TIG-1 Cells Research Articles

Genome-wide identification and annotation of HIF-1α binding sites in two cell lines using massively parallel sequencing

We identified 531 and 616 putative HIF-1α target sites by ChIP-Seq in the cancerous cell line DLD-1 and the non-cancerous cell line TIG-3, respectively. We also examined the positions and expression levels of transcriptional start sites (TSSs) in these cell lines using our TSS-Seq method. We observed that 121 and 48 genes in DLD-1 and TIG-3 cells, respectively, had HIF-1α binding sites in proximal regions of the previously reported TSSs that were up-regulated at the transcriptional level. In addition, 193 and 123 of the HIF-1α target sites, respectively, were located in proximal regions of previously uncharacterized TSSs, namely, TSSs of putative alternative promoters of protein-coding genes or promoters of putative non-protein-coding transcripts. The hypoxic response of DLD-1 cells was more significant than that of TIG-3 cells with respect to both the number of target sites and the degree of induced changes in transcript expression. The Nucleosome-Seq and ChIP-Seq analyses of histone modifications revealed that the chromatin formed an open structure in regions surrounding the HIF-1α binding sites, but this event occurred prior to the actual binding of HIF-1α. Different cellular histories may be encoded by chromatin structures and determine the activation of specific genes in response to hypoxic shock. The online version of this article (doi:10.1007/s11568-011-9150-9) contains supplementary material, which is available to authorized users.

Abstract 1605: A possible target key molecule for treatment of pancreatic cancer, screened as cancer-specific immortalization associated gene

Abstract [Background] Pancreatic caner shows a poor prognosis, and thus development of an efficient therapy is needed. Last year, we have reported that 13 genes were screened in relation to an event “cancer cell immortalization” by means of comprehensive gene expression analyses of normal, mortal and immortal cells and tissue specimen, and that these might be potent candidates for novel targets of anticancer chemotherapy. [Purpose] GTSE1 gene has been selected as one of the 13 genes that expressed at higher levels in immortal cancer cells, though its biological function relative to growth regulation of cancer cells are yet to be determined. To clarify its roles, we performed in vitro experiments to validate its possibility as a target key molecule for treatment of cancer, specifically pancreatic one. [Materials and Methods] Quantitative analyses of gene expression levels were conducted by real-time RT-PCR method using gene specific-primers and Universal Probe Library probes (Roche). Whole genome gene expression analysis was conducted with 4×44K Whole Human Genome Microarray (Agilent). Knockdown experiments were performed in various cancer cells using Stealth RNAi and RNAiMax reagent (Invitrogen). Cell proliferation was measured with MTT assay. Caspase activities were analyzed using ApoAlert Caspase Profiling Plate (Clontech). Cell cycle profile was obtained by measuring PI-stained cells with FACS “Calibur” (BD Biosciences). [Results] GTSE1 expressed at much higher levels in primary pancreatic and endometrial cancer tissue specimens as compared with matched normal ones: it expressed at higher levels in pancreatic, esophageal, and lung cancer cell lines than normal cells: higher expression levels of GTSE1 were observed in transformants of lung fibroblast TIG-1 cells with SV40 and TERT as compared with TIG-1 cells harboring TERT alone. Treatment with GTSE1 siRNA revealed a significant inhibition of growth of various cancer cells including paclitaxel-resistant ovarian cancer KFr13Tx, cisplatin-resistant lung cancer PC-9/CDDP, and gemcitabine-resistant pancreatic cancer MIAPaCa-2 cells. GTSE1 knockdown induced alteration of cell cycle profile and apoptosis of pancreatic cancer cells: it enhanced caspase 2 and 3 activities. Moreover, to seek for downstream genes, we performed microarray analysis of KLM-1, MIAPaCa-2 and DLD-1 cells treated with GTSE1 siRNA for 72 hrs: We found a gene A that showed less than 1/8 gene expression levels as compared with negative control ones in all of the tested cells by using microarray: This was confirmed by real-time RT-PCR. Furthermore, knockdown of the selected gene also altered cell cycle profile and apoptosis of pancreatic cancer cells. [Conclusion] These results indicate that GTSE1 may be involved in cancer-specific immortalization and may be useful as a molecular target for cancer therapy including pancreatic one. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1605.

