Correlation between luminescence intensity and cytotoxicity in cell-based cytotoxicity assay using luciferase.

The luciferase reporter assay has become one of the conventional methods for cytotoxicity evaluation. Typically, the decrease of luminescence expressed by a constitutive promoter is used as an index of cytotoxicity. However, to our knowledge, there have been no reports of the correlation between cytotoxicity and luminescence intensity. In this study, to accurately verify the correlation between them, beetle luciferase was stably expressed in human hepatoma HepG2 cells harboring the multi-integrase mouse artificial chromosome vector. We showed that the cytotoxicity assay using luciferase does not depend on the stability of luciferase protein and the kind of constitutive promoter. Next, HepG2 cells in which green-emitting beetle luciferase was expressed under the control of CAG promoter were exposed to 58 compounds. The luminescence intensity and cytotoxicity curves of cells exposed to 48 compounds showed similar tendencies, whereas those of cells exposed to 10 compounds did not do so, although the curves gradually approached each other with increasing exposure time. Finally, we demonstrated that luciferase expressed under the control of a constitutive promoter can be utilized both as an internal control reporter for normalizing a test reporter and for monitoring cytotoxicity when two kinds of luciferases are simultaneously used in the cytotoxicity assay.

Refined human artificial chromosome vectors for gene therapy and animal transgenesis.

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.

Correction of a genetic defect in multipotent germline stem cells using a human artificial chromosome.

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including regulatory elements. Multipotent germline stem (mGS) cells have a great potential for gene therapy because they can be generated from an individual's testes, and when reintroduced can contribute to the specialized function of any tissue. As a proof of concept, we herein report the functional restoration of a genetic deficiency in mouse p53-/- mGS cells, using a HAC with a genomic human p53 gene introduced via microcell-mediated chromosome transfer. The p53 phenotypes of gene regulation and radiation sensitivity were complemented by introducing the p53-HAC and the cells differentiated into several different tissue types in vivo and in vitro. Therefore, the combination of using mGS cells with HACs provides a new tool for gene and cell therapies. The next step is to demonstrate functional restoration using animal models for future gene therapy.

SIRT2, a tubulin deacetylase, acts to block the entry to chromosome condensation in response to mitotic stress.

We previously identified SIRT2, an nicotinamide adenine dinucleotide (NAD)-dependent tubulin deacetylase, as a protein downregulated in gliomas and glioma cell lines, which are characterized by aneuploidy. Other studies reported SIRT2 to be involved in mitotic progression in the normal cell cycle. We herein investigated whether SIRT2 functions in the mitotic checkpoint in response to mitotic stress caused by microtubule poisons. By monitoring chromosome condensation, the exogenously expressed SIRT2 was found to block the entry to chromosome condensation and subsequent hyperploid cell formation in glioma cell lines with a persistence of the cyclin B/cdc2 activity in response to mitotic stress. SIRT2 is thus a novel mitotic checkpoint protein that functions in the early metaphase to prevent chromosomal instability (CIN), characteristics previously reported for the CHFR protein. We further found that histone deacetylation, but not the aberrant DNA methylation of SIRT2 5'untranslated region is involved in the downregulation of SIRT2. Although SIRT2 is normally exclusively located in the cytoplasm, the rapid accumulation of SIRT2 in the nucleus was observed after treatment with a nuclear export inhibitor, leptomycin B and ionizing radiation in normal human fibroblasts, suggesting that nucleo-cytoplasmic shuttling regulates the SIRT2 function. Collectively, our results suggest that the further study of SIRT2 may thus provide new insights into the relationships among CIN, epigenetic regulation and tumorigenesis.

PI3K-Akt pathway: its functions and alterations in human cancer.

Phosphatidylinositol-3-kinase (PI3K) is a lipid kinase and generates phosphatidylinositol-3,4,5-trisphosphate (PI(3, 4, 5)P3). PI(3, 4, 5)P3 is a second messenger essential for the translocation of Akt to the plasma membrane where it is phosphorylated and activated by phosphoinositide-dependent kinase (PDK) 1 and PDK2. Activation of Akt plays a pivotal role in fundamental cellular functions such as cell proliferation and survival by phosphorylating a variety of substrates. In recent years, it has been reported that alterations to the PI3K-Akt signaling pathway are frequent in human cancer. Constitutive activation of the PI3K-Akt pathway occurs due to amplification of the PIK3C gene encoding PI3K or the Akt gene, or as a result of mutations in components of the pathway, for example PTEN (phosphatase and tensin homologue deleted on chromosome 10), which inhibit the activation of Akt. Several small molecules designed to specifically target PI3K-Akt have been developed, and induced cell cycle arrest or apoptosis in human cancer cells in vitro and in vivo . Moreover, the combination of an inhibitor with various cytotoxic agents enhances the anti-tumor efficacy. Therefore, specific inhibition of the activation of Akt may be a valid approach to treating human malignancies and overcoming the resistance of cancer cells to radiation or chemotherapy.

