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. 2003 May 15;17(10):1201–1206. doi: 10.1101/gad.1088003

Mice lacking a transcriptional corepressor Tob are predisposed to cancer

Yutaka Yoshida 1,8, Takahisa Nakamura 1,8, Masato Komoda 4, Hitoshi Satoh 2, Toru Suzuki 1, Junko K Tsuzuku 1, Takashi Miyasaka 1, Eri H Yoshida 1, Hisashi Umemori 1, Reiko K Kunisaki 3, Kenzaburo Tani 4, Shunsuke Ishii 5, Shigeo Mori 2, Masami Suganuma 4, Tetsuo Noda 6,7, Tadashi Yamamoto 1,9
PMCID: PMC196063  PMID: 12756225

Abstract

tob is a member of antiproliferative family genes. Mice lacking tob are prone to spontaneous formation of tumors. The occurrence rate of diethylnitrosamine-induced liver tumors is higher in tob−/− mice than in wild-type mice. tob−/−p53−/− mice show accelerated tumor formation in comparison with single null mice. Expression of cyclin D1 mRNA is increased in the absence of Tob and is reduced by Tob. Tob acts as a transcriptional corepressor and suppresses the cyclin D1 promoter activity through an interaction with histone deacetylase. Levels of tob mRNA are often decreased in human cancers, implicating tob in cancer development.

Keywords: Tob, liver cancer, corepressor, cyclin D1 expression

Supplemental material is available at http://www.genesdev.org.

There is accumulating evidence that genes involved in the negative control of cell growth can function as tumor suppressors. In humans, tob, tob2, ana, pc3b, btg1, and btg2 comprise a family (tob family) of antiproliferative genes (Bradbury et al. 1991; Fletcher et al. 1991; Rouault et al. 1992; Matsuda et al. 1996; Guehenneux et al. 1997; Yoshida et al. 1998; Ikematsu et al. 1999; Buanne et al. 2000). Exogenous expression of Tob family proteins suppresses growth of NIH-3T3 cells by inhibiting G1 progression of the cell cycle (Yoshida et al. 1998; Ikematsu et al. 1999; Guardavaccaro et al. 2000; Maekawa et al. 2002; Suzuki et al. 2002). We showed previously that Tob is a substrate of Erk MAPK, and unphosphorylated Tob suppresses cell-cycle entry of quiescent cells. Erk phosphorylation of Tob blocks the antiproliferative activity (Maekawa et al. 2002; Suzuki et al. 2002), which, at least in part, describes the importance of Erk activation in the cells stimulated by growth factors. When Tob is depleted, Cyclin D1 continues to be expressed and readily progress into S phase during serum starvation (Suzuki et al. 2002). In addition, the antiproliferative activity of Tob is impaired in the presence of exogenously coexpressed Cyclin D1 (Suzuki et al. 2002). These data suggest that tob functions as a tumor suppressor. However, possible involvement of Tob in tumorigenesis and roles of Tob in the control of cyclin D1 expression are unclear.

Tob family proteins associate with transcription factors. Virtually all of the Tob family members interact with Caf1 (Rouault et al. 1998; Ikematsu et al. 1999; Yoshida et al. 2001), whose yeast homolog is a component of the CCR4–NOT transcriptional complex (Albert et al. 2000). The CCR4–NOT complex participates in the control of specific sets of genes such as those involved in the late mitotic phase of the cell cycle (Liu et al. 1997). Both BTG1 and BTG2 associate with HoxB9 and estrogen receptor α, and modulate their transcription activity (Prevot et al. 2000, 2001). Tob associates with Smads transcription complex and affects Smad-mediated gene expression (Yoshida et al. 2000; Tzachanis et al. 2001). This suggests that Tob family proteins are regulators of gene transcription, functioning as either coactivators or corepressors.

Here, we report that mice lacking tob are prone to spontaneous formation of tumors in various tissues. Intriguingly, we find that levels of tob mRNA are often decreased in human cancers. We further show that Tob is a transcriptional corepressor and suppresses the promoter activity of genes, such as cyclin D1, relevant to cell growth control through an interaction with histone deacetylase.