Therapeutic efficacy of Kangen-karyu against H2O2-induced premature senescence

Abstract The anti-aging potential of Kangen-karyu extract was investigated using the mechanisms of the cellular aging model of stress-induced premature senescence (SIPS) in TIG-1 human fibroblasts. SIPS was induced by a sublethal dose of H2O2 and chronic oxidative stress with repeat treatment of low-dose H2O2. Reactive oxygen species generation, lipid peroxidation, and senescence-associated β-galactosidase activity were elevated in TIG-1 cells under SIPS induced by H2O2. However, Kangen-karyu extract led to significant declines in these parameters, suggesting its role in ameliorating oxidative stress-related aging. It was also observed that SIPS due to H2O2 treatment led to the loss of cell viability, whereas Kangen-karyu extract improved cell viability by attenuating H2O2-induced oxidative damage. TIG-1 cells under the condition of SIPS caused by sublethal and chronic low doses of H2O2 showed nuclear factor-κB (NF-κB) translocation to the nucleus from the cytosol, while Kangen-karyu extract inhibited NF-κB nuclear translocation, implying that Kangen-karyu extract could exert an anti-aging effect through NF-κB modulation. In addition, treatment with Kangen-karyu extract under H2O2-induced chronic oxidative stress normalized the cell cycle by reducing the number of cells in the G0/G1 phase and elevating the proportion of those in the S phase, indicating the role of Kangen-karyu extract in cell cycle regulation. On the other hand, Kangen-karyu extract did not exert such an effect on cell cycle regulation under acute oxidative stress induced by sublethal H2O2. Furthermore, treatment with Kangen-karyu extract prolonged the lifespan of TIG-1 cells under SIPS. The present study suggests that Kangen-karyu might play a therapeutic role against the aging process caused by oxidative stress.

Protein Kinase C Plays a Key Role in Cellular Senescence Programs of Human Normal Diploid Cells

In the present study, we clarified that transforming growth factor beta (TGF-beta) induces cellular senescence in human normal diploid cells, TIG-1, and identified protein kinase Cs (PKCs) as downstream mediators of TGF-beta-induced cellular senescence. Among PKCs, we showed that PKC-delta induced cellular senescence in TIG-1 cells and was activated in replicatively and prematurely senescent TIG-1 cells. The causative role of PKC-delta in cellular senescence programs was demonstrated using a kinase negative PKC-delta and small interfering RNA against PKC-delta. Furthermore, PKC-delta was shown to function in human telomerase reverse transcriptase (hTERT) gene repression. These results indicate that PKC-delta plays a key role in cellular senescence programs, and suggest that the induction of senescence and hTERT repression are coordinately regulated by PKC-delta.

Therapeutic efficacy of Kangen-karyu against H<SUB>2</SUB>O<SUB>2</SUB>-induced premature senescence

The anti-aging potential of Kangen-karyu extract was investigated using the mechanisms of the cellular aging model of stress-induced premature senescence (SIPS) in TIG-1 human fibroblasts. SIPS was induced by a sublethal dose of H2O2 and chronic oxidative stress with repeat treatment of low-dose H2O2. Reactive oxygen species generation, lipid peroxidation, and senescence-associated beta-galactosidase activity were elevated in TIG-1 cells under SIPS induced by H2O2. However, Kangen-karyu extract led to significant declines in these parameters, suggesting its role in ameliorating oxidative stress-related aging. It was also observed that SIPS due to H2O2 treatment led to the loss of cell viability, whereas Kangen-karyu extract improved cell viability by attenuating H2O2-induced oxidative damage. TIG-1 cells under the condition of SIPS caused by sublethal and chronic low doses of H2O2 showed nuclear factor-kappaB (NF-kappaB) translocation to the nucleus from the cytosol, while Kangen-karyu extract inhibited NF-kappaB nuclear translocation, implying that Kangen-karyu extract could exert an anti-aging effect through NF-kappaB modulation. In addition, treatment with Kangen-karyu extract under H2O2-induced chronic oxidative stress normalized the cell cycle by reducing the number of cells in the G0/G1 phase and elevating the proportion of those in the S phase, indicating the role of Kangen-karyu extract in cell cycle regulation. On the other hand, Kangen-karyu extract did not exert such an effect on cell cycle regulation under acute oxidative stress induced by sublethal H2O2. Furthermore, treatment with Kangen-karyu extract prolonged the lifespan of TIG-1 cells under SIPS. The present study suggests that Kangen-karyu might play a therapeutic role against the aging process caused by oxidative stress.