Expression of RUNX3 protein in human gastric mucosa, intestinal metaplasia and carcinoma.

BACKGROUND: Human runt-related transcription factor gene 3 (RUNX3) is considered as a possible candidate of tumour suppressor gene in human gastric carcinoma. MATERIALS AND METHODS: To investigate the RUNX3 protein expression in human gastric mucosa and carcinoma, we generated a polyclonal antibody, AS251, which recognized amino acid sequences from 251 to 266 of human RUNX3. The AS251 antibody was immunoreactive with only RUNX3 protein, but not with RUNX1 and RUNX2. The AS251-antibody was available for Western blotting and immunohistochemistry using paraffin-embedded specimens. RESULTS: Western-blot analysis revealed that three (MKN-1, -7 and -45) of six human gastric carcinoma cell lines variably expressed RUNX3 protein, consistent with the expression pattern of RUNX3 mRNA reported previously by Li et al. (Cell 2002;109:113-24). Immunohistochemistry disclosed RUNX3 protein in most chief cells and a few gastrin-containing G cells in normal mucosa, but not in intestinal metaplasia and carcinoma cells. CONCLUSIONS: These data suggest that RUNX3 may play a physiologic role in chief cells and G cells in gastric mucosa, and that suppression of RUNX3 expression in intestinal metaplasia and carcinoma of human stomach may be implicated in gastric carcinogenesis.

Targeted regulation of imprinted genes by synthetic zinc-finger transcription factors.

Epigenetic control of transcription is essential for mammalian development and its deregulation causes human disease. For example, loss of proper imprinting control at the IGF2-H19 domain is a hallmark of cancer and Beckwith-Wiedemann syndrome, with no targeted therapeutic approaches available. To address this deficiency, we engineered zinc-finger transcription proteins (ZFPs) that specifically activate or repress the IGF2 and H19 genes in a domain-dependent manner. Importantly, we used these ZFPs successfully to reactivate the transcriptionally silent IGF2 and H19 alleles, thus overriding the natural mechanism of imprinting and validating an entirely novel avenue for 'transcription therapy' of human disease.

Induction of cellular senescence in a telomerase negative human immortal fibroblast cell line, LCS-AF.1-3, by human chromosome 6.

In immortal cells (LCS-AF.1-3) which originated from cultured skin fibroblasts from a patient with Li-Fraumeni syndrome, a specific deletion has been identified on chromosome 6. To examine the relationship between this deletion and the loss of function in aging genes, we performed microcell mediated chromosome transfer (MMCT) of normal human chromosome 6 into LCS-AF.1-3 cells and analyzed their characteristics. Transferred human chromosome 6 induced morphological changes in the cells and cellular senescence with growth suppression. In a revertant clone that had escaped from induced senescence, a specific deletion was found at D6S309 in the transferred chromosome 6. High molecular weight telomeric sequences were lost in a clone that had been induced to senescence by introduction of human chromosome 6. On the other hand, human chromosome 7 induces senescence in immortal cells by suppressing the alternative lengthening of telomere (ALT) mechanism. We also performed MMCT of chromosome 7 and discuss the effect of chromosome transfer by comparing the two chromosomes. LCS-AF.1-3 cells containing a transferred chromosome 7 showed no changes in immortality. These results suggest that genes which are new candidates for aging and which suppress the ALT mechanism are located in the region D6S309 in human chromosome 6.

hTR repressor-related gene on human chromosome 10p15.1.

