鈴木 敏和

5-Bromodeoxyuridine (BrdU) induces a phenomenon similar to cellular senescence in mammalian cells. To address an underlying molecular mechanism in this phenomenon, we assessed the role of AT-hook proteins that bind to the minor grooves of specific AT-rich sequences. We expressed DsRed-tagged HMGI, MATH2, and MATH20 proteins in HeLa cells in a doxycycline dependent manner. Modest expression of these proteins revealed no apparent effect on the cells although high levels of expression were toxic to the cells. In contrast, their modest expression in the presence of low concentrations of BrdU similarly and dose-dependently induced senescence markers examined, although the same concentrations of BrdU alone showed no obvious effect. In both cases, DsRed fluorescence was mainly observed as foci or intense dots on Hoechst 33342-staining regions. These distribution patterns were not changed by addition of BrdU. Since AT-hook domains can displace chromatin compacting proteins pre-bound on AT-rich sequences, these results suggest that chromatin unpacking is one of the factors stimulating expression of the senescence markers in human cells. (C) 2003 Elsevier Inc. All rights reserved.

5-Bromodeoxyuridine (BrdU) immediately and clearly suppresses expression of the mouse Myod1 and human MYOD1 genes in myoblastic cells. Despite various studies, its molecular mechanism remains unknown. We failed to identify a BrdU-responsive element of the genes in experiments in which reporter constructs containing known regulatory sequences were transferred to mouse C2C12 myoblasts. Therefore, we transferred human chromosome 11 containing the MYOD1 gene to the cells by microcell-mediated chromosome transfer. In the resulting microcell hybrids, BrdU suppressed expression of the transgene, as determined by quantitative real-time RT-PCR analysis. We then transfected human PAC clones containing the MYOD1 gene to the cells. In the resulting transfectants, BrdU suppressed the transgene similarly. Deletion analysis suggested that a BrdU-responsive element or chromatin structure exists between 24 and 47 kb upstream of the gene. These results are the first demonstrating BrdU-responsiveness of a transgene for the known BrdU-responsive genes and facilitating determination of its precise responsible structure.

5-Chlorodeoxyuridine (CldU) immediately induces a senescence-like phenomenon in any type of mammalian cells probably due to a change in nuclear matrix structure. We thus examined nuclear matrix proteins in HeLa cells cultured with CIdU by high-resolution two-dimensional gel electrophoresis and peptide mass spectrometry. Three proteins were found to be down-regulated and one protein up-regulated by addition of CIdU. In addition, one acidic protein accumulated in the nuclear matrix, although not quantitatively changed in the nuclei. Since these alterations were observed within 24 It after addition of CIdU, these proteins may be involved in an early step of the senescence-like phenomenon. (C) 2002 Elsevier Science Inc. All rights reserved.

5-Bromodeoxyuridine induces a senescence-like phenomenon in mammalian cells. This effect was dramatically potentiated by AT-binding ligands such as distamycin A, netropsin, and Hoechst 33258. The genes most remarkably affected by these ligands include the widely used senescence-associated genes and were located on or nearby Giemsa-dark bands of human chromosomes. We hypothesize that AT-rich scaffold/nuclear matrix attachment region sequences are involved in this phenomenon. In fact, upon substitution of thymine with 5-bromouracil, a rat S/MAR sequence reduced its degree of bending and became insensitive to cancellation of the bending by distamycin A The S/MAR sequence containing 5-bromouracil also bound more tightly to nuclear scaffold proteins in vitro and this binding was not inhibited by distamycin A. Under the same conditions, the S/MAR sequence containing thymine easily dissociated from the nuclear scaffold proteins. Taken together, the synergistic induction of the genes may be explained not only by opening of condensed chromatin by distamycin A but also by increase in the binding of 5-bromouracil-containing S/MAR sequences to the nuclear scaffolds. (C) 2002 Elsevier Science (USA).

We tested various thymidine analogues for induction of a senescence-like phenomenon in HeLa cells. CldU, BrdU, and IdU similarly induced the morphology of senescent cells and typical senescence markers. Thymidine analogues other than 5-halogenated forms caused only cell death. BrdU efficiently killed the cells in cooperation with irradiation with light and a brief treatment with Hoechst 33258, but CldU did not at all. 5-Halogenated thymidine analogues were thus shown to be specific inducers of cellular senescence in mammalian cells.

We established a thymidine-auxotrophic strain of the yeast Saccharomyces cerevisiae to examine biological effects of thymidine analogues. 5-Bromo-2'-deoxyuridine efficiently suppressed the division potential of yeast showing morphology similar to senescent cells.