Effects of radical scavenger protein from broad beans on glutathione status in human lung fibroblasts

Human diploid cells are more susceptible to oxidative stress at late passage than at early passage, presumably because of the decrease in cellular-reduced glutathione (GSH) concentration. Water-soluble protein (WSP) from broad beans scavenges free radicals. The effects of WSP on the glutathione system were examined in PDL 20 (early passage) and PDL 50 (late passage) human lung fibroblasts (TIG-1). To determine cytosolic glutathione peroxidase (GSH-Px) activities, glutathione reductase (GR) activities, oxidized glutathione (GSSG) concentrations, and GSSG/reduced glutathione (GSH) ratios, WSP and hydrocortisone (HC) treatments of TIG-1 cells (PDL 20→50 and PDL 50→75) were performed for 40 days. We also investigated the GSSG concentrations and GR activities in PDL 20 cells that were continuously treated with WSP until PDL 39 and 55. GSSG concentrations decreased in WSP- and HC-treated PDL 50→75 cells. The GSSG/GSH ratios in PDL 50→75 cells became low after the treatments. Increases in GR activities were observed in treated PDL 50→75 cells. The decline in the GSSG concentration of PDL 50→75 cells correlated with the increase in GR activity. The GSSG levels in control cells were higher following cellular age, whereas the levels in treated cells were lower than those in the control. The studies on cellular age-related changes indicated that greater increases in GR activity were found in treated cells than in the control. These results indicated that WSP influences the GSSG concentration that is associated with cellular aging, but the mechanism of GSSG reduction by WSP remains unknown. The enhancement of glutathione status following WSP treatment may be related to the delay in the cellular aging.

Pro‐inflammatory cytokine production from normal human fibroblasts is induced by Tannerella forsythia detaching factor

Tannerella forsythia is a periodontal pathogen. Recently, we have reported that the cytopathic component of T. forsythia contains two distinct factors. One arrests the cell cycle at the G2 phase and the other, named forsythia detaching factor, detaches adhesion-dependent immortalized human cells. In this study, we investigated the biological function of forsythia detaching factor using human normal fibroblasts. A recombinant forsythia detaching factor, reported previously, was used. TIG-3 cells, cultured in the absence or presence of forsythia detaching factor, were lysed and the supernatant was analyzed by western blotting with polyclonal forsythia detaching factor antibodies. The cells were subsequently fractionated to isolate the cytoplasmic, mitochondrial and remaining fractions. In order to measure the activity of mitochondria using nicotinamide adenine dinucleotide-linked reductase, the water-soluble tetrazolium method was used. The mitochondrial oxidative membrane potential was estimated by measuring the oxidization-dependent fluorogenic conversion of dihydrotetramethylrosamine using flow cytometry. The concentration of interleukin-8 in the culture supernatant was assayed using a Human IL-8 ELISA kit. Forsythia detaching factor-treated cells detached from the substratum and aggregated from 3 to 24 h. Then, the detached cells resumed adhesion and proliferated after 48 h. The western blot analysis revealed that most forsythia detaching factor trans-located into the mitochondrial fraction. Forsythia detaching factor suppressed the nicotinamide adenine dinucleotide-linked reductase activity in a dose-dependent manner and consequently increased the mitochondrial oxidative membrane potential. The production of interleukin-8 was reinforced in forsythia detaching factor-treated cells at 72 h through an increase of the mitochondrial oxidative membrane potential. The forsythia detaching factor might be involved in the virulence of T. forsythia through induction of the pro-inflammatory cytokine interleukin-8.