Somatic cells express genes that suppress telomerase activity and these genes may be inactivated in tumour cells. We postulated that cancer cells acquire immortality by activation of telomerase by the loss of such a gene. We have reported recently that a telomerase repressor gene may be located on 10p15.1 by deletion mapping using microcell-mediated chromosome transfer (MMCT), radiated microcell fusion (RMF), fluorescent in situ hybridization (FISH) and STS analysis. To independently confirm this result, we correlated expression of RNA component of telomerase (hTR) as a marker of telomerase expression by in situ hybridization with allelic loss in pulmonary carcinoid tumours. Unlike most malignant tumours, pulmonary carcinoids (which are low-grade malignant tumours) are heterogeneous for telomerase expression. Loss of 5 closely spaced polymorphic markers on 10p15.1, especially D10S1728, were highly correlated with hTR expression. In an additional experiment, 10p15.1 showed higher and more significant correlation than any region of 3p where it has been predicted as another chromosomal location of telomerase repressor with allelic loss of the region. Our findings strongly suggest that 10p15.1 harbours a gene involved in repression of telomerase RNA component in human somatic cells and each putative repressor (on 3p and 10p) may act independently.

Frequent loss of imprinting of IGF2 and MEST in lung adenocarcinoma.

Genomic imprinting is a parental origin-specific chromosomal modification that causes differential expression of maternal and paternal alleles of a gene. Accumulating evidence suggests that deregulation of imprinted genes, including loss of imprinting (LOI), plays a role in oncogenesis. In the present study, we investigated allelic expression of six imprinted genes in human lung adenocarcinomas as well as in matched normal lung tissue. Informative cases showing heterozygosity for the gene of interest were selected from 35 patients. LOI of the insulin-like growth factor 2 gene (IGF2) and mesoderm-specific transcript (MEST, also known as paternally expressed gene 1) was noted in 47% (seven of 15) and 85% (11 of 13) of informative cases, respectively. Monoallelic expression was maintained in all the matched normal tissues examined. LOI of IGF2 was seen more frequently in moderately to poorly differentiated adenocarcinomas. In contrast, H19, small nuclear ribonucleoprotein-associated polypeptide N gene (SNRPN), necdin gene (NDN), and long QT intronic transcript 1 (LIT1) exhibited consistent monoallelic expression in all the informative samples. These findings indicated that independent deregulation took place in imprinted genes and suggested that aberrant imprinting of IGF2 and MEST was involved in the development of lung adenocarcinoma.

Epigenetic silencing of PEG3 gene expression in human glioma cell lines.

Genomic imprinting, the phenomenon in which alleles of genes are expressed differentially depending on their parental origins, has important consequences for mammalian development, and disturbance of normal imprinting leads to abnormal embryogenesis and some inherited diseases and is also associated with various cancers. In the context of screening for novel imprinted genes on human chromosome 19q13.4 with mouse A9 hybrids, we identified a maternal allele-specific methylated CpG island in exon 1 of paternally expressed imprinted gene 3 (PEG3), a gene that exhibits paternal allele-specific expression. Because PEG3 expression is downregulated in some gliomas and glioma cell lines, despite high-level expression in normal brain tissues, we investigated whether the loss of PEG3 expression is related to epigenetic modifications involving DNA methylation. We found monoallelic expression of PEG3 in all normal brain tissues examined and five of nine glioma cell lines that had both unmethylated and methylated alleles; the remaining four glioma cell lines exhibited gain of imprinting with hypermethylated alleles. In addition, treatment of glioma cell lines with the DNA demethylating agent 5-aza-2'-deoxycytidine reversed the silencing of PEG3 biallelically. In this article, we report that the epigenetic silencing of PEG3 expression in glioma cell lines depends on aberrant DNA methylation of an exonic CpG island, suggesting that PEG3 contributes to glioma carcinogenesis in certain cases.

Epigenetic heterogeneity at imprinted loci in normal populations.

Genomic imprinting is the phenomenon by which the two alleles of certain genes are differentially expressed according to their parental origin. Extensive analysis of allelic expression at multiple imprinted loci in a normal population has not performed so far. In the present study, we examined the allelic expression pattern of three imprinted genes in a panel of 262 Japanese normal individuals. We observed differences in the extent of maintenance of allele-specific expression of the three genes. The allelic expression of small nuclear ribonucleoprotein N (SNRPN) was stringently regulated while that of multimembrane-spanning polyspecific transporter-like gene 1 (IMPT1) showed a large degree of variation. Significant biallelic expression of insulin-like growth factor II (IGF2) was observed in about 10% of normal individuals. Our findings add to the accumulating evidence for variable allelic expression at multiple loci in a normal human population. This epigenetic heterogeneity can be a stable trait and potentially influence individual phenotypes.

Loss of imprinting of long QT intronic transcript 1 in colorectal cancer.

