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In vitro aging research in Japan
Experimental Gerontology 35 (2000) 291–298
In vitro aging research in Japan Toshiya Tsuji, Masahiro Miyazaki, Masakiyo Sakaguchi, Masayoshi Namba* Department of Cell Biology, Institute of Molecular and Cellular Biology, Okayama University Medical School, Shikata-cho 2-5-1, Okayama 700-8558 Received 27 September 1999; accepted 23 December 1999
1. Introduction For the past two decades, we have been engaged in transforming normal human cells into neoplastic cells by treatment with either chemical carcinogens or irradiation (Namba et al., 1996). Since beginning these experiments, we have had great difficulty in converting normal human cells into neoplastic cells, because there is hardly any immortalization of cells even after extensive treatment of normal cells with carcinogenic agents, and the cells eventually yield cellular senescence (Hayflick & Moorhead, 1961). In other words, normal human cells can not overcome the senescent stage so easily as the cells from rodents such as mice, rats and hamsters. However, once normal human cells are immortalized, it is relatively easily for them to be transformed into neoplastic cells with carcinogenic agents such as chemical carcinogens, physical agents and so-called oncogenes (Namba et al., 1988; Rhim et al., 1990). These facts indicate that the immortalization of the cells is a critical step for neoplasmic transformation of human cells. Therefore, we have become interested in the study of mechanisms of the immortalization of human cells (Iijima et al., 1998). We have focused on cellular senescence mechanisms, because immortalization and senescence must be a mirror image. Thus, if the mechanism of senescence are elucidated, the mechanism of immortalization will be known, and vice versa. Although the mechanism of immortalization is not completely known at present, the following elements of the possible mechanisms have been elucidated (Namba and Tsuji, 1999): 1) dysfunction of p53- and Rb-cascade, resulting in deregulated functions of the cell cycle-related genes; 2) loss of function in putative senescence genes located on chromosomes 1, 4, 6, 7, 11, 13, 17, 18, and x; and 3) acquisition by immortalized cells of the ability to maintain a constant telomere length, for instance, activation of telomerase activity.
* Corresponding author. Tel.: ⫹81-86-235-7393; fax: ⫹81-86-235-7400. E-mail address: [email protected] (M. Namba). 0531-5565/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 0 0 ) 0 0 0 8 5 - 1
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On the other hand, one of the mechanisms of cellular senescence is due to telomere shortening. Because the majority of normal human somatic cells have no telomerase activity, the cells progressively shorten their chromosome ends (telomeres) with each round of cell division (Harley et al., 1990). However, telomere shortening in itself is not sufficient to explain cellular senescence. For instance, normal mouse cells have telomerase activity and long telomeres, but they still show cellular senescence (Prowse and Greider, 1995). When two different types of cancer cells having an unlimited life span and telomerase activity are fused, the fused cells undergo cellular senescence (Pereira–Smith and Smith, 1988). These results indicate that the senescent phenotype is dominant and that the immortal phenotype is recessive. With these facts in mind, searches for genes for which the expression can be detected in senescent cells but can hardly be detected in immortalized cells have been pursued worldwide. As a result, senescence-related genes such as ING1 (Garkavtsev et al., 1996), SEN 6A (Sandhu et al., 1996), and SEN6 (Banga et al., 1997) have been discovered. In this review, we describe searches for senescencerelated genes that have been conducted in Japan. 2. Hydrogen peroxide-inducible clone 5 (hic-5) Sibanuma et al. (1994) cloned the hic-5 (hydrogen peroxide-inducible clone 5) gene whose expression was enhanced in the mouse osteoblastic MC3T3-E1 cell line by H2O2 and by the transforming growth factor (TGF) 1. Thereafter, it has been found that hic-5 is highly expressed in human senescent fibroblasts and that its expression is downregulated in several human tumor cell lines. A transient expression of exogenous hic-5 reduced the colony-forming efficiency of human immortal KMST-6 cells that had been immortalized in culture by treatment with 60Co-gamma rays (Namba et al., 1985). Furthermore, some human immortalized cell lines transfected with the hic-5 gene displayed not only a decrease in the growth rate and saturation density but also senescent morphology after 20 to 40 population doublings (Sibanuma et al., 1997). These transfectants showed an increased expression of senescence-related