Nuclear localization of barrier-to-autointegration factor is correlated with progression of S phase in human cells

Barrier-to-autointegration factor (BAF) is a conserved metazoan protein that plays a critical role in retrovirus infection. To elucidate its role in uninfected cells, we first examined the localization of BAF in both mortal and immortal or cancerous human cell lines. In mortal cell lines (e.g. TIG-1, WI-38 and IMR-90 cells) BAF localization depended on the age of the cell, localizing primarily in the nucleus of >90% of young proliferating cells but only 20-25% of aged senescent cells. In immortal cell lines (e.g. HeLa, SiHa and HT1080 cells) BAF showed heterogeneous localization between the nucleus and cytoplasm. This heterogeneity was lost when the cells were synchronized in S phase. In S-phase-synchronized populations, the percentage of cells with predominantly nuclear BAF increased from 30% (asynchronous controls) to approximately 80%. In HeLa cells, RNAi-induced downregulation of BAF significantly increased the proportion of early S-phase cells that retained high levels of cyclin D3 and cyclin E expression and slowed progression through early S phase. BAF downregulation also caused lamin A to mislocalize away from the nuclear envelope. These results indicate that BAF is required for the integrity of the nuclear lamina and normal progression of S phase in human cells.

Bladder Cancer Cell Invasion Is Enhanced by Cross-Talk with Fibroblasts Through Hepatocyte Growth Factor

To examine the effects of fibroblast-derived humoral factors, especially hepatocyte growth factor (HGF), on the invasive potential of bladder cancer cells. Stromal cells in cancer tissue are thought to play an important role in the transformation and invasion of cancer cells. The influence of fibroblast cells (TIG-1 cells) and HGF on the invasive potential of bladder cancer cells (5637, T24, J82, HT1376, and MGHU-1 cells) was evaluated by in vitro cell invasion assay. The expression of HGF and c-Met, which is the receptor of HGF, was examined by reverse transcriptase-polymerase chain reaction. To clarify the relationship between the serum HGF level and invasive bladder cancer, we measured the serum concentrations of HGF in patients with bladder cancer without metastatic disease and normal controls, using an enzyme-linked immunosorbent assay. The in vitro cell invasion assay showed that the number of invading bladder cancer cells was significantly increased by the conditioned medium (CM) of the fibroblast cells. HGF neutralization antibody partially inhibited the enhancement of invasiveness by fibroblast CM. The CM of fibroblasts cultured with bladder cancer CM stimulated cancer cell invasion more strongly (with increased HGF secretion) than did the CM of fibroblasts cultured without bladder cancer CM. The serum HGF levels were significantly greater in patients with muscle-invasive bladder cancer (regardless of tumor size) than in patients with non-muscle-invasive bladder cancer. The present results have suggested that bladder cancer cell invasion is enhanced by cross-talk with fibroblasts through humoral factors, including HGF. Elucidation of this mechanism could lead to novel therapeutic strategies for bladder cancer.

H2O2 Accelerates Cellular Senescence by Accumulation of Acetylated p53 via Decrease in the Function of SIRT1 by NAD+ Depletion

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD⁺-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H₂O₂ and examined DNA cleavage, depletion of intracellular NAD⁺, expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated β-galactosidase (SA-β-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H₂O₂, though no cell death was observed. NAD⁺ levels in TIG-3 cells treated with H₂O₂ were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD⁺ levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H₂O₂ treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H₂O₂ induced cell cycle arrest at 24h and increased SA-β-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.