Results and Discussion

Tumor development in mice lacking tob

tob−/− mice show no apparent phenotypic abnormalities in their early lives except development of osteopetrotic phenotype (Yoshida et al. 2000). Therefore, we conducted a long-term study of spontaneous tumor development in wild-type and tob−/− mice. By 18 mo of age, 16% (5/31) of wild-type mice developed tumors, primarily malignant lymphomas and lung adenomas, whereas 77% (20/26) of tob−/− mice had developed a variety of tumors, including hemangiosarcomas, lung carcinomas, and hepatocellular adenomas. The spectrum of tumors observed in tob−/− mice between 6 and 22 mo is shown in Table 1. Typical histopathological findings for some of these tumors are shown in Figure 1A–I. Despite extensive systematic studies of tumor suppressor genes, there are presently no mouse models of increased susceptibility to liver tumors. Because tob−/− mice often developed liver tumors, we further assessed tumor susceptibility of tob−/− mice by treatment with a liver-specific carcinogen, diethylnitrosamine (DEN). Intraperitoneal injection of DEN into 2-week-old tob−/− mice led to earlier onset of liver tumors; at 6–9 mo after DEN administration, liver tumors were observed more frequently in tob−/− mice than in wild-type mice (Fig. 1J,K). These findings suggest that tob−/− mice are predisposed to cancer and may serve as a model of liver cancer.

Table 1.

Spontaneous tumors in tob−/− mice

Animal
Sex/age (wk)
Site
Histology
 1 M/52 Liver Hepatocellular adenoma
 2 M/99 Liver Hepatocellular adenoma
 3 M/95 Liver Hepatocellular adenoma
 4 M/78 Liver Hepatocellular adenoma
 5 M/95 Liver Hepatocellular adenoma
Lung Adenoma
 6 M/26 Lymph node Malignant lymphoma
 7 M/78 Lymph node Malignant lymphoma
 8 M/78 Lymph node Malignant lymphoma
 9 M/86 Lymph node Malignant lymphoma
Lung Adenoma
10 M/52 Liver Hepatocellular adenoma
Lung Carcinoma
11 M/52 Liver Hepatocellular adenoma
Lung Carcinoma
12 M/72 Liver Hepatocellular adenoma
13 M/68 Liver Hepatoblastoma
14 F/78 Subcutis Hemangioma
Lung Adenoma
15 F/78 Pancreas Hemangiosarcoma
16 F/43 Subcutis Hemangiosarcoma
17 F/73 Liver Hemangiosarcoma
18 F/86 Liver Hemangioma
19 F/40 Liver Hepatocellular adenoma
20 F/78 Lymph node Malignant lymphoma
21 F/95 Lymph node Malignant lymphoma
22 F/60 Liver Hepatocellular adenoma
23 F/24 Pancreas Acinar cell carcinoma
Lymph node/Liver/Kidney Malignant lymphoma
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Figure 1.

Figure 1

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Development of a spontaneous tumor in tob−/− mice and DEN-induced liver tumors in wild-type and tob−/− mice. Images of a hemangioma in subcutis (A,B), a hemangiosarcoma in the pancreas (C,D), a hepatocellular adenoma (E), an acinar cell carcinoma in the pancreas (F), a lung carcinoma (G), a malignant lymphoma in a lymph node (H), and a malignant lymphoma in the pancreas (I) that were observed in tob−/− mice at the ages of 6 mo (F–I), 12 mo (G), and 18 mo (A–E). Panels B and D are high-magnification images of the histopathological sections in A and C, respectively. (J,K) Percentage of mice with DEN-induced liver tumors. Wild-type and tob−/− mice are shown in open and closed boxes, respectively. Bars: B,E–I, 0.2 mm; C, 3 mm; D,H, 0.1 mm.