Loss of imprinting (LOI) of the insulin-like growth factor 2 (IGF2) and H19 genes on human chromosome 11 has been found not only in childhood tumors but also in common adult cancers including colorectal cancer. Recently, a transcript called LIT1 (long QT intronic transcript 1) has been identified within the KvLQT1 locus on chromosome 11. LIT1 is expressed preferentially from the paternal allele and is transcribed in most human tissues. LOI of LIT1 was found in a considerable number of Beckwith-Wiedemann syndrome (BWS) patients, suggesting that it is associated with the etiology of BWS. Since LOI of IGF2 was observed in association with overexpression of IGF2 in colorectal cancer in our previous study, we examined the status of genomic imprinting of LIT1 and H19 in comparison with IGF2 in colorectal cancer. We examined 44 surgically dissected colorectal cancer tissues. Ten of them represented informative cases for LIT1. None of these patients exhibited loss of heterozygosity (LOH) of LIT1, and LOI of LIT1 was observed in 4 of the 10 (40%) informative patients, but not in non-cancerous tissues. Neither LOH nor LOI of H19 was observed. LOI of IGF2 was observed in 4 of 18 (22%) informative patients. These results suggest that LOI of LIT1 is frequently observed in colorectal cancer and may be a useful marker for diagnosis of colorectal cancer.

Functional evidence for the presence of tumor suppressor gene on chromosome 10p15 in human prostate cancers.

Loss of heterozygosity on chromosome 10p was observed frequently in human prostate cancers. Studies have demonstrated that the introduction of the short arm of human chromosome 10 into a human prostate cancer cell line, PPC-1, by microcell-mediated chromosome transfer (MMCT), suppressed the malignant phenotype, suggesting the presence of a prostate tumor suppressor gene(s) within a region of 17 cM at distal 10p. To narrow down the candidate region harboring the tumor suppressor gene, a series of 10p fragments were transferred into PPC-1 cells by MMCT using a panel of hamster-human hybrid cells containing various portions of 10p. Four of the six hybrid cells obtained showed decreased tumorigenicity when injected subcutaneously into athymic nude mice. Tumors developed only at six of 40 injection sites for these four hybrid cells. In contrast, the other two hybrid cells, as well as parental PPC-1 cells, were judged to be fully tumorigenic because tumors appeared at a total 26 of 32 sites for the two hybrid cells and 15 of 16 sites for PPC-1. Allelotyping of 10p combined with fluorescence in situ hybridization in these hybrid cells suggested that a prostate tumor suppressor gene was located within a fragment of approximately 1.2 Mb flanked by D10S1172 and D10S226 on 10p15.1.

Functional evidence for a telomerase repressor gene on human chromosome 10p15.1.

Based on the sites of frequent allelic loss in hepatocellular carcinoma, five normal human chromosomes (2, 4, 5, 10 and 16) were transferred individually into a telomerase-positive human hepatocellular carcinoma cell line, Li7HM, by microcell-mediated chromosome transfer (MMCT). Chromosome 10, but not the others, repressed telomerase activity immediately and stopped cell growth after 50 population doublings (PDs). Loss of the transferred 10p loci resulted in the emergence of revertant cells that continued to proliferate and expressed telomerase activity, suggesting the presence of a telomerase repressor gene on this chromosomal arm. Transfer of a series of defined fragments from chromosome 10p successfully narrowed down the responsible region: a 28.9-cM region on 10p15 (between WI-4752 and D10S249), but not a 26.2-cM region (between D10S1728 and D10S249), caused repression of telomerase activity and progressive telomere shortening. A strong correlation between the expression level of telomerase catalytic subunit gene (hTERT) and telomerase activity was observed. These findings suggest that a novel telomerase repressor gene which controls the expression of hTERT is located on the 2.7-cM region (between WI-4752 and D10S1728) on chromosome 10p15.1.

Construction of 700 human/mouse A9 monochromosomal hybrids and analysis of imprinted genes on human chromosome 6.

