, TSLC1, whose expression is reduced or absent in A549 and several other NSCLC, hepatocellular carcinoma (HCC) and pancreatic cancer (PaC) cell lines. TSLC1 expression or suppression is correlated with promoter methylation state in these cell lines. Restoration of TSLC1 expression to normal or higher levels suppresses tumor formation by A549 cells in nude mice. Only 2 inactivating mutations of TSLC1 were discovered in 161 tumors and tumor cell lines, both among the 20 primary tumors with LOH for 11q23.2. Promoter methylation was observed in 15 of the other 18 primary NSCLC, HCC and PaC tumors with LOH for 11q23.2. Thus, attenuation of TSLC1 expression occurred in 85% of primary tumors with LOH. Hypermethylation of the TSLC1 promoter would seem to represent the 'second hit' in NSCLC with LOH.

High-frequent microsatellite instability (MSI-H) was detected in two of the 80 gliomas examined, whlie the other 78 gliomas showed microsatellite stable (MSS) phenotype. Both of the two MSI-H tumors were glioblastomas which developed in teenage patients. One of the patient was diagnosed as having Turcot's syndrome and had a germline mutation in the hMLH1 gene. Loss of expression due to promoter methylation was selectively observed in the wild type allele of the hMLH1 gene in the tumor of this patient. The other patient had neither a family history nor a past personal history of malignancy. Although no mutation in the mismatch repair genes was detected in the tumor of this patient, the level of expression of the hMLH1 gene was markedly decreased and the promoter sequence of the gene was highly methylated. In the tumor of this patient, the PTEN1 gene, one of the genes carrying microsatellite sequences in their coding regions, was altered by a slippage mutation within five adenine repeat sequences. These findings indicate that the genetic or epigenentic inactivation of the hMLH1 gene is involved in a subset of early-onset gliomas and the PTEN1 gene could be a downstream target for mutation as observed in glioblastoma without MSI.

DNA from 10 human glioma cell lines was analyzed by arbitrarily primed polymerase chain reaction. By fingerprinting of the DNA fragments obtained, the presence of fragment Qx with an abnormal signal was detected in one of the glioblastoma cell lines, CCF-STTG1. The nucleotide sequence of this fragment of 387 base pairs showed no homology with any known sequences. Southern-blot analysis using Qx as a probe revealed that the abnormal signal was caused by amplification of DNA by about 50-fold. By analysis of radiation hybrid panels, the fragment was shown to be derived from a chromosomal region on 6p21. The cyclin D3 (ccnd3) gene and an EST locus, H40682, both of which were located in this region, were amplified by about 50-fold in this cell line. Two other loci, R75654 and M78872, flanking the Qx, CCND3 and H40682 loci, were not amplified, suggesting that the size of the amplicon was less than 62 cR. Since over-expression of the ccnd3 gene, but not the H40682 locus, was detected in the cell line CCF-STTG1, the increased amounts of cyclin D3 caused by gene amplification could be involved in the development and/or progression of this glioblastoma., (Copyright 2000 Wiley-Liss, Inc.)

BCNT, named after Bucentaur, is a protein that contains a 324-amino-acid region derived from part of a long interspersed DNA sequence element (LINE) in Ruminantia. However, the unique portion is completely missing in human and mouse BCNTs. Since no significant information on their function has been obtained by homology search, we at first examined cellular localization and biochemical characteristics of bovine BCNT to get a hint on its function. Subcellular fractionation and immunohistochemical analyses using a normal bovine epithelial cell line and bovine brain revealed that a significant amount of bovine BCNT is localized in the nuclei, while the major portion is present in the cytosol. Furthermore, it was shown that bovine BCNT is a phosphoprotein and that both bovine and human BCNTs are phosphorylated by casein kinase II in vitro. These results show that BCNTs consist of a unique family, probably a substrate of casein kinase II, which may contribute further to the understanding of gene evolution.

Loss of heterozygosity on chromosome 11q23 is observed at high frequency in human nonsmall cell lung carcinomas (NSCLCs), suggesting the presence of a tumor suppressor gene. Previous analysis of DNA from 79 patients identified a commonly deleted segment of 5 centimorgans. Complementation analysis was used to further localize a putative tumor suppressor gene. Three yeast artificial chromosome (YAC) clones spanning the minimal loss of heterozygosity region were modified, and spheroplast fusion was used to transfer them into human A549 NSCLC or murine Lewis lung carcinoma (LLC) cell lines. The resulting yeast x human hybrid cell lines containing an intact copy of a 1.6-Mb YAC, 939b12, showed reduced growth in vitro. Injection of parental A549 cells into athymic (nu/nu) mice resulted in tumor formation at 27 of 28 injection sites. In contrast, two independent 939b12-containing cell lines formed tumors at only 3 of 20 injection sites. 939b12 also suppressed tumor formation by LLC NSCLC cells in nude mice, but YACs 785e12 and 911f2, which flank 939b12, had no suppressor activity. Further localization of tumor suppression activity on 939b12 was accomplished by introduction of defined fragmentation derivatives into A549 cells and by analysis of YACs that were broken on transfer into LLC cells. This complementation approach localized tumor suppression activity to the central 700 kb of 939b12 and provides a functional assay for positional cloning of this tumor suppressor gene.