Oncogene (2004) 23, 3670–3680
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Cancer predisposition in mice deficient for the metastasis-associated Mts1(S100A4) gene Christina EL Naaman1,5, Birgitte Grum-Schwensen1,5, Ahmed Mansouri2, Mariam Grigorian1, Eric Santoni-Rugiu3, Thomas Hansen1, Marina Kriajevska1, Beat W Schafer4, Claus W Heizmann4, Eugene Lukanidin1 and Noona Ambartsumian1,* 1
Department of Molecular Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK2100 Copenhagen, Denmark; Department of Molecular Cell Biology, Max-Planck Institute for Biophysical Chemistry, Am Fassberg 11, D37077 Go¨ttingen, Germany; 3Department of Pathology, Rigshospitalet, Blegdamsvej 9, DK2100 Copenhagen, Denmark; 4Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zu¨rich, Steinwiesstrasse 75, CH-8032 Zu¨rich, Switzerland 2
Metastasis-promoting Mts1(S100A4) protein belongs to the S100 family of Ca2 þ -binding proteins. A mouse strain with a germ-line inactivation of the S100A4 gene was generated. The mice were viable and did not display developmental abnormalities in the postnatal period. However, an abnormal sex ratio was observed in the litters with the S100A4/ genotype, raising the possibility of a certain level of embryonic lethality in this strain. In all, 10% of 10–14-month-old S100A4-null animals developed tumors. This is a characteristic feature of mouse strains with inactivated tumor suppressor genes. Spontaneous tumors of S100A4/ mice were p53 positive. Recently, we have shown that S100A4 interacts with p53 tumor suppressor protein and induces apoptosis. We proposed that impairment of this interaction could affect the apoptosis-promoting function of p53 that is involved in its tumor suppressor activity. The frequency of apoptosis in the spleen of S100A4/ animals after whole-body c-irradiation was reduced compared to the wild-type animals. The same was true for the transcriptional activation of the p53 target genes – waf/p21/cip1 and bax. Taken together, these observations indicate that spontaneous tumors in S100A4/ mice are a result of functional destabilization of p53 tumor suppressor gene. Oncogene (2004) 23, 3670–3680. doi:10.1038/sj.onc.1207420 Keywords: animal models; Mts1(S100A4); p53; tumor development; tumor suppressor activity
Introduction Metastasis-inducing S100A4 protein belongs to the S100 family of Ca2 þ -binding proteins. Members of this family function both intra- and extracellularly. Inside a cell, they are implicated in a variety of activities such as cell proliferation and differentiation, cytoskeleton dynamics *Correspondence: N Ambartsumian; E-mail:
[email protected] 5 These authors contributed equally to the work Received 26 August 2003; revised 3 November 2003; accepted 28 November 2003
and apoptosis. When released into extracellular space, S100 proteins stimulate neuronal differentiation, astrocyte proliferation, modulate activity of inflammatory cells and stimulate angiogenesis (for a review, see Donato, 2001; Heizmann et al., 2002). Some of the S100 family members are involved in cancer development (Weterman et al., 1993; Pedrocchi et al., 1994; Al-Haddad et al., 1999; Guerreiro Da Silva et al., 2000; Feng et al., 2001). In this respect, much interest has been focused on S100A4 since its expression was strongly associated with stimulation of metastasis. The S100A4 gene was isolated as a gene differentially expressed in metastatic mouse mammary adenocarcinoma cells (Ebralidze et al., 1989). Introduction of the S100A4 gene into a number of nonmetastatic tumor cell lines and suppression of its activity in metastatic ones proved its involvement in metastasis formation (Mazzucchelli, 2002). Stimulation of tumor metastatic activity was demonstrated in two transgenic mouse models with overexpression of the S100A4 gene (Ambartsumian et al., 1996; Davies et al., 1996). In a number of human cancers, the enhanced expression of S100A4 was associated with poor prognosis (Platt-Higgins et al., 2000; Yonemura et al., 2000; Davies et al., 2002; Rosty et al., 2002). The exact mechanism by which S100A4 stimulates metastasis formation is poorly understood. When applied extracellularly, S100A4 stimulates neurite