Because mutations in the p53 tumor suppressor gene are the most frequently observed genetic lesions in human cancers, we investigated the relation between tob and p53 in tumorigenesis by generating mice carrying null mutations of both genes. Eight percent (3/39) of tob−/− mice and 59% (17/29) of p53−/− mice showed tumor development within 6 mo, but the tumor incidence increased to 81% (30/37) in tob−/−p53−/− mice. The difference between tob−/− mice and tob−/−p53−/− mice was statistically significant (χ2 test, p < 0.05). As in p53−/− mice, malignant lymphomas were found most frequently (80%; 24 of 30 tumors) in tob−/−p53−/− mice. However, several pathological changes, such as a glioblastoma, were uniquely observed in tob−/−p53−/− mice (see Supplemental Material). These data suggest that tob and p53 contribute synergistically to tumor suppression.

Growth aberration of tob−/− MEFs

Primary mouse embryonic fibroblasts (MEFs) of tob−/− animals were morphologically indistinguishable from wild-type MEFs and grew at a rate similar to that of wild-type MEFs. Upon successive passages with the defined 3T3 protocols, wild-type and tob−/− MEFs initially underwent approximately four population doublings with each passage. Growth virtually ceased around passages 5–7 due to senescence crisis (Fig. 2A). tob−/− MEFs weathered the senescence crisis around passage 10, whereas wild-type MEFs did so after passage 15. Furthermore, established tob−/− MEFs had a shorter doubling time than did wild-type cells (Fig. 2B). The saturation densities of the established tob−/− MEF lines were higher than those of wild-type cells, suggesting that contact inhibition of cell growth was hampered, at least in part, in the absence of Tob. MEFs lacking a tumor suppressor gene, such as p19ARF, proliferate continuously and never undergo an obvious senescence crisis (Harvey et al. 1993; Kamijo et al. 1997). Secondary genetic alterations might have occurred in the established tob−/− MEF lines, and the Tob deficiency could contribute to such alterations. Supportingly, tob−/− MEFs at passage 4 displayed an increased number of chromosome aberrations (χ2 test, p < 0.005; Fig. 2C). The number of chromosome aberrations in DEN-treated MEFs were increased (approximately twofold) in the absence of Tob (Fig. 2D). MEFs lacking DNA repair genes XRCC4 and Ku80 show marked genomic instability (Difilippantonio et al. 2000; Gao et al. 2000). Because expression of tob is induced in response to DNA damage, such as that caused by adriamycin treatment or γ-irradiation exposure (Cortes et al. 2000), Tob may contribute to genome stability.

Figure 2.

Figure 2

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Characterization of tob−/− MEFs. (A) Cell proliferation on a 3T3 protocol. At 3-d intervals, the total numbers of cells per culture were determined prior to dilution of the cells to 7.5 × 105 cells per 10-cm dish for repassage. (B) Growth properties of established tob−/− cells. Cells from wild-type and tob−/− mice at passage 20 were seeded at 1 × 105 cells per culture in 6-cm dishes. Duplicate dishes were harvested at daily intervals, and the total numbers of cells per culture were determined. (C) Typical chromosome aberrations found in tob−/− MEFs at passage 4. (Left) A representative chromosome spread from tob−/− MEFs. chromatid gap (ctg; top, left), chromosome gap (csg; top, right), chromatid break (ctb; bottom, left), and chromatid exchange, quadriradial (cte, qr; bottom, right). (D) Percentage of cells with chromosome aberrations. Wild-type and tob−/− MEFs at passage 4 were treated with or without DEN, and each metaphase spread was assessed for the frequency of chromosome abnormalities. The total number of cells counted were as follows: Untreated, 624 for wild-type and 726 for tob−/− MEFs; DEN-treated, 365 for wild-type and 372 for tob−/− MEFs.