genes, such as p21/SDI1/ WAF1, collagenase, and fibronectin, and a decreased expression of c-fos. Hic-5 protein has four zinc finger-like LIM motifs in its C-terminal region. Its LIM motifs show striking similarity to paxillin, a focal adhesion protein. In addition, hic-5 protein is mainly localized to focal adhesions and binds to focal adhesion-related molecules such as FAK (focal adhesion kinase) and CAK- (cell adhesion kinase-) (Fujita et al., 1998; Matsuya et al., 1998). Taken together, there is a possibility that one of the functions of hic-5 is to inhibit tyrosine phosphorylation of paxillin by binding to proteintyrosine kinases such as FAK in a competitive manner to paxillin (Fujita et al., 1998). Accordingly, hic-5 inhibits the integrin signal transduction, leading to growth retardation and to morphological changes characteristic of cellular senescence. On the other hand, LIM domains of the hic-5 protein can bind to a DNA sequence that contains a high amount of G⫹A and/or a long A/T tract in a zinc-dependent manner in vitro (Nishiya et al., 1998). Thus, hic-5 may affect gene transcription through its binding to DNA. 3. Pluto and orpheus (Oct-1): transcriptional factors of type I collagenase gene expression Type I collagenase gene is highly expressed in human senescent cells, but its expression is very low or not detectable in young mortal and immortal cells (Imai and Takano,
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1992). Imai et al. (1994) identified two immortalization-susceptible cis-acting elements (ISE1 and ISE2) in the upstream region of the collagenase gene, which up-regulate the expression of the type I collagenase gene in senescent cells and down-regulate the expression of the gene in immortalized cells. In addition, they also found two transcriptional regulators, Pluto and Orpheus, which bind to ISE2 (Imai et al., 1994). Pluto functions as a transcriptional activator for predominant expression of the collagenase gene in senescent cells and seems to be a novel NFAT (nuclear factor of activated T-cells)— related transcriptional factor. On the other hand, Orpheus may function as a transcriptional repressor of the collagenase gene expression in young mortal and immortalized cells. Subsequently, Orpheus was found to be identical to Oct-1, a member of the POU domain family (Imai et al., 1997). In addition, Orpheus proteins are mainly localized in the perinuclear region during the young stage, whereas this subspecies of Orpheus disappears from there during the senescent stage. These transcriptional factors may also regulate expression of other genes involved in the mechanism of cellular senescence. 4. Mortalins (mot-1 and mot-2) as mortality markers It is very important to identify the mortality markers for cellular senescence research. Senescent-associated (SA) -galactosidase is expressed by human senescent cells but is not associated with quiescence, terminal differentiation, or immortality (Dimri et al., 1995). Wadhawa et al. (1993a, 1993b) discovered a mortality marker, which contained mortalins mot-1 and mot-2, novel members of the HSP70 (heat shock protein 70) family proteins. The mot-1 protein showed a pancytosolic cellular distribution in normal murine and human cells, whereas the mot-2 protein was found in the perinuclear region of murine and human immortal cells (Wadhawa et al., 1993c). The two types of murine mortalins differ only in two amino acids, arise from difficult genes, and have contrasting biological activities (Wadhawa et al., 1996). The mot-2 protein exhibited four types of cellular distribution, which were found to be correlated with four complementation groups in human immortalized cells (Pereira–Smith and Smith, 1988; Wadhawa et al., 1995). Microinjection of anti-mot-1 antibody could transiently release murine senescent cells from growth arrest, whereas transfection of mot-1 cDNA induced cellular senescence in the murine immortal cell line, indicating that mot-1 has antiproliferative activity (Wadhawa et al., 1993a; 1993b). In contrast, transfection of mot-2 cDNA to primary mouse fibroblasts could not induce immortality of the cells. However, stable expression of mot-2.1 mRNA, a mot-2-variant with a long 3⬘-noncoding region having highly translational efficiency, induced a transformed-morphology and anchorage-independent growth in a murine immortal cell line, and the mot-2.1-transfected cells produced tumors in nude mice (Kaul et al., 1998). Interestingly, the mot-2 protein interacted with the C terminus of wild-type p53 in transformed cells (Wadhawa et al., 1998). As a result, the wild-type p53 associated with mot-2 could not enter the nucleus, resulting in down-regulation of p53-responsible genes such as p53 itself, p21/SDI1/WAF1, and MDM2. Thus, mot-2 is closely related to neoplastic transformation rather than immortalization. 5. A putative senescent gene on chromosome 7 in the complementation D group Studies on cell fusion of normal cells with immortal ones demonstrated that the phenotype of senescence is dominant (Stein et al., 1985). Furthermore, complementation