outgrowth (Novitskaya et al., 2000) and exhibits angiogenic activity (Ambartsumian et al., 2001). This implies that S100A4 could contribute to tumor progression by stimulating neovascularization of a tumor. On the other hand, a number of intracellular interacting partners, such as heavy chain of nonmuscle myosin (Kriajevska et al., 1994; Ford and Zain, 1995), liprinb-1 (Kriajevska et al., 2002), p53 (Grigorian et al., 2001) and recently methionine aminopeptidase (Endo et al., 2002) were described for the S100A4 protein, suggesting its participation in cell motility, adhesion and proliferation. This raised the possibility that S100A4 participates in metastasis by interfering with any of these processes. One of the interacting partners of S100A4 in the cell is p53 tumor suppressor protein (Chen et al., 2001; Grigorian et al., 2001). The wild-type p53 plays a central role in reducing the probability of a cell
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becoming cancerous. It controls the induction of apoptosis and growth arrest at cell cycle checkpoints. This function of p53 leads to elimination of damaged cells from a population and is believed to be a basis of its tumor suppressor function (Vousden, 2002). Transactivation of p53 target genes is regarded as one of the mechanisms of regulation of p53 response (Bourdon et al., 1997; El-Deiry, 1998). In addition, wild-type p53 regulates expression of several metastasis-related genes, such as several matrix metalloproteinases, cathepsin D, thrombospondin-1 and KAI1 (Dameron et al., 1994; Bian and Sun, 1997; Mashimo et al., 1998; Wu et al., 1998; Sun et al., 1999, 2000). By interacting with p53, S100A4 enhances p53-dependent apoptosis and differentially modulates p53 target gene expression (Grigorian et al., 2001). Although S100 genes are structurally and evolutionarily related, the intracellular location, expression pattern and localization of each individual S100 protein is distinct (Mandinova et al., 1998; Hsieh et al., 2002). Expression of the S100A4 in an adult organism is restricted mainly to the cells of the lymphoid system, presumably T lymphocytes and macrophages (Lukanidin and Georgiev, 1996). In embryonic development, S100A4 also demonstrates a very distinct pattern of expression in several mesenchymal tissues and in fetal macrophages (Klingelhofer et al., 1997). The important role of S100A4 in embryogenesis could also be postulated from its high level of expression in mouse trophoblast (Ford and Zain, 1995). S100A4 gene is located in a cluster of S100 genes on human chromosome 1q21 and in a syntenic region on mouse chromosome 3 (Ridinger et al., 1998). Despite cluster organization, there are no indications of a coordinated regulation of transcription of S100 genes, each member of the family being under a complex transcriptional regulation (Chen and Barraclough, 1996; Cohn et al., 2001). Cell- and tissue-specific expression of the S100A4 protein is supported by a complex regulation on a post-transcriptional level (Ambartsumian et al., 1998–1999). Moreover, the S100A4 gene is differently expressed in the tissues of related species, such as rat and mouse (Davies et al., 1995). To address the questions of the role of S100A4 in normal mouse organism and in cancer development, we generated a mouse strain with a germ-line inactivation of the S100A4 gene. Here we show that deletion of S100A4 in the mouse does not affect embryonic development. However, a part of S100A4-deficient mice was prone to spontaneous tumor development, suggesting the participation of S100A4 in tumor suppressor function. Whole-body girradiation of mice revealed impairment of p53 function in the S100A4/ animals, which results in decreased apoptosis and suppression of p53 target gene transcription. We suggest that disrupted interaction of S100A4 with p53 affects its function as a guardian of the genome raising the probability of tumor formation in S100A4/ animals. Analysis of the expression of the neighboring S100 genes – S100A3 and S100A5 – in S100A4-null animals
demonstrated alterations in the expression of S100A5 raising the possibility of a compensatory mechanism of the S100A4 activity in mice.