Involvement of Tob in regulation of cyclin D1 transcription

Tob family proteins are involved in transcriptional regulation. To identify target genes whose transcription might be regulated by Tob, we examined transcripts that were affected by exogenous Tob expression (gain of function) and by depletion of Tob (loss of function). Microarray analyses of ∼500 cancer-related genes revealed that expression of several genes appeared to be regulated either directly or indirectly by Tob (data not shown). The genes whose expression was suppressed to approximately half or less by gain-of-function and induced more than twofold by loss-of-function include the cyclin D1, E2F5, RalA, and RalBP1 genes.

The cyclin D1 gene is relevant to G1 progression, and expression of the gene is often abrogated in human tumors (Prober and Edgar 2001). Because partial hepatectomy provides an in vivo model for the study of G0 progression, RNAs prepared from partially hepatectomized liver of 10-week-old tob−/− and wild-type mice were analyzed for cyclin D1 expression. As shown in Figure 3A, expression of cyclin D1 mRNA in both untreated and partially hepatectomized liver was increased in the absence of Tob, suggesting that Tob suppresses cyclin D1 expression in both resting and growing cells. The level of cyclin D1 mRNA was reduced in 293T cells that overexpress Tob (Fig. 3B). These observations are consistent with our previous findings that significant levels of Cyclin D1 are present in serum-starved tob−/− MEFs, and the levels are reduced by re-expression of Tob (Suzuki et al. 2002). Luciferase assay with a reporter plasmid containing the promoter region of the cyclin D1 gene (Matsumura et al. 1999) revealed that overexpression of Tob suppressed activity of the cyclin D1 promoter (Fig. 3C). Interestingly, the Tob-mediated repression of transcription from the cyclin D1 promoter was reduced significantly by increasing concentrations of trichostatin A (TSA), an inhibitor of HDAC activity (Fig. 3D). The results suggested that HDAC is involved in Tob-mediated repression of transcription. Anti-Flag immunocomplexes prepared from lysates of Flag-Tob-transfected cells contained significantly higher HDAC activities than those from control cells (Fig. 3E), indicating that Tob complexes contain HDAC proteins. The interaction of Tob with HDAC1 was confirmed by coimmunoprecipitation experiments with COS7 cells transfected with the 6Myc-Tob construct and an expression plasmid encoding Flag-HDAC1 protein (Fig. 3F). Therefore, overexpression of Tob could suppress transcription of the cyclin D1 gene via recruitment of HDAC1. Analysis of the cyclin D1 promoter by chromatin immunoprecipitation assay showed that lower levels of the acetylated histones, acetyl H3 and acetyl H4, were associated with the promoter in Tob-transfected HeLa cells than in control cells (Fig. 3G). Again, the data suggest that Tob recruits HDAC to the cyclin D1 promoter region. Analysis of a series of deletion mutants of the cyclin D1 promoter suggested that the Tob/HDAC complex acted through several cis-acting elements in the promoter region (data not shown). In addition, Tob is likely to be involved in suppression of expression of other genes such as RalA, E2F5 (see above), and IL-2 (Tzachanis et al. 2001), as well as the genes regulated by Smad downstream of bone morphogenetic protein (Yoshida et al. 2000). Taken together, these data indicate that Tob may recruit HDAC to different transcription factors.

Figure 3.