Preferential reduction of the α-2-6-sialylation from cell surface N-glycans of human diploid fibroblastic cells by in vitro aging

Human diploid fibroblastic cell line, TIG-3, has a finite life span of about 80 population doubling levels (PDL), and is used for in vitro aging studies. Young cells (PDL 23) grew to higher cell densities at a higher growth rate than aged cells (PDL 77). When the electrophoretic mobility of cells was determined, the negative surface charge of the aged cells decreased significantly when compared to that of young cells. Lectin blot analysis of membrane glycoproteins showed that the alpha-2-6-sialylation but not the alpha-2-3-sialylation of N-glycans decreases markedly in the aged cells when compared to the young cells. In support of this observation, the cDNA microarray assay and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the gene expression of the alpha-2,6-sialyltransferase I (ST6Gal I), which transfers sialic acid to galactose residues of N-glycans, decreases in the aged cells. These results indicate that the concordant decrease of the alpha-2,6-sialylation of N-glycans with the ST6Gal I gene expression is induced in TIG-3 cells by in vitro aging.

Cytotoxicity of Flavonoids toward Cultured Normal Human Cells

The cytotoxicity of flavonoids, including apigenin, eriodictyol, 3-hydroxyflavone, kaempherol, luteolin, naringenin, quercetin, rutin, and taxifolin, toward cultured human normal cells, i.e., human lung embryonic fibroblasts (TIG-1) and human umbilical vein endothelial (HUVE) cells, was examined. When these normal human cells were incubated with each flavonoid in culture medium for 24 h, some of the flavonoids showed considerable cytotoxicity at relatively high concentrations and in a dose-dependent manner. 3-Hydroxyflavone, luteolin, and apigenin were more toxic toward TIG-1 cells than the other flavonoids, and luteolin, 3-hydroxyflavone, and quercetin were more toxic toward HUVE cells. HUVE cells were more vulnerable to flavonoid cytotoxicity than TIG-1 cells. Using 2',7'-dichlorofluorescin diacetate (DCF-DA), the intracellular reactive oxygen species (ROS) level of flavonoid-treated TIG-1 cells was examined. The ROS level increased significantly in the presence of the flavone apigenin or luteolin or the flavonol 3-hydroxyflavone, quercetin, or kaempherol. These results suggest that these flavones and flavonols exert cytotoxicity through increasing intracellular ROS levels. Further, the incorporation of apigenin, 3-hydroxyflavone, luteolin, and quercetin, which are more toxic, into TIG-1 cells during 24-h incubation was examined. These flavonoids were incorporated into them and the order of their incorporation efficiency was similar to that of their cytotoxicity. In conclusion, some flavonoids are cytotoxic at higher concentrations toward human normal cells. Further, it is suggested that they are incorporated into cells, increase intracellular ROS levels, and then exert cytotoxicity.

Reconstituted type V collagen fibrils as cementing materials in the formation of cell clumps in culture

Previous studies have reported that type V collagen is an anti-adhesive substrate for cultured cells in that the cells detach from culture dishes coated with type V collagen molecules or polypeptides derived from them. We have noticed that human fetal lung fibroblasts (TIG-1) initially show no reduction in adherence to and spreading on a dish coated with reconstituted type V collagen fibrils but eventually detach from the dish and form cell clumps. To determine the way in which reconstituted type V collagen fibrils are involved in cell clump formation, we have followed the fate of the fluorescence of type V collagen fibrils pre-labeled with fluorescein isothiocyanate. Essentially, all the fluorescence disappeared from the dish surface as the cells detached and was condensed in the cell clumps. The cells that were recovered from clumps and dissociated into separate cells by trypsin treatment proliferated normally after they were seeded on a bare culture dish. This result and those from gel electrophoresis, fluorescence microscopy, and a cell proliferation assay indicate that the cell detachment from the dish is not caused by cell necrosis or apoptosis but by cellular motility together with the unique features of type V collagen fibrils. Not only the adherence of type V collagen fibrils to TIG-1 cells is much stronger than that to the culture dish, but the fibrils are retained on the cellular surface. The strong adherence of type V collagen fibrils to cells plays a role in cementing TIG-1 cells together.