5-Bromodeoxyuridine (BrdU) induces or suppresses senescence-associated genes in any types of mammalian cells. From a cDNA library upregulated by BrdU in HeLa cells, we identified the gene encoding VDUP1 as a senescence-associated gene in normal human fibroblasts. To address a role of VDUP1 in senescence, we established HeLa cell clones. V7 and V27. which express its mRNA in a doxycycline-dependent manner. Although their growth in liquid culture was moderately retarded, colony formation on semi-solid medium was strongly inhibited by overexpression of the mRNA. We also examined susceptibility of these clones to various reagents. Consequently, colony formation in liquid culture was strongly inhibited by paraquat in these clones. Their superoxide dismutase activity was normal. (C) 2002 Elsevier Science (USA). All rights reserved.

Substitution of thymine with 5-bromouracil in DNA is known to change interaction between DNA and proteins, thereby inducing various biological phenomena. We hypothesize that A/T-rich scaffold/nuclear matrix attachment region (S/MAR) sequences are involved in the effects of 5-bromodeoxyuridine. We examined an interaction between DNA containing an intronic S/MAR sequence of the immunoglobulin heavy chain gene and nuclear halos prepared from HeLa cells. Upon substitution with 5-bromouracil, the S/MAR DNA bound more tightly to the nuclear halos. The multi-functional nuclear matrix protein YY1 was also found to bind more strongly to 5-bromouracil-substituted DNA containing its recognition motif. These results are consistent with the above hypothesis.

An ectopic gene integrated in the host genome is occasionally silenced due to a position effect of its adjacent chromatin structure. We found that 5-bromodeoxyuridine clearly activated such a transgene in HeLa cells. The transgene was also activated to various degrees by inhibitors of histone deacetylase, DNA topoisomerases, or DNA methyltransferase. The peptide antibiotic distamycin A potentiated markedly the effect of 8-bromodeoxyuridine. Transient expression of an artificial AT-hook protein termed MATH20 also potentiated its effect although significantly activated the transgene alone. Since distamycin A and MATH20 are able to displace histone H1 and other DNA-binding proteins bound to specific AT-rich sequences by a dominant, mutually exclusive fashion, these results suggest that 5-bromodeoxyuridine targets such an AT-rich sequence located adjacent to the silenced transgene, resulting in chromatin accessibility. (C) 2001 Academic Press.

5-Bromodeoxyuridine (BrdU) universally induces a senescence-like phenomenon in mammalian cells. To assess this phenomenon at the level of gene expression, we constructed a PCR-based subtractive cDNA library enriched for mRNA species that immediately increase by administration of BrdU to HeLa cells. Candidate cDNA clones were isolated by differential colony hybridization, and then positive clones were identified by Northern blot analysis. Sequencing analysis revealed that the identified cDNA species were classified into three groups: widely used senescence-markers, known species whose relevance to senescence is yet to be reported, and known or novel ESTs. As expected, the majority of them showed an increase in expression in senescent human diploid fibroblasts. These results suggest that similar mechanisms operate in the regulation of BrdU-induced genes and senescence-associated genes. (C) 2001 Elsevier Science Inc. All rights reserved.

5-Bromodeoxyuridine was found to induce flat and enlarged cell shape, characteristics of senescent cells, and senescence-associated beta-galactosidase in mammalian cells regardless of cell type or species, In immortal human cells, fibronectin, collagenase I, and p21(wafl/sdi-1) mRNAs were immediately and very strongly induced, and the mortality marker mortalin changed to the mortal type from the immortal type. Human cell lines lacking functional p21(wafl/sdi-1), p16(ink4a), or p53 behaved similarly. The protein levels of p16(ink4a) and p53 did not change uniformly, while the level of p21(wafl/sdi-1) was increased by varying degrees in positive cell lines, Telomerase activity was suppressed in positive cell lines, but accelerated telomere shortening was not observed in tumor cell lines; These results suggest that 5-bromodeoxyuridine activates a common senescence pathway present in both mortal and immortal mammalian cells.