Results Generation of S100A4-deficient mice The murine S100A4 gene was inactivated by homologous recombination in ES cells, using the strategy depicted in Figure 1a. The entire coding sequence of the S100A4 gene from exons 2 and 3 was replaced by a cassette containing selective markers flanked by loxP sites of bacteriophage P1. Next, the cassette was excised by means of the Cre/Lox recombination system of bacteriophage P1 (Sauer, 1993). Selected clones of ES cells were aggregated with the morulae and transferred into a foster mother. Mice bearing the deletion of S100A4 were identified by Southern blot analysis (Figure 1b). The S100A4/ mice were born viable, demonstrating that S100A4 is not required for embryonic development. Histological analysis of the tissues obtained from 12- and 24-week-old animals did not reveal any abnormalities as compared to the control mice (data not shown). S100A4 RNA and protein were not detected in the tissues of these mice (Figure 1c and d). Breeding data from the strain carrying deletion of the S100A4 gene revealed a deficiency of S100A4/ female progeny (Table 1). The reason for abnormal sex ratio is not clear and is currently under investigation. Spontaneous tumor development in S100A4-null mice We followed a cohort of animals bearing homozygous deletion of S100A4 gene for an extended period of time for a possible appearance of pathological manifestations. Quite unexpectedly, 12% of the 47–59-week-old S100A4-null animals developed tumors. Control mice of matching genetic background were tumor free at this age (Table 2). Moreover, a tumor was also detected in one of the S100A4/ animals of the 21–33-week-old group. Two of the knockout animals developed two independent tumors. A total of eight tumors were detected and subjected to histopathological analysis. The tumors were predominantly of epithelial origin, most frequently carcinomas in the lung (Table 3). The latter were represented by bronchioalveolar carcinomas of solid type (Turusov and Mohr, 1994), composed of dense tumor masses infiltrating the alveolar spaces with undefined cellular borders and relatively mild nuclear pleomorphism (Figure 2a). The tumor in the mammary gland was diagnosed as an adenocarcinoma type B (Turusov and Mohr, 1994) composed of solid undifferentiated structures with a minor tubular component (Figure 2b) similar to invasive ductular carcinomas in humans. The other tumors detected were a hepatocellular carcinoma surrounded by severe liver cell dysplasia, a fibrosarcoma in the uterus and lymphoma with metastases to the liver. We did not observe Oncogene
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Figure 1 Generation of S100A4 knockout mice. (a) Targeting strategy at the S100A4 locus. Exons 2 and 3 are replaced by the neomycin- and thymidin kinase-containing cassette in a recombinant allele generated by homologous recombination. Probe I was used to identify homologous recombinant ES cells. Selection cassette was excised by transfection of selected clone with Cre-recombinase bearing plasmid. Probe II was used to identify ES clones bearing deletion of the S100A4 gene sequences. (b) Southern blot analysis of genomic DNA. A restriction digest with ScaI when hybridized with probe II shows two bands, 8 kb represents the knockout allele whereas 6kb represents the wild type. (c) Northern blot analysis of total RNA isolated from different organs of þ / þ , þ / and / animals. Hybridization with S100A4 probe. (d) Immunoprecipitation of S100A4 protein from primary embryonic fibroblasts isolated from þ / þ and / animals. CSML100 cell lysate expressing S100A4 was used as a positive control
Table 1 Numbers of S100A4/ progeny at weaning Genotype +/+ /
No. of litters
No. at weaning
No. of females
No. of males
No. females/ no. males
13 18
139 160
72 60
67 101
1.09 0.59
metastases in the animals bearing tumors of epithelial origin. A high rate of spontaneous tumor incidence is a characteristic feature of knockout mouse strains with inactivated tumor suppressor genes (Hakem and Mak, 2001). To our knowledge, S100A4 does not exhibit tumor suppressor function. Previously we have shown that S100A4 interacts with p53 tumor suppressor Oncogene
protein, stimulating p53-dependent apoptosis and affecting the transactivation of p53-dependent genes (Grigorian et al., 2001). Development of the tumors in S100A4/ mice only in some of the animals and with a substantial delay in the life together with the previously established interaction of S100A4 with p53 protein allowed us to speculate that spontaneous tumor development in the S100A4/ mice is a result of modulation of p53 tumor suppressor function. To test this hypothesis, we first determined the status of p53 in spontaneous tumors developed in the S100A4null animals. Under physiological circumstances, the wild-type p53 protein is detectable by immunohistochemical methods only in solitary cells in the tissues (Greenblatt et al.,
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S100A4(/)
S100A4(+/+)
P-value*
No. of animals
No. with tumors
No. of animals
No. with tumors
10 12 12 40
0 1 0 5
10 10 12 34
0 0 0 0
8–20 21–33 34–46 47–59
— 0.17 — 0.015
*P value was determined using the Wilcoxon rank sum test Table 3 Spectrum of spontaneous tumors in S100A4-null mice Type of the tumor Bronchioalveolar carcinoma Mammary gland carcinoma Hepatocellular carcinoma Fibrosarcoma Lymphoma
No. of tumors