As an in vitro assay system for the identification of human imprinted genes, a library of human/mouse A9 monochromosomal hybrids containing a single, intact bsr-tagged human chromosome of known parental origin, derived from normal human fibroblasts, has been previously generated by microcell-mediated chromosome transfer (MMCT). To supplement this assay system, we constructed additional 700 A9 monochromosomal hybrids, using a pSTneo or pPGKneo selection marker. To validate the A9 hybrids, we screened them with chromosome-specific polymorphic markers, and identified the hybrids containing either human chromosome 6, 7, 14, 18, or 21 of known parental origin. Matching paternal and maternal chromosome pairs of A9 hybrids were identified for chromosomes 6, 7, 14, and 18. The paternal-specific expression of ZAC (zinc finger protein, which regulates apoptosis and cell cycle arrest) and HYMAI (hydatidiform mole-associated and imprinted transcript), and the maternal-specific methylation of a CpG island within an imprinted domain on human chromosome 6q24, were maintained in A9 hybrids. For an example, we profiled the expression of expressed sequence tags (ESTs) and the methylation of CpG islands in the 300-kb imprinted domain around 6q24, which may be associated with cancers and transient neonatal diabetes mellitus (TNDM). Thus, the 700 A9 hybrids should be useful for various aspects of imprinting studies.

A novel maternally expressed gene, ATP10C, encodes a putative aminophospholipid translocase associated with Angelman syndrome.

Lack of a maternal contribution to the genome at the imprinted domain on proximal chromosome 15 causes Angelman syndrome (AS) associated with neurobehavioral anomalies that include severe mental retardation, ataxia and epilepsy. Although AS patients have infrequent mutations in the gene encoding an E6-AP ubiquitin ligase required for long-term synaptic potentiation (LTP), most cases are attributed to de novo maternal deletions of 15q11-q13. We report here that a novel maternally expressed gene, ATP10C, maps within the most common interval of deletion and that ATP10C expression is virtually absent from AS patients with imprinting mutations, as well as from patients with maternal deletions of 15q11-q13.

14-3-3 is involved in p75 neurotrophin receptor-mediated signal transduction.

The low affinity neurotrophin receptor (p75NTR) has been shown to mediate the apoptosis signaling to neural cells. However, the specific mechanisms of intracellular signal transduction of this process are largely unknown. To understand p75NTR-mediated signal transduction, we previously identified a protein that interacts with the intracellular domain of p75NTR, and we named it p75NTR-associated cell death executor (NADE). To elucidate further the signaling mechanisms utilized by p75NTR and NADE, we screened for NADE-binding protein(s) with the yeast two-hybrid method, and we identified 14-3-3epsilon as a NADE-binding protein in vivo. To examine whether 14-3-3epsilon affects the induction of p75NTR-mediated apoptosis, wild type or various deletion mutant forms of 14-3-3epsilon were co-expressed in HEK293, PC12nnr5, and oligodendrocytes. Interestingly, transient expression of the mutant form of 14-3-3epsilon lacking the 208-255 amino acid region blocked nerve growth factor-dependent p75NTR/NADE-mediated apoptosis, although this mutant form of 14-3-3epsilon continued to associate with NADE. These results suggest that 14-3-3epsilon plays an important role in the modulation of nerve growth factor-dependent p75NTR/NADE-mediated apoptosis.

Identification of a mutated DNA ligase IV gene in the X-ray-hypersensitive mutant SX10 of mouse FM3A cells.

The mouse carcinoma cell line SX10 is a hypersensitive mutant to x-rays and bleomycin. An earlier complementation test suggests that SX10 would belong to x-ray-cross complementing group (XRCC) 4. However, in this study, a human XRCC4 expression vector failed to complement the SX10 phenotype. Consistent with the previous report, SX10 showed the same level of DNA-dependent protein kinase activity as the wild-type SR-1. We isolated and analyzed hybrids between SX10 and human diploid fibroblast cells and found that human chromosome 13 conferred the x-ray resistance to the hybrids, suggesting that a candidate gene would be located on this chromosome. Polymerase chain reaction analysis with these hybrids and x-ray-resistant transformants obtained by introducing human chromosomes into SX10 indicated that the mutant was likely to be defective in DNA ligase IV. Sequence analysis of the DNA ligase IV gene confirmed that a defect in SX10 was attributed to a transition of G to A at nucleotide position 1413 of the gene, leading to an amino acid substitution from Trp at residue 471 to a stop codon. Revertant clones (Rev1-3) derived from SX10 showed a restored x-ray resistance; Rev1 reverted to the original nucleotide G at position 1413, whereas Rev2 and Rev3 to C. Transfection of a mouse DNA ligase IV cDNA vector into SX10 restored the resistance to both x-rays and bleomycin. SX10 showed a reduced frequency of chromosomal integration of transfected DNA, but the revertants restored the frequency found in the wild-type cells. These results suggest a possible involvement of DNA ligase IV in the integration event of foreign DNA as well as a crucial role in DNA double-strand break repair.