Previous studies on human cell hybrids between HeLa and normal human fibroblasts have indicated that the tumorigenicy may be: controlled by a putative tumor suppressor gene on chromosome 11. We previously demonstrated a twofold increase in glucose uptake with a reduced K-m by tumorigenic HeLa cell hybrids which expressed a highly glycosylated GLUT1. In this study, we reported that a tumorigenic cell hybrid, CGL4, also expressed a glucose transporter isoform, GLUT3, that was undetectable in nontumorigenic CGL1 cells. The expression of GLUTS together with GLUT1 of 70 kDa was also evident in three gamma-ray-induced tumorigenic clones isolated from CGL1 cells, while control nontumorigenic irradiated cells expressed 50 kDa GLUT1 alone. In accordance with this, GLUT3 mRNA was specifically expressed in tumorigenic cell hybrids. To examine the role of GLUT3, clones which stably overexpress GLUT3 were developed from both CGL1 and CGL4 cells. In these transfectants, the affinity for 2-deoxyglucose markedly increased, in parallel with the amount of expressed GLUT3 irrespective of its N-glycosylation state. These results suggest that the enhanced GLUT3 expression in HeLa cell hybrids associated with the tumorigenic phenotypes may account for the increased affinity for 2-deoxyglucose. Possible roles of the putative tumor suppressor in control of gene expression and glucose uptake is discussed.

Inhibitors of DNA topoisomerases I and II induced arrest in cell division in normal human fibroblasts depending on cell divisions. Arrested cells showed morphology similar to those of normally senesced cells and strongly induced senescence-associated P-galactosidase. In these cells, p16(ink4a) was upregulated, whereas p21(waf1) or p53 was not altered. Upon removal of the inhibitors, the cells resumed growth but their cumulative population doublings were reduced dose dependently. Accelerated telomere shortening was not observed in the arrested cells. These results suggest that DNA topoisomerase inhibitors are efficient and reversible inducers of premature senescence in normal human cells. (C) 1998 Academic Press.

Studies on human cell hybrids of a cervical carcinoma cell line, HeLa, and normal fibroblasts have indicated that the tumorigenicity of these cells is under the control of a putative tumor suppressor on chromosome 11, although the nature of this suppressor remains unknown. We examined the expression of caveolin-1, a protein component of caveolae of the plasma membrane in these cell hybrids. The non-tumorigenic cell hybrid, CGL1, and normal fibroblast WI38 cells expressed 21-24kDa caveolin-1, whereas in tumorigenic hybrid CGL4 as well as in the parental HeLa cells, the level of caveolin-1 was markedly reduced. Caveolin-l expression was also reduced in gamma-ray-induced tumorigenic clones (GIMs) isolated from CGL1 cells, whereas non-tumorigenic irradiated cells expressed the same level of caveolin-1 as CGL1 cells. In accordance with these changes, the cellular level of caveolin-1 mRNA was reduced in the tumorigenic CGL4 cells and GIMs without any detectable changes in the caveolin-1 gene. However, the in vivo tumor growth of CGL4 cells was not altered when caveolin-1 was stably overexpressed through the transfection of a human caveolin-1 cDNA, These results suggest that reduction of caveolin-1 expression is necessary but not sufficient for emergence of the tumorigenic phenotypes of HeLa cell hybrids. Possible roles of the putative tumor suppressor in the control of gene expression are also discussed.

Some immortal human cell lines lack telomerase activity. These cell lines were found to contain small dispersed DNA hybridizing to TTAGGG: repeats. Such DNA was located in their cytoplasm and nuclei, Normal human fibroblasts or telomerase-positive cell lines did not contain such DNA. Upon cloning and sequencing, it was shown to consist of TTAGGG repeats. When electrophoresed on neutral and alkaline agarose gels, it behaved as double-stranded and linear DNA. These results suggest that telomeric DNA is released from chromosomes in association with maintenance of telomeres in telomerase-negative cell lines. (C) 1998 Academic Press.

Pieces of metaphase chromosomes prepared from mouse cells containing neo-tagged human chromosome 7 were transferred to mouse cells with calcium phosphate to isolate G418-resistant clones. FISH analysis revealed that the majority of them contained human DNA at a single site on their genome. These transformants contained STS markers mapped to various regions of chromosome 7. It is thus suggested that pieces of human chromosomes tend to assemble and integrate on the mouse genome.

In mouse FM3A ornithine decarboxylase (ODC) overproducing cells (EXOD-1), the amount of ODC protein was approximately 100-fold that of normal cells. Since it is well known that the translational efficiency of ODC mRNA is very low and that eIF-4E is a limiting factor for the mRNA recognition and the scanning of 40 S ribosomal subunits, we measured the amount and phosphorylation of eIF-4E in EXOD-1 cells. An increase in the phosphorylation of eIF-4E, its association with p220 protein, and an enhancement of RNA helicase activity were observed in the cells. These results support the hypothesis that phosphorylation of eIF-4E enhances RNA helicase activity through eIF-4F (4A, SE, and p220) complex formation. (C) 1996 Academic Press, Inc.