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analyses with different immortal cell lines revealed that immortality is derived from a limited number of recessive genetic alterations (Pereira–Smith and Smith, 1988). They described four complementation groups, A, B, C, and D. Ogata et al. (1993) genetically compared two immortalized human cell lines (SUSM-1 and KMST-6) in the complementation D group with their normal counterparts. They found chromosomal rearrangements on the long arm of chromosome 7 in both immortalized cell lines. Introduction of chromosome 7 into these immortalized cell lines specifically suppressed the indefinite division potential of the immortal cells and induced senescent morphology in these cells. These cells also exhibited remarkable shortening of their telomeric sequences and senescent-specific markers such as SA -galactosidase activity and pancytosolic mortalin (Nakabayashi et al., 1997). Similarly, intracellular introduction of chromosome 7 also induced senescence in the human hepatoma HepG2 cell line assigned to the complementation D group (Ogata et al., 1995). However, chromosome 7 does not affect the cell lines belonging to other (A, B, and C) complementation groups. These findings indicate that a senescent gene(s) that commonly mutates in the cells of complementation D is located on chromosome 7q31-32.
6. Premature aging disorder Werner’s syndrome (WRN) is a rare autosomal recessive disorder that shows several premature aging features such as arteriosclerosis, osteoporosis and malignant neoplasms. Japan has the largest percentage of WRN patients in the world. Recently, Japanese and USA groups working in collaboration succeeded in isolating a gene responsible for WRN by using the positional cloning method (Yu et al., 1996). This gene codes for Escherichia coli RecQ-type DNA helicase. Furthermore, 19 different kinds of WRN gene mutation were found in Japanese patients, and these mutations were nonsense mutation or frame shift, which resulted in trancated WRN proteins (Yu et al., 1996; Oshima et al., 1996; Matsumoto et al., 1997). The WRN protein, which was proven to be a helicase, may be associated with DNA replication, recombination, chromosome segmentation, DNA repair, transcription, and other functions requiring DNA unwinding. Therefore, loss of functions in the WRN protein may lead to accumulation of DNA damage, resulting in premature aging.
7. Identification of senescence-related genes using human immortalized cells and their normal counterparts We previously succeeded in establishing three human immortalized cell lines (KMST-6, SUSM-1, and OUMS-24F) by treatment either with 60Co ␥-rays or a chemical carcinogen, 4-nitroquinoline 1-oxide (Namba and Tsuji, 1996). Since then, we have started to isolate the senescence-related genes using these immortalized cell lines and their normal counterparts. By 2-dimensional gel electrophoresis (2D-PAGE), we identified two kinds of proteins that are down-regulated in these immortalized cell lines. One of them was identified as transferrin, a carrier protein for iron (Kondo et al., 1996). Transferrin acts as a chelator for free iron and reduces the iron-mediated generation of free radicals that are harmful to cells. Thus, decreased expression of transferrin in the immortalized cells induces accu-
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Table 1 Identification of cellular senescence-related genes using the RDA method Cloned genes Extracellular matrix ␣(2) type I collagen Fibrin 2 Extracellular protein S1-5 Bone proteoglycan II Signal transduction WS 9-14 TGF masking protein SDI-1 MAPK phosphatase-1 Glycoprotein/Thy-1 IGF binding protein 5 Others Lamin A Coatmer epsilon subunit Unknown EST1 EST2 EST3 EST4 EST5 EST6 EST7
mulation of DNA damage by free radicals. However, it is not clear at present that this down-regulation of transferrin is the cause or effect of immortalization of human cells. The other such protein identified was S100C, a member of the family of EF-hand type Ca2⫹-binding proteins. Interestingly, expression of the S100C protein increased when cells reached confluence. In addition, S100C moved from the cytoplasm to the nucleus of normal cells at confluence, while it remained in the cytoplasm of immortalized cells even after confluence. Furthermore, microinjection of S100C-antibody into normal confluent cells caused resumption of BrdU incorporation into their nuclei. Thus, S100C may contribute to contact inhibition of growth of normal cells (Sakaguchi et al., in