Relative incidence (%)
4 1 1 1 1
50 12.5 12.5 12.5 12.5
1994). An alteration of p53 stabilizes the protein and makes it detectable by immunohistochemical analysis. Detection of p53 protein is regarded as a prognostic marker in a number of human cancers (Greenblatt et al., 1994). Such alterations of p53 are a rare event in spontaneous and chemically induced cancers in rodents (Buzard, 1996; Cazorla et al., 1998; Duddy et al., 1999). Immunohistochemical staining of the spontaneous tumors of the S100A4-null mice (n ¼ 5) revealed p53positive nuclei present in all the tumors analysed (see Figure 2c and d), indicating stabilization of p53 protein. p53-positive nuclei were also revealed with anti-p53 antibodies that specifically recognize a mutant form of p53 protein (clone 240, data not shown). Moreover, spontaneous tumors of the S100A4-null mice expressed high levels of proliferating cell nuclear antigen (PCNA), a negatively regulated target of wild-type p53 (Mercer et al., 1991) (Figure 2e and f). In contrast, expression of p21Waf, a gene that is positively regulated by p53 was not detected in these tumors (Figure 2g and h). Therefore, assay of the activity of p53 responsive genes together with enhanced expression of the p53 protein itself suggests that p53 function is impaired in these tumors. The functional status of p53 was tested by measuring the level of apoptosis in the thymuses of 2- and 7-monthold knockout animals and compared it with the basal level of apoptosis in the wild-type animals. The level of apoptosis was measured by TUNEL assay. We have observed a certain decrease in the level of apoptosis in the thymuses of 2-month-old S100A4/ mice compared to the control, but this difference was not statistically significant (Table 4). Alteration of p53-dependent functions in S100A4/ mice after whole-body g-irradiation Following g-irradiation in vivo, a number of p53regulated genes are strongly activated in a p53-dependent manner (Komarova et al., 2000); p53dependent apoptosis is induced shortly after irradiation
in radiation-sensitive organs (Bouvard et al., 2000). Different responses to whole-body irradiation could reflect changes in the functional status of p53 and can be used as a test for p53 activity in vivo (Komarova et al., 2000). We studied the effect of the whole-body g-irradiation in tissues of S100A4/ and wild-type mice. Normally, Waf/p21/cip1 and bax, p53-regulated genes are induced in response to whole-body g-irradiation in radiationsensitive organs, such as the spleen and thymus (Bouvard et al., 2000). The expression of these genes in mouse organs from irradiated wild-type and null mice was analysed by Northern blot hybridization (Figure 3a). The waf1/p21/cip1 gene was highly induced in the thymus and spleen of both wild-type and knockout animals in response to ionizing radiation; bax was also induced, but to a lesser extent. Notably, the induction of transcription of both genes was less in the spleen of S100A4 knockout animals. Western blot analysis of spleen lysates with anti-p21Waf antibodies confirmed this observation (Figure 3b). The average level of induction of waf1/p21/cip1 and bax was comparable in the thymus, whereas in the spleen of S100A4/ mice it was two times less than in the control (Figure 3c). We also compared the level of apoptosis in the spleen of the animals after irradiation by TUNEL assay. Strong apoptosis was detected in the white pulp of both knockout and the wild-type animals. Virtually all the cells contain apoptotic nuclei making the quantification impossible. Apoptosis was also observed in the red pulp of the spleen and in this case the amount of apoptotic nuclei in the TUNEL assay was quantified (Figure 4a). The amount of apoptotic nuclei in the spleen was approximately two times less in the g-irradiated S100A4/ animals compared to the wild-type controls. In conclusion, evaluation of the results obtained by Northern blot hybridization analysis of p53 target genes and assessment of the level of apoptosis in the spleen of S100A4/ g-irradiated animals supports the hypothesis that p53-dependent functions are altered in these mice. Expression of S100A4 adjacent genes in the S100A4-null animals We studied the possibility of alterations in the expression of neighboring S100 genes in response to germ-line deletion of S100A4-specific sequences. Oncogene
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Figure 2 Tumors in the S100A4/ mice. (a, b) Bronchioalveolar carcinoma in the lung (a) and mammary gland carcinoma (b) H&E ( 20 objective). (c, d) (Immunohistochemical staining of lung carcinoma (c) and mammary gland carcinoma (d) with anti-p53 antibodies ( 40 objective). Note the positive nuclei indicated by arrows. (e, f) Immunohistochemical staining of lung carcinoma (e) and mammary gland carcinoma (f) with anti-PCNA antibodies ( 40 objective). Note the positive nuclei indicated by arrows. (g, h) Immunohistochemical staining of lung carcinoma (g) and mammary gland carcinoma (h) with anti-p21 antibodies ( 40 objective). Note the expression of p21Waf only in some of the stromal cells, indicated by arrows, but not in the tumor cells. Positive nuclei are indicated by arrows
Northern blot hybridization analysis of RNA isolated from different organs of wild-type ( þ / þ ), heterozygous ( þ /) and S100A4-null (/) mice with probes specific for the S100 genes lying in the close vicinity of the S100A4 gene revealed that the expression profile of upstream S100A3 was not changed. The expression of Oncogene