The effect of repented cold stress (RCS) on both mRNA level and enzyme activity of stomach histidine decarboxylase (HDC) was studied in ddY mice. Following 1-day treatment of RCS, stomach HDC activity, but not its mRNA level, increased two fold. Following 3-day treatment of RCS, which is the essential period for the induction of hyperalgesia in mice, HDC mRNA level and enzyme activity increased in the stomach. After cessation of RCS treatment HDC mRNA level decreased and reached the level of non-RCS treated mice, but HDC activity did not. The stomach from the 1-day RCS-treated mouse contained proteolytic activity, which converts the in vitro-translated 74 kD HDC species into the 53 kD HDC species. These data demonstrate that RCS-treated mouse stomach induces both the de novo synthesis of the 74 kD HDC species and its proteolytic cleavage to 53 kD HDC species.

Exposure of ornithine decarboxylase (ODC; L ornithine carboxy-lyase, EC 4.1.1.17)-overproducing mouse FM3A cells to micromolar levels of spermine or spermidine caused abnormal accumulation and toxicity of polyamines. This was apparently due to the inefficiency of negative feedback control of polyamine transport by polyamines in ODC-overproducing cells. Since antizyme is the only protein thus far recognized that can interact with ODC, depletion of free antizyme was regarded as the reason for the abnormal accumulation of polyamines. Accordingly, ODC-overproducing cells were transfected with pMAMneoZ1 possessing rat antizyme cDNA under the control of a glucocorticoid-inducible promoter. In the transfected cells, the addition of dexamethasone caused an increase in the amount of antizyme with an apparent molecular mass of 27 kDa, a decrease in the amount of ODC, a decrease in the polyamine transport activity, and the recovery of growth inhibition or cell death. The results indicate that antizyme can regulate not only the amount of ODC but also the activity of polyamine transport.

Effects of antizyme on polyamine transport and spermine restoration of the growth of polyamine-deficient cells were examined by using mouse FM3A cells transfected with pMAMneoZ1 possessing rat antizyme cDNA under the control of glucocorticoid-inducible promoter. Treatments of the transfected cells with a-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), and dexamethasone, an inducer of antizyme, both caused a decrease in ODC activity and polyamine contents and inhibition of cell growth. However, spermine uptake of the transfected cells was repressed by dexamethasone but stimulated by DFMO. The decrease in the rate of spermine uptake in dexamethasone-treated cells was attributed to an increase in K-m value and a slight decrease in V-max value. Accordingly, restoration of cell growth by spermine was less effective in dexamethasone-treated cells than DFMO-treated cells. These results clearly indicate that antizyme has dual functions: one for ODC degradation and the other for negative regulation of polyamine transport. (C) 1994 Academic Press, Inc.

The antiproliferating effect of nine kinds of bis(ethyl)polyamine analogues [three kinds each of bis(ethyl)triamine, bis(ethyl)tetraamine and bis(ethyl)pentaamine] was compared using FM3 A cells. The inhibitory effect was in the order BE4444 > BE3443 > BE4334 greater than or equal to BE444 > BE343 > BE333 > BE44 > BE34 > BE33. Our results indicate that not only polyamine deficiency but also the accumulation of polyamine analogues is involved in the inhibition of cell growth. Accumulation of bis(ethyl)polyamine analogues caused the inhibition of protein synthesis and the decrease in the ATP content. The protein synthetic system in mitochondria was more strongly inhibited by bis(ethyl)polyamine analogues than that in the cytoplasm. Under conditions such that cytoplasmic protein synthesis was inhibited by 50% by bis(ethyl)polyamine analogues, mitochondrial protein synthesis was almost completely inhibited. Mitochondrial Ile-tRNA formation was inhibited by bis(ethyl)polyamine analogues at the concentrations that cytoplasmic Ile-tRNA formation was stimulated. This may be one of the reasons for the selective inhibition of mitochondrial protein synthesis. This inhibition was followed by the decrease in ATP content, swelling of mitochondria and depletion of mitochondrial DNA. These results suggest that the early event of metabolic change caused by bis(ethyl)polyamine analogues in cells is the inhibition of protein synthesis, especially of mitochondrial protein synthesis.