preparation). By the representational difference analysis (RDA) method, we isolated 19 different genes whose expressions were low in the three immortalized cell lines as compared with expression levels in their normal counterparts. Among 19 genes, seven genes are novel, and twelve genes have already been reported to be senescence-related. They include ␣(2) type 1 collagen, p21/SDI1/WAF1, WS9-14, and IGF binding protein 5 (Table 1). One of the seven novel genes, which was named REIC, was not only down-regulated in three human immortalized cell lines but also in eight human tumor-derived cell lines (Hep3B and HuH-7 hepatocellular carcinomas, HuH-6 Clone 5 hepatoblastoma, HuCCT-1 cholangiocarcinoma, A549 lung cancer, HaCaT immortalized keratinocyte, HeLa cervical carcinoma, and Saos-2 osteosarcoma) (Tsuji et al., 2000). In contrast, among the human tissues examined, the heart and brain, which contain a large number of post-mitotic cells, showed the highest expression of REIC. Furthermore, expression of the REIC mRNA gradually decreased in the cells stimulated with the addition of serum to the culture medium after the 120-hour-culture of the cells under serum-free conditions, and reached the lowest level at 12 hours after the serum stimulation, which is consistent with the G1/S
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phase in the cell cycle. Moreover, an enhanced expression of the REIC mRNA was observed in senescent cells and the cells treated with TGF-. A transient expression of endogenous REIC cDNA significantly reduced the DNA-synthesis of the human osteosarcoma Saos-2 cell line, indicating the the REIC gene has an antiproliferative activity (Tsuji et al., in preparation). REIC gene localized in chromosome 11p15 whose genomic imprinting frequently found in many kind of cancer (Feinberg et al., 1999). The full-length REIC cDNA indicated that the predicted protein contains 350 amino acids and possesses two coiled-coil tertiary structures which function protein-protein interaction in each of the amino- and carboxyl-termini. The REIC gene belongs to a Dkk gene family which plays a role in induction of amphibian head structures by inhibiting of Wnt signal pathways (Glinka et al., 1998). Since there is a lot of evidence that activation of the Wnt signaling contributes to the neoplastic process, aberrations of Wnt signal pathways may be related to the development of some human cancers (Nusse and Varmus, 1992). Taken together, the REIC gene cloned by us may function as a tumor suppressor gene.
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In vitro aging research in Japan Toshiya Tsuji, Masahiro Miyazaki, Masakiyo Sakaguchi, Masayoshi Namba* Department of Cell Biology, Institute of Molecular and Cellular Biology, Okayama University Medical School, Shikata-cho 2-5-1, Okayama 700-8558 Received 27 September 1999; accepted 23 December 1999
1. Introduction For the past two decades, we have been engaged in transforming normal human cells into neoplastic cells by treatment with either chemical carcinogens or irradiation (Namba et al., 1996). Since beginning these experiments, we have had great difficulty in converting normal human cells into neoplastic cells, because there is hardly any immortalization of cells even after extensive treatment of normal cells with carcinogenic agents, and the cells eventually yield cellular senescence (Hayflick & Moorhead, 1961). In other words, normal human cells can not overcome the senescent stage so easily as the cells from rodents such as mice, rats and hamsters. However, once normal human cells are immortalized, it is relatively easily for them to be transformed into neoplastic cells with carcinogenic agents such as chemical carcinogens, physical agents and so-called oncogenes (Namba et al., 1988; Rhim et al., 1990). These facts indicate that the immortalization of the cells is a critical step for neoplasmic transformation of human cells. Therefore, we have become interested in the study of mechanisms of the immortalization of human cells (Iijima et al., 1998). We have focused on cellular senescence mechanisms, because immortalization and senescence must be a mirror image. Thus, if the mechanism of senescence are elucidated, the mechanism of immortalization will be known, and vice versa. Although the mechanism of immortalization is not completely known at present, the following elements of the possible mechanisms have been elucidated (Namba and Tsuji, 1999): 1) dysfunction of p53- and Rb-cascade, resulting in deregulated functions of the cell cycle-related genes; 2) loss of function in putative senescence genes located on chromosomes 1, 4, 6, 7, 11, 13, 17, 18, and x; and 3) acquisition by immortalized cells of the ability to maintain a constant telomere length, for instance, activation of telomerase activity.