downstream gene, S100A5, although was altered (Figure 5a and b). In the wild-type mice, S100A5 RNA is mostly expressed in certain areas of the brain, but not in the other organs (Schafer et al., 2000). In the RNA extracted from the organs of S100A4-null animal, the S100A5 gene expression was detected in the thymus,
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2-month-old
7-month-old
Wild type S100A4-null
15.874.15* 10.772.88
11.5571.79 9.874.29
*Amount of apoptotic nuclei per field7s.d. (Po0.094)
Figure 4 Comparison of levels of apoptosis (TUNEL assay) in the spleen of S100A4/ and wild-type ( þ / þ ) mice 6 h after girradiation. (a) TUNEL assay in the red pulp of the spleen. Magnification 400. (b) Quantification of the amount of apoptotic nuclei in the red pulp of wild-type and S100A4/ mice. The mean amount of apoptotic nuclei per field (7s.d.) is compared for groups of three animals (Po0.02)
Figure 3 Expression of waf/p21/cip1waf and bax mRNA in S100A4/mouse organs after whole-body g-irradiation. (a) Representative Northern blots hybridized with p21/waf/cip and bax probes. (b) Western blot analysis of whole-cell lysates of spleen from wild-type and knockout mice with probed with anti-p21 antibodies. Anti-a´-tubulin antibodies were used as a loading control. (c) Comparison of normalized mRNA levels in the spleen and thymus of wild-type and S100A4/ mice after irradiation. The mean expression level in the wild-type animals was taken as 100% and compared to the mean value (7s.d.) of expression in the organs of S100A4/ animals
S100A4-null animals. Immunohistochemical staining of tumors of S100A4-null animals with anti-S100A5 antibodies revealed that tumor cells did not express S100A5. We observed single positive cells in the stroma of all the tumors tested (Figure 5b). We also tested whether the S100A5 expression is activated in mouse tumor cell lines and compared it with the expression of S100A3 and S100A4. S100A4 and S100A3 were expressed in a number of mouse tumor cells, but S100A5 was silent in the entire tumor cell lines tested (Table 5). Similar results were obtained both with Northern blot analysis and RT–PCR. These data do not therefore support the assumption of the oncogenic function of S100A5. Discussion
spleen, skin, but absent from the liver (Figure 3b). The profile of the S100A5 expression corresponded to the profile of the S100A4 gene expression in adult wild-type animals (Figure 5a, compare with Figure 1c). This result suggests that inactivation of S100A4 leads to activated expression of neighboring S100A5 gene in some of the cells, normally expressing S100A4 and could partially compensate the S100A4 activity. Activation of S100A5 in the S100A4/ animals raised the possibility that spontaneous tumors detected in these animals might be due to putative oncogenic activity of S100A5. To explore this possibility, we tested S100A5 expression in the spontaneous tumors of
We have inactivated the S100A4 gene in order to clarify its role in mouse development and promotion of metastatic disease. Knockout animal models were applied to study the functions of S100 proteins (Heizmann et al., 2002). While S100B is not essential for life, glial cells from S100B-null mice display an altered Ca2 þ homeostasis (Xiong et al., 2000). S100A1-deficient mice did not display any abnormalities in growth and development under baseline conditions, but demonstrate reduced Ca2 þ -sensitivity and impaired cardiac contractility in response to hemodynamic stress (Du et al., 2002). Oncogene
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Oncogene
a
Mammary carcinoma bLung carcinoma cImmortalized fibroblasts dNorthern blot hybridization analysis eRT–PCR analysis
NB PCR S100A5
+ + + + + + + + + + + + + + + + + + + + + + NB PCR S100A4
+ +
+ +
+ + + + + + + + + + + + + + + + + NBd PCRe S100A3
+
+ +
RAC34E MC MT1TC3 MC MT1TC1 MC 10T 1/2 IFc LL-met LC Line-1 LCb CSML-100 MC CSML-0 MC VMR-liver MC VMR-lymph MCa
S100A9-deficient neutrofils demonstrated altered migratory behavior and did not affect myeloid cell function (Hobbs et al., 2003; Manitz et al., 2003). On the other hand, germ-line inactivation of S100A8 indicates that it is required for embryonic development of the mouse embryo (Passey et al., 1999). Targeted deletion of S100A4 does not largely affect embryonic and postnatal development of the mice. However, misbalance in the ratio of sexes in the progeny of S100A4-null crosses indicates that S100A4 deletion causes some female-related embryonic lethality. Developmental abnormalities leading to a disordered sex ratio were observed in particular in the p53-null mice. A subset of the p53/ female embryos developed defects in neural tube closure and exencephaly (Armstrong et al., 1995; Sah et al., 1995). Another interesting finding is the propensity of S100A4/ mice to develop tumors in different organs. A wide range of tumors of different origin were diagnosed in the S100A4-null animals, which is characteristic of germ-line inactivated tumor suppressor genes (Hakem and Mak, 2001; Meuwissen et al., 2001). This raises the possibility that S100A4 could contribute to tumor suppression. Participation of S100 proteins in tumor suppression was postulated for S100A2, given its downregulation in progressed forms of mammary and lung carcinomas (Feng et al., 2001).