A variant cell line, termed SAM-1, which overproduced S-adenosylmethionine decarboxylase (AdoMetDC), was isolated by treatment of mouse FM3A cells with N-methyl-N'-nitro-N-nitrosoguanidine and subsequent incubation with ethylglyoxal bis(guanylhydrazone), an inhibitor of the enzyme. The cells were resistant to ethylglyoxal bis(guanylhydrazone), and showed AdoMetDC activity approximately five-times higher than control cells. The rate of AdoMetDC synthesis and the amount of AdoMetDC existing in SAM-1 cells were about five-times those in control cells. The amount of AdoMetDC mRNA existing in SAM-1 cells was five-times more than that in control cells. The amount of 5'-{[(Z)-4-amino-2-butenyl]methylamino}-5'-deoxyadenosine, an irreversible inhibitor of AdoMetDC, necessary to inhibit cell growth was also five-times more in SAM-1 cells than in control cells. However, the following were the same in both SAM-1 and control cells; the amount of genomic DNA for AdoMetDC, the size and nucleotide sequence of 5' untranslated region of AdoMetDC mRNA, the deduced amino acid sequence (334 residues) from the nucleotide sequence of AdoMetDC cDNA and the degradation rate (t1/2 = about 4 h) of AdoMetDC. In addition, AdoMetDC mRNA in control cells was slightly more stable than that in SAM-1 cells. The results indicate that the overproduction of AdoMetDC in SAM-1 cells was caused by the increase of AdoMetDC mRNA. The variant cell line is convenient for studying the regulation of AdoMetDC and the physiological function of polyamines.

Nucleolin is a major nucleolar phosphoprotein and is presumably involved in rDNA transcription and ribosome biosynthesis. This protein is known to be very labile and to be cleaved by endogenous proteases into many small peptides. We found that, when rat liver nucleolar suspension (Nu-1) or nucleolin-rich extract (Nu-2) was incubated under conventional conditions, polyamines and histones interacted with the nucleolin to lead to its preferential degradation to 60 kDa phosphopeptide (p60). The peptide p60 was identified as a peptide containing the N-terminal half of the nucleolin molecule, as judged from peptide-map analysis. Whereas spermine binding to the purified nucleolin was decreased by KCl concentrations above 50 mM, histones (H1, H2B and H3) were able to bind to the nucleolin in the presence of up to 300 mM KCl. A distinct difference between H1 and other histones was found in that H1 could produce p60 from nucleolin in both Nu-1 and Nu-2, whereas H2B and H3 stimulated the degradation of nucleolin to p60 only when Nu-2 was used for the source of nucleolin. A possible relationship between p60 formation and rRNA synthesis is discussed. but its exact role remains to be studied.

Ribosomal RNA (rRNA) synthesis in murine P1798 lymphosarcoma cells is reversibly inhibited by glucocorticoids. The effects of dexamethasone upon nucleolin phosphorylation and upon the amount and activity of casein kinase II have been examined. P1798 cells were exposed to 0.1-mu-M dexamethasone for 36 h. Cells were labeled in vivo with [P-32]orthophosphate followed by immunoprecipitation with anti-nucleolin antibody. Nucleolin phosphorylation was reduced by 60% in dexamethasone-treated cells. Nucleoli were isolated and labeled with [gamma-P-32]ATP in vitro. Nucleolin protein was reduced to 40% of control in nuclei from dexamethasone-treated cells. Nucleolin phosphorylation was reduced to 20% of control. Nucleolar casein kinase II activity and protein were also reduced (30-55% and 35-50% of control, respectively) by treatment with dexamethasone. Cycloheximide (10-mu-g/ml for 3 h) reduced the amount and activity of casein kinase II, but did not cause a decrease in nucleolin protein. These observations are discussed relative to the hypothesis that glucocorticoids regulate the amount or activity of proteins of short biological half-life that are involved in the regulation of rRNA synthesis.

Effects of dexamethasone, EGF and insulin on the synthesis of rRNA and phosphorylation of nucleolin in primary cultures of adult rat hepatocytes were studied. Hepatocytes were incubated for 8 h with EGF (20 ng/ml) plus insulin (0.1-mu-M) and/or for 20 h with dexamethasone (1-mu-M) before the end of incubation. The incorporation of [H-3]uridine into acid-insoluble materials and the nuclear activity of RNA polymerase I were stimulated approx. 2-fold with EGF plus insulin and these were further enhanced 2-3-times by dexamethasone, although dexamethasone alone exerted no stimulation. When hepatocytes were incubated with [P-32]orthophosphate, similar enhancement by these hormones was also observed in the phosphorylation of a nucleolar protein, nucleolin, which was detected by immunoprecipitation with anti-nucleolin antibodies. The amount of nucleolin was slightly increased by EGF plus insulin in the presence of dexamethasone, but scarcely changed by treatment with EGF plus insulin or dexamethasone alone. Cycloheximide inhibited RNA synthesis to a greater or lesser degree in the case of all hepatocytes which were cultured with or without these hormonal treatments. These results indicate that the in vivo effect of glucocorticoid on rRNA synthesis and nucleolin phosphorylation in liver is primarily a direct action on parenchymal cells and requires other growth factors such as EGF and insulin.