* Corresponding author. Tel.: ⫹81-86-235-7393; fax: ⫹81-86-235-7400. E-mail address: [email protected] (M. Namba). 0531-5565/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 0 0 ) 0 0 0 8 5 - 1
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On the other hand, one of the mechanisms of cellular senescence is due to telomere shortening. Because the majority of normal human somatic cells have no telomerase activity, the cells progressively shorten their chromosome ends (telomeres) with each round of cell division (Harley et al., 1990). However, telomere shortening in itself is not sufficient to explain cellular senescence. For instance, normal mouse cells have telomerase activity and long telomeres, but they still show cellular senescence (Prowse and Greider, 1995). When two different types of cancer cells having an unlimited life span and telomerase activity are fused, the fused cells undergo cellular senescence (Pereira–Smith and Smith, 1988). These results indicate that the senescent phenotype is dominant and that the immortal phenotype is recessive. With these facts in mind, searches for genes for which the expression can be detected in senescent cells but can hardly be detected in immortalized cells have been pursued worldwide. As a result, senescence-related genes such as ING1 (Garkavtsev et al., 1996), SEN 6A (Sandhu et al., 1996), and SEN6 (Banga et al., 1997) have been discovered. In this review, we describe searches for senescencerelated genes that have been conducted in Japan. 2. Hydrogen peroxide-inducible clone 5 (hic-5) Sibanuma et al. (1994) cloned the hic-5 (hydrogen peroxide-inducible clone 5) gene whose expression was enhanced in the mouse osteoblastic MC3T3-E1 cell line by H2O2 and by the transforming growth factor (TGF) 1. Thereafter, it has been found that hic-5 is highly expressed in human senescent fibroblasts and that its expression is downregulated in several human tumor cell lines. A transient expression of exogenous hic-5 reduced the colony-forming efficiency of human immortal KMST-6 cells that had been immortalized in culture by treatment with 60Co-gamma rays (Namba et al., 1985). Furthermore, some human immortalized cell lines transfected with the hic-5 gene displayed not only a decrease in the growth rate and saturation density but also senescent morphology after 20 to 40 population doublings (Sibanuma et al., 1997). These transfectants showed an increased expression of senescence-related genes, such as p21/SDI1/ WAF1, collagenase, and fibronectin, and a decreased expression of c-fos. Hic-5 protein has four zinc finger-like LIM motifs in its C-terminal region. Its LIM motifs show striking similarity to paxillin, a focal adhesion protein. In addition, hic-5 protein is mainly localized to focal adhesions and binds to focal adhesion-related molecules such as FAK (focal adhesion kinase) and CAK- (cell adhesion kinase-) (Fujita et al., 1998; Matsuya et al., 1998). Taken together, there is a possibility that one of the functions of hic-5 is to inhibit tyrosine phosphorylation of paxillin by binding to proteintyrosine kinases such as FAK in a competitive manner to paxillin (Fujita et al., 1998). Accordingly, hic-5 inhibits the integrin signal transduction, leading to growth retardation and to morphological changes characteristic of cellular senescence. On the other hand, LIM domains of the hic-5 protein can bind to a DNA sequence that contains a high amount of G⫹A and/or a long A/T tract in a zinc-dependent manner in vitro (Nishiya et al., 1998). Thus, hic-5 may affect gene transcription through its binding to DNA. 3. Pluto and orpheus (Oct-1): transcriptional factors of type I collagenase gene expression Type I collagenase gene is highly expressed in human senescent cells, but its expression is very low or not detectable in young mortal and immortal cells (Imai and Takano,
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1992). Imai et al. (1994) identified two immortalization-susceptible cis-acting elements (ISE1 and ISE2) in the upstream region of the collagenase gene, which up-regulate the expression of the type I collagenase gene in senescent cells and down-regulate the expression of the gene in immortalized cells. In addition, they also found two transcriptional regulators, Pluto and Orpheus, which bind to ISE2 (Imai et al., 1994). Pluto functions as a transcriptional activator for predominant expression of the collagenase gene in senescent cells and seems to be a novel NFAT (nuclear factor of activated T-cells)— related transcriptional factor. On the other hand, Orpheus may function as a transcriptional repressor of the collagenase gene expression in young mortal and immortalized cells. Subsequently, Orpheus was found to be identical to Oct-1, a member of the POU domain family (Imai et al., 1997). In addition, Orpheus proteins are mainly localized in the perinuclear region during the young stage, whereas this subspecies of Orpheus disappears from there during the senescent stage. These transcriptional factors may also regulate expression of other genes involved in the mechanism of cellular senescence. 