Tumor type
Figure 5 Expression of S100A3 and S100A5 in tissues of S100A4/ mice. (a) Northern blot of total RNA isolated from different organs of þ / þ , þ / and / animals. Hybridization with S100A3and S100A5 probes. Hybridization of the same filter with polyU was used as a loading control. Immunohistostaining of mammary gland carcinoma (b) and bronchioalveolar carcinoma of the lung (c) with anti-S100A5 antibodies. Arrows point to S100A5positive fibroblasts ( 400 objective). (d) Antibody blocking control
Table 5 Expression of S100A3, S100A4 and S100A5 in mouse tumor cell lines
RAC5E MC
RAC10P MC
RAC311C MC
NIH3T3 IF
LL-G2 LC
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Based on the following arguments, we propose here that spontaneous tumor development in S100A4/ mice is a secondary event that emerges because of disruption of S100A4–p53 interaction in the S100A4null mice. Spontaneous tumors in the S100A4/ mice developed with a long latency and only in a part of the animals. This indicates that besides the S100A4 inactivation another event, or events could occur in the cells in order to reveal S100A4 tumor suppressor activity. Staining of tumors developed in the S100A4-null mice revealed accumulation of immunohistochemically detectable p53 protein in the tumor cells. Under physiological circumstances, the wild-type p53 protein is not detected in the cells by immunohistochemistry. Detection of p53 in the nuclei of tumor cells is regarded as an indicator of functionally altered or mutated p53 (Greenblatt et al., 1994). S100A4-null tumors express elevated levels of PCNA and suppressed levels of p21Waf proteins. PCNA is negatively regulated by wild-type p53, whereas the expression of p21Waf is upregulated by active wildtype p53. Detection of elevated levels of p53 together with upregulation of PCNA and downregulation of p21Waf expression in the spontaneous tumors of the S100A4-null mice denotes a functional inactivation of the p53 protein. Immunochemical detection of p53positive cells in human tumors is suggested as a marker for advanced stage of a number of human cancers (Borresen-Dale, 2003; Iacopetta, 2003; Schuijer and Berns, 2003; Staib et al., 2003). In contrast, alteration of p53 status is a rare event in spontaneous and chemically induced cancers in rodents (Buzard, 1996; Cazorla et al., 1998; Duddy et al., 1999). We can propose that in the spontaneous tumors observed in the S100A4/ mice, p53 gene is either mutated or functionally altered. Tumor suppressor protein p53/ homozygous deletion causes early development of lymphomas in mice (Donehower et al., 1992). The heterozygous p53 þ / animals also developed a wide range of spontaneous tumors with a longer average latency (Jacks et al., 1994; Kuperwasser et al., 2000). Analysis of the tumor spectrum in the p53 þ / mice revealed mostly sarcomas; lung carcinomas were also among the tumors generated in the p53 þ / mice (Jacks et al., 1994). The predominant type of tumor developed in the S100A4/ mice was adenocarcinoma of the lung. Long latency, together with a wide spectrum of tumors observed in the S100-null animals, reflects the phenotype of p53 þ / animals. It is proposed that another mutation event (possibly inactivation of the wild-type allele of p53) is necessary for triggering the development of tumors in the p53 þ / mice (Jacks et al., 1994; Kuperwasser et al., 2000). The functional activity of p53 protein in the S100A4/ animals is altered. After whole-body girradiation of the mice, the level of apoptosis and transactivation of p53 target genes in the spleen of S100A4/ mice was suppressed. It has been shown that a number of p53 target genes, including waf/p21/ cip1 and bax, are strongly activated in the spleen of