12(S)-Hydroxyeicosatetraenoic acid induces cAMP production via increasing intracellular calcium concentration

We have found that a 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12-HETE), induces cAMP production in human normal fibroblast TIG-1 cells. This phenomenon was not observed in other cells tested including human embryonic kidney HEK293 cells. We have speculated that this specific response might be influenced by the kinds of isoform of adenylyl cyclase (AC) present in cells. We found that TIG-1 cells specifically expressed type VIII AC. As type VIII AC is known to be activated by an increase of calcium concentration, we determined the change of intracellular Ca2+ concentration after the addition of 12-HETE. It was elevated not only in TIG-1 cells, but also HEK293 cells, which did not respond to 12-HETE to produce cAMP. The addition of a calcium ionophore elevated the concentration of intracellular cAMP in TIG-1 cells, but it was without effect in HEK293 cells. To show that the expression of this particular isoform of AC is responsible for the positive response to 12-HETE, we transfected this AC isoform into HEK293 cells. The type VIII AC-transfected cells, in contrast to the mock-transfected ones, became very responsive to 12-HETE to produce cAMP. Taken all together the data would strongly suggest that 12-HETE specifically activates type VIII AC via increasing intracellular Ca2+ concentration.

Cellular aging-dependent decrease in cholesterol in membrane microdomains of human diploid fibroblasts

Recently it has been shown that cholesterol plays indispensable roles in the function of cholesterol-rich microdomains (rafts), such as in ligand-mediated signal transduction. Using a perfringolysin O derivative (BCθ) that binds selectively to cholesterol in rafts without causing membrane damage (Proc. Natl. Acad. Sci. USA 98 (2001) 4926), we have investigated the effect of in vitro replicative aging of human diploid fibroblasts, TIG-1, on the distribution of plasma membrane cholesterol. The amount of BCθ-labeled membrane cholesterol decreased during replicative aging of TIG-1 cells, whereas total cholesterol increased somewhat. The relationship was confirmed by double staining with BCθ and senescence-associated-β-galactosidase, a biomarker of senescent cells. Cell fractionation experiments revealed decreases in both cholesterol in rafts and a raft marker, flotillin, during replicative aging. In addition, hydroxyurea-induced prematurely senescent cells also showed a lower level of BCθ-labeled cholesterol than untreated cells, despite maintaining the total amount of cholesterol. When TIG-1 cells were cultured in cholesterol-deficient medium, BCθ labeling was first diminished and then premature senescence was induced. Taken together with the reduced signaling capacity of aged cells, these results suggest that plasma membrane cholesterol in raft microdomains is more sensitive to senescence than total cholesterol and is a primary target in aging.

Telomerase upregulates expression levels of interleukin (IL)-1α, IL-1β, IL-6, IL-8, and granulocyte–macrophage colony-stimulating factor in normal human fibroblasts

Expression of human telomerase reverse transcriptase (hTERT) in normal human fibroblast cell strain, TIG-3, extends their replicative life span. We found that expression levels of interleukin (IL)-1α, IL-1β, IL-6, IL-8, and granulocyte–macrophage colony-stimulating factor (GM-CSF) mRNA were up-regulated in hTERT-induced fibroblasts irrespective of population doubling level (PDL). Expression levels of these cytokines were low in growing young TIG-3 cells and in control vector-transfected TIG-3 cells but were up-regulated in growth-arrested young cells maintained at high cell density. In senescent TIG-3 cells, expression of IL-1β, IL-6, and GM-CSF was moderately increased. These results indicate that the introduction of hTERT into normal fibroblasts up-regulates the expression of some inflammatory cytokines, and caution should be paid when introducing the hTERT gene to establish cell lines with normal phenotype.

Expression of cholesteryl glucoside by heat shock in human fibroblasts

We investigated the heat-induced alteration of glycolipids in human cultured cells, TIG-3 fibroblasts, to show the expression of steryl glucoside by heat shock. A glycolipid band was detected on a thin-layer chromatography plate in lipid extracts from TIG-3 cells exposed to high temperature (42 degrees C) for 15 and 30 minutes, while it was hardly detectable without heat shock. Both cholesterol and glucose were almost exclusively detected by gas liquid chromatography as degradation products of the lipid. The structure of the lipid molecule was elucidated by electrospray mass spectrometry to be a cholesteryl glucoside. This is the first report to show the occurrence of a steryl glucoside in mammalian cells, and this substance is considered to have a significant role in heat shock responses in mammalian cells.