4. Mortalins (mot-1 and mot-2) as mortality markers It is very important to identify the mortality markers for cellular senescence research. Senescent-associated (SA) -galactosidase is expressed by human senescent cells but is not associated with quiescence, terminal differentiation, or immortality (Dimri et al., 1995). Wadhawa et al. (1993a, 1993b) discovered a mortality marker, which contained mortalins mot-1 and mot-2, novel members of the HSP70 (heat shock protein 70) family proteins. The mot-1 protein showed a pancytosolic cellular distribution in normal murine and human cells, whereas the mot-2 protein was found in the perinuclear region of murine and human immortal cells (Wadhawa et al., 1993c). The two types of murine mortalins differ only in two amino acids, arise from difficult genes, and have contrasting biological activities (Wadhawa et al., 1996). The mot-2 protein exhibited four types of cellular distribution, which were found to be correlated with four complementation groups in human immortalized cells (Pereira–Smith and Smith, 1988; Wadhawa et al., 1995). Microinjection of anti-mot-1 antibody could transiently release murine senescent cells from growth arrest, whereas transfection of mot-1 cDNA induced cellular senescence in the murine immortal cell line, indicating that mot-1 has antiproliferative activity (Wadhawa et al., 1993a; 1993b). In contrast, transfection of mot-2 cDNA to primary mouse fibroblasts could not induce immortality of the cells. However, stable expression of mot-2.1 mRNA, a mot-2-variant with a long 3⬘-noncoding region having highly translational efficiency, induced a transformed-morphology and anchorage-independent growth in a murine immortal cell line, and the mot-2.1-transfected cells produced tumors in nude mice (Kaul et al., 1998). Interestingly, the mot-2 protein interacted with the C terminus of wild-type p53 in transformed cells (Wadhawa et al., 1998). As a result, the wild-type p53 associated with mot-2 could not enter the nucleus, resulting in down-regulation of p53-responsible genes such as p53 itself, p21/SDI1/WAF1, and MDM2. Thus, mot-2 is closely related to neoplastic transformation rather than immortalization. 5. A putative senescent gene on chromosome 7 in the complementation D group Studies on cell fusion of normal cells with immortal ones demonstrated that the phenotype of senescence is dominant (Stein et al., 1985). Furthermore, complementation
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analyses with different immortal cell lines revealed that immortality is derived from a limited number of recessive genetic alterations (Pereira–Smith and Smith, 1988). They described four complementation groups, A, B, C, and D. Ogata et al. (1993) genetically compared two immortalized human cell lines (SUSM-1 and KMST-6) in the complementation D group with their normal counterparts. They found chromosomal rearrangements on the long arm of chromosome 7 in both immortalized cell lines. Introduction of chromosome 7 into these immortalized cell lines specifically suppressed the indefinite division potential of the immortal cells and induced senescent morphology in these cells. These cells also exhibited remarkable shortening of their telomeric sequences and senescent-specific markers such as SA -galactosidase activity and pancytosolic mortalin (Nakabayashi et al., 1997). Similarly, intracellular introduction of chromosome 7 also induced senescence in the human hepatoma HepG2 cell line assigned to the complementation D group (Ogata et al., 1995). However, chromosome 7 does not affect the cell lines belonging to other (A, B, and C) complementation groups. These findings indicate that a senescent gene(s) that commonly mutates in the cells of complementation D is located on chromosome 7q31-32.
6. Premature aging disorder Werner’s syndrome (WRN) is a rare autosomal recessive disorder that shows several premature aging features such as arteriosclerosis, osteoporosis and malignant neoplasms. Japan has the largest percentage of WRN patients in the world. Recently, Japanese and USA groups working in collaboration succeeded in isolating a gene responsible for WRN by using the positional cloning method (Yu et al., 1996). This gene codes for Escherichia coli RecQ-type DNA helicase. Furthermore, 19 different kinds of WRN gene mutation were found in Japanese patients, and these mutations were nonsense mutation or frame shift, which resulted in trancated WRN proteins (Yu et al., 1996; Oshima et al., 1996; Matsumoto et al., 1997). The WRN protein, which was proven to be a helicase, may be associated with DNA replication, recombination, chromosome segmentation, DNA repair, transcription, and other functions requiring DNA unwinding. Therefore, loss of functions in the WRN protein may lead to accumulation of DNA damage, resulting in premature aging.