irradiated mice (Bouvard et al., 2000). Activation of these genes in the irradiated S100A4/ mice was substantially downregulated. Moreover, the amount of cells that undergo apoptosis in the spleen of knockout mice after irradiation was significantly lower than in the controls. p53 plays a major role in determining the radiation response in mouse tissues in vivo (Bourdon et al., 1997; Komarova et al., 2000; Fei et al., 2002). P53 function helps to maintain the genome integrity and is regarded as a basis of the tumor suppressor function of p53 protein (Hanahan and Weinberg, 2000). Carcinogenesis is a complex multistep process, involving a number of events occurring at molecular, cellular and morphological levels. The same molecules may exhibit, depending on the context, opposing functions. The most striking example of such a diverse behavior is E2F1, which can act as an oncogene and tumor suppressor gene (Johnson, 2000). Interaction of wildtype p53 protein with S100A4 leads, as it has been shown, to stimulation of apoptosis in tumor cells (Grigorian et al., 2001). When the S100A4 protein is absent in the cells of knockout animals, the cells damaged by genotoxic stress are not eliminated by apoptosis. This leads to a selection of cells bearing different mutations, and eventually to development of cancer. In developed cancer, activity of S100A4 either in the tumor cells or in the surrounding stroma could activate functions stimulating angiogenesis or invasion of tumor cells thereby stimulating metastasis (Ambartsumian et al., 2001). The important point is that the spontaneous tumors developed in the S100A4 knockout animals were nonmetastatic. The only exception was the lymphoma developed in one of the animals. This fits with the ability of S100A4 to stimulate metastasis in epithelial tumors (Ambartsumian et al., 1996). Germ-line inactivation of the S100A4 gene led to alterations in transcription of one of the neighboring genes in S100 gene cluster – S100A5. Normally, the S100A5 protein is expressed in restricted areas of wildtype mouse brain (Schafer et al., 2000). Expression of S100A5 was recently detected in epidermoid lesions and in the human kidney (Teratani et al., 2002; Pelc et al., 2003). Activation of S100A5 might be a compensatory reaction for S100A4 inactivation in certain cell types. Alternatively, the activation of S100A5 transcription might be a result of deletion of a section of the DNA that either bears a repressor of S100A5, or physically brings the S100A5 gene sequences under the influence of S100A4 enhancer. The spontaneous tumors developed in S100A4-null animals may be a result of unknown oncogenic activity of S100A5. Two observations argue against this proposition. Firstly, S100A5 is not expressed in the spontaneous tumors developed in the S100A4-null mice. Secondly, in the panel of mouse tumor cell lines, we were unable to detect S100A5-specific transcripts. In our opinion, these observations reduce the probability for S100A5 to act as an oncogene. Oncogene
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Besides p53, S100A4 interacts with a number of target proteins, such as nonmuscle myosin or liprinb-1. Disruption of the interaction with these target proteins also could lead to changes in the physiology of the mice, which are not yet revealed. We cannot exclude also that these interactions are compensated by activation of other members of S100 family proteins. Complex studies of S100 family proteins are required to determine the phenotypic outcome of inactivation of single member of this multigenic family.