Human CDC45 protein binds to minichromosome maintenance 7 protein and the p70 subunit of DNA polymerase alpha.

Budding yeast CDC45 encodes Cdc45p, an essential protein required to trigger initiation of DNA replication in late G1 phase. We cloned four and one species of the human Cdc45p homolog cDNA, resulting from different splicing patterns, from HeLa cell and human placenta cDNA libraries, respectively. A comparison of the cDNAs and the genomic sequence showed that the longest encoding a 610-amino acid protein was comprised of 20 exons. One species, which lacks exon 7 and contains the shorter of two exons 18, was identical with the previously reported CDC45L cDNA and constituted 24 out of 28 clones from HeLa cells. Splicing was different in HeLa cells and TIG-1 cells, a human diploid cell line. Human CDC45 protein was found to bind directly in vitro to human minichromosome maintenance 7 protein (hMCM7) and to the p70 subunit of DNA polymerase alpha. The data support a thesis that human CDC45 acts as a molecular tether to mediate loading of the DNA polymerase alpha on to the DNA replication complex through binding to hMCM7.

Age-dependent changes in DNA polymerase fidelity and proofreading activity during cellular aging

DNA polymerase α and the 3′→5′ exonuclease involved in the proofreading of DNA synthesis were isolated from human diploid fetal lung fibroblast (TIG-1) cells at various population doubling levels (PDL). The final PDL of the TIG-1 cells used in these experiments was 70. The fidelity of DNA polymerase α remained high until late passage and fell suddenly just before the end of the life span between 65 and 69 PDL. The activities of the 3′→5′ exonuclease related to proofreading remained unchanged from 21 to 61 PDL, but the activity decreased rapidly in more aged cells. The 3′→5′ exonuclease activity at 69 PDL was about 50% of that in TIG cells at 21 PDL. In vitro DNA synthesis by DNA polymerase α from TIG-1 cells harvested at 69 PDL showed the amount of non-complementary nucleotides incorporated to be decreased by the addition of the 3′→5′ exonuclease from the same cells. However, not all errors were edited out since the ratio of DNA polymerase activity to 3′→5′ exonuclease activity was adjusted to reflect that in vivo and the infidelity of DNA synthesis by error-prone DNA polymerase α from aged cells was improved by the addition of the highly active 3′→5′ exonuclease from cells at 41 PDL. These results suggested that the mutation frequency rises just before the end of the life span of TIG-1 cells.

Cyclin D1 Inhibits Cell Proliferation through Binding to PCNA and Cdk2

Cyclin D1 is known as a promoting factor for cell growth. We previously showed, however, that the expression of cyclin D1 increases markedly in senescent human fibroblastsin vitro.Here we investigate whether the overexpression of cyclin D1 inhibits cell proliferation. Colony formation after transfection with the cyclin D1 expression vector was repressed in NIH-3T3, TIG-1, CHO-K1, and HeLa cells, compared with those with mock and cyclin E expression vectors. A transient transfection assay demonstrated that the overexpression of cyclin D1 inhibited DNA synthesis of TIG-1 cells. The complexes of cyclin D1 with PCNA and cdk2 increased remarkably in senescent cells, compared with young counterparts. Excessive glutathioneS-transferase (GST)–cyclin D1 inhibited DNA replication and repressed cdk2-dependent kinase activityin vitro.DNA synthesis of NIH-3T3 transfectants with PCNA or cdk2 expression vectors was not inhibited by the overexpression of cyclin D1. These results indicate that an excessive level of cyclin D1 represses cell proliferation by inhibiting DNA replication and cdk2 activity through the binding of cyclin D1 to PCNA and cdk2, as it does in senescent cells.