7. Identification of senescence-related genes using human immortalized cells and their normal counterparts We previously succeeded in establishing three human immortalized cell lines (KMST-6, SUSM-1, and OUMS-24F) by treatment either with 60Co ␥-rays or a chemical carcinogen, 4-nitroquinoline 1-oxide (Namba and Tsuji, 1996). Since then, we have started to isolate the senescence-related genes using these immortalized cell lines and their normal counterparts. By 2-dimensional gel electrophoresis (2D-PAGE), we identified two kinds of proteins that are down-regulated in these immortalized cell lines. One of them was identified as transferrin, a carrier protein for iron (Kondo et al., 1996). Transferrin acts as a chelator for free iron and reduces the iron-mediated generation of free radicals that are harmful to cells. Thus, decreased expression of transferrin in the immortalized cells induces accu-
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Table 1 Identification of cellular senescence-related genes using the RDA method Cloned genes Extracellular matrix ␣(2) type I collagen Fibrin 2 Extracellular protein S1-5 Bone proteoglycan II Signal transduction WS 9-14 TGF masking protein SDI-1 MAPK phosphatase-1 Glycoprotein/Thy-1 IGF binding protein 5 Others Lamin A Coatmer epsilon subunit Unknown EST1 EST2 EST3 EST4 EST5 EST6 EST7
mulation of DNA damage by free radicals. However, it is not clear at present that this down-regulation of transferrin is the cause or effect of immortalization of human cells. The other such protein identified was S100C, a member of the family of EF-hand type Ca2⫹-binding proteins. Interestingly, expression of the S100C protein increased when cells reached confluence. In addition, S100C moved from the cytoplasm to the nucleus of normal cells at confluence, while it remained in the cytoplasm of immortalized cells even after confluence. Furthermore, microinjection of S100C-antibody into normal confluent cells caused resumption of BrdU incorporation into their nuclei. Thus, S100C may contribute to contact inhibition of growth of normal cells (Sakaguchi et al., in preparation). By the representational difference analysis (RDA) method, we isolated 19 different genes whose expressions were low in the three immortalized cell lines as compared with expression levels in their normal counterparts. Among 19 genes, seven genes are novel, and twelve genes have already been reported to be senescence-related. They include ␣(2) type 1 collagen, p21/SDI1/WAF1, WS9-14, and IGF binding protein 5 (Table 1). One of the seven novel genes, which was named REIC, was not only down-regulated in three human immortalized cell lines but also in eight human tumor-derived cell lines (Hep3B and HuH-7 hepatocellular carcinomas, HuH-6 Clone 5 hepatoblastoma, HuCCT-1 cholangiocarcinoma, A549 lung cancer, HaCaT immortalized keratinocyte, HeLa cervical carcinoma, and Saos-2 osteosarcoma) (Tsuji et al., 2000). In contrast, among the human tissues examined, the heart and brain, which contain a large number of post-mitotic cells, showed the highest expression of REIC. Furthermore, expression of the REIC mRNA gradually decreased in the cells stimulated with the addition of serum to the culture medium after the 120-hour-culture of the cells under serum-free conditions, and reached the lowest level at 12 hours after the serum stimulation, which is consistent with the G1/S
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phase in the cell cycle. Moreover, an enhanced expression of the REIC mRNA was observed in senescent cells and the cells treated with TGF-. A transient expression of endogenous REIC cDNA significantly reduced the DNA-synthesis of the human osteosarcoma Saos-2 cell line, indicating the the REIC gene has an antiproliferative activity (Tsuji et al., in preparation). REIC gene localized in chromosome 11p15 whose genomic imprinting frequently found in many kind of cancer (Feinberg et al., 1999). The full-length REIC cDNA indicated that the predicted protein contains 350 amino acids and possesses two coiled-coil tertiary structures which function protein-protein interaction in each of the amino- and carboxyl-termini. The REIC gene belongs to a Dkk gene family which plays a role in induction of amphibian head structures by inhibiting of Wnt signal pathways (Glinka et al., 1998). Since there is a lot of evidence that activation of the Wnt signaling contributes to the neoplastic process, aberrations of Wnt signal pathways may be related to the development of some human cancers (Nusse and Varmus, 1992). Taken together, the REIC gene cloned by us may function as a tumor suppressor gene.
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