Materials and methods Generation of S100A4-deficient mice Genomic clones of the S100A4 gene were obtained by screening of a mouse 129/SvJ genomic library with a mouse S100A4 cDNA fragment (Ebralidze et al., 1989). A clone carrying a 10 kb BamH1 fragment containing the entire S100A4 gene and 50 and 30 flanking sequences was selected for generation of a targeting vector. The entire coding part of the S100A4 gene was excised by PCR using primers: 50 -AACTCGAGTGCCATGGTAACCGTTGAGAC-30 and 50 -AACTCGAGAGACTCCTCAGATGAAGTGTT-30 . This generated deletion of the S100A4-coding sequences and introduced the Xho1 recognition sequence. The tk/neo (Mombaerts et al., 1996) cassette was inserted into the generated Xho1 site. The targeting vector was introduced by electroporation into the embryonic stem cell line R1 (The ES cells were kindly provided by Dr A Nagy, Mount Sinai Hospital, Toronto) and selected as described (Mansouri, 1998). Genomic DNA of the selected colonies was screened for homologous recombination by Southern blot hybridization of EcoR1 digested DNA with a probe 1 (Figure 1a). The selection cassette was excised from the clones by transient transfection with PGK-Cre plasmid (kindly provided by Klaus Rajewsky, Institute for Genetics, University of Cologne, Cologne, Germany). Selected colonies were screened for excision of the cassette by Southern blot hybridization of EcoR1 with the probe II (Figure 1a). Clones containing deletion of the S100A4 gene were aggregated with the eight-cell embryos of NMR1 mice following by implantation into pseudo-pregnant females. High degree chimeras were mated with NMR1 mice. Germ-line transmission was identified by Southern blotting of DNA extracted from tail biopsies digested by Sca1. The S100A4/ mice were maintained by intercrossing of littermates. The wildtype littermates of the þ / intercrosses were maintained as a matching control group. Gene expression analysis S100A4, S100A3 and S100A5 expression was assayed by Northern blot hybridization and RT–PCR. S100A3- and S100A5-specific probes were obtained by PCR amplification of genomic sequences corresponding to the exon 2 of S100A3 and exon 3 of S100A5. The primers were 50 -CAGGTGGG CAGCTCCTTCT-30 and 50 -ATGACCCGGCCCCTGGAG CA-30 for S100A3, and 50 -GTAGCCTCTGATCCAAAG-30 and 50 -GGTGGGTTAGCACATCAA-30 for S100A5. S100A4-specific probe was obtained as in Ebralidze et al. (1989). RNA extraction from mouse organs and Northern blot hybridization were performed as described (Ambartsumian et al., 1996). The same filter was reprobed with probes specific Oncogene
to S100A4, S100A3 and S100A5. The above-mentioned primers were used for RT–PCR analysis of RNA extracted from different tumor cell lines. A description of the tumor cell lines used could be found in Christensen et al. (1998). Filters containing RNA extracted from mouse organs after g-irradiation was sequentially hybridized with waf/p21/cip1 and bax-specific probes. The amounts of mRNA on the filters were calibrated by hybridization with [g-32P]ATP- poly(U) probe. Membranes were scanned using Fujifilm FLA-3000 computing densitometer with Image Gauge software. Western blotting and immunoprecipitation were performed as described (Ambartsumian et al., 2001). Rabbit anti-S100A4 antibodies, mouse anti-p21 antibodies (NeoMarkers, USA) and mouse anti-alpha-tubulin antibodies (Sigma, USA) were used. Animal handling Groups of S100A4-null mice (129/SvJxNMR1 background) and genetically matching controls were observed for 12–14 months after birth for possible pathological manifestations. For the whole-body irradiation experiments, S100A4/ mice (A/Sn genetic background) and matching in age A/Sn control mice were used. Animals were maintained and killed according to the FELACA guidelines. Irradiation of mice Mice (8 weeks old) were given whole-body irradiation (8 Gy) from an HF160 Pantak X-ray unit at 1.77 Gy/min (150 kV, 15 mA). Five animals were used per time point. The animals were killed at 0 and 6 h postirradiation and their tissues were analysed for mRNA expression and in situ apoptosis. Histological analyses and immunohistochemical staining Animals killed at the indicated time periods were subjected to macroscopic examination. Tissues were fixed in 10% buffered formalin, embedded in paraffin and analysed by light microscopy and immunohistochemistry. Tumors were classified according to the morphological criteria described by Turusov and Mohr (1994). Tumor tissues were stained with p53 (CM-5, Novocastra Laboratories, Newcastle, UK), PCNA (Dako A/S) and p21 (Neo-Markers, AB9) antibodies followed by biotinylated goat anti-rabbit antibodies and amplification of the signal with the TCA-biotin system (NEN Life Sciences, Boston, USA) according to the protocol of the manufacturer. Immunohistochemical staining with anti-S100A5 rabbit polyclonal antibodies was performed as described (Schafer et al., 2000). The antigen blocking control was used for testing the specificity of the antibodies. TUNEL assay for in situ apoptosis was performed on paraffin-embedded tissues of mice using the ApopTag Peroxidase in situ apoptosis detection kit (Intergen, USA). TUNEL assay was quantified by counting the amount of apoptotic nuclei in 10 randomly selected fields by a blinded investigator. Acknowledgements We thank Ingrid Fossar-Larsen, Birgitte Kaas, Dorrit Lu¨tzh ft and Sharif Mahsur for excellent technical assistance. We also thank Anne-Marie Hansen for the help in the preparation of the manuscript. This work was supported by grants from Danish Cancer Society, Danish Research Council and Dansk Kraeftforsknings Fond.
Spontaneous tumors in the S100A4-null mice C El-Naaman et al
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