Figure 3

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Inhibition of cyclin D1 promoter activity by Tob. (A) Increased expression of cyclin D1 mRNA in the absence of Tob. RNAs were prepared from liver cells of wild-type and tob−/− mice before (Liver) and 3.5 h after partial hepatectomy (PH3.5). (B) Suppression of cyclin D1 mRNA by Tob. RNAs were prepared from 293T cells transfected with pME18S (vector) or pMETob-Flag (Tob) and incubated for 24 h. In A and B, RNAs were subjected to Northern blot hybridization with the cyclin D1 cDNA probe. (C) Inhibitory effect of Tob on cyclin D1 promoter activity. HeLa cells were transfected with increasing amounts (0.025–0.25 μg DNA/well in 12-well tissue culture plates) of pMETob-Flag together with pRL-TK and –1745-CD1-Luc. As a control, pMEβ-galactosidase was used instead of pMETob-Flag. After 24 h, the cells were subjected to luciferase assays with a Dual-Luciferase Reporter System. (D) Effect of TSA on Tob-mediated inhibition of cyclin D1 promoter activity. HeLa cells were transfected with pMETob-Flag, pRL-TK, and –1745-CD1-Luc. Six hours after transfection, TSA was added at the indicated concentration, incubated for another 18 h, and luciferase activity was measured. (E) Tob is associated with histone deacetylase activity. The 293T cells were transfected with pME18S or pMETob-Flag. Twenty-four hours after transfection, proteins in the lysates were immunoprecipitated with anti-Flag antibody. The HDAC activity in immunocomplexes was measured as described in the Materials and Methods. (F) Interaction between Tob and HDAC1. COS-7 cells were transfected with Flag-HDAC1 and 6Myc-Tob plasmids. The interaction between the two proteins was examined by immunoprecipitation (IP) followed by immunoblotting (Blot). (Top) Interaction. (Bottom) Expression of each indicated protein. (G) ChIP analysis of the cyclin D1 promoter. Chromatin preparations from HeLa cells or HeLa cells stably transfected with pMETob-Flag were subjected to ChIP assay with anti-acetyl H3 (top panel) and anti-acetyl H4 (middle panel) antibodies. DNAs in the immunoprecipitates were PCR amplified with the primers shown in the illustration of the promoter region. DNAs from equal amounts of the chromatin preparations were also PCR amplified before immunoprecipitation (Input).

Suppression of tob in human cancers

Analysis of more than 50 human tumors did not reveal any point mutations or gross aberrations of the tob gene. In contrast, reverse transcriptase PCR (RT–PCR) analysis revealed that the level of tob mRNA was decreased to 4.7%–87.3% (mean, 30.1%) of the normal level in 13 of 18 human lung cancers (Table 2). The decrease was not related to the type of lung cancer. The data suggest that suppression of Tob expression contributes to tumor progression. It is theoretically possible that Tob expression was suppressed in the lung tumors as a consequence of growth stimulation. However, levels of tob transcript in an EBC1 human lung tumor cell line, in which the level of endogenous tob mRNA expression is low, were increased upon treatment of the cells with 5-aza-2‘-deoxycytidine and/or TSA (data not shown). Further analysis of the methylation and acetylation of the tob promoter might clarify this issue.

Table 2.

Reduced expression of tob mRNA in human lung cancers

Type
Sex/age (years)
Percent of tob mRNAa
Squamous cell carcinoma Male/60 8.2
Squamous cell carcinoma Male/72 23.1
Squamous cell carcinoma Male/78 96.0
Large cell carcinoma Male/57 9.0
Adenocarcinoma Male/77 28.4
Adenocarcinoma Male/76 10.5
Adenocarcinoma Female/43 29.6
Adenocarcinoma Female/66 39.8
Adenocarcinoma Female/67 100.4
Adenocarcinoma Male/53 65.9
Adenocarcinoma Male/65 4.7
Adenocarcinoma Male/52 104.2
Adenocarcinoma Male/51 87.3
Adenocarcinoma Male/74 384.4
Adenocarcinoma Female/77 26.5
Adenocarcinoma Male/80 131.5
Adenocarcinoma Female/53 28.2
Adenocarcinoma Male/71 30.6
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a

Levels of mRNAs from tumor and normal tissues of each patient were compared by RT–PCR. 

Conclusion

Tob family proteins associate with transcription factors that could interact with HDAC (Yoshida et al. 2000; Prevot et al. 2001) and protein arginine methyl transferase that could regulate transcription through methylation of histones (Tirone 2001), suggesting that the Tob family proteins function as transcriptional coregulators. Consistently, we show here that Tob negatively regulates the cyclin D1 gene by recruiting HDAC to cyclin D1 promoter. Amplification and overexpression of cyclin D1 have been reported in various human tumors, including non-small-cell lung carcinomas, and hepatocellular carcinomas (Betticher et al. 1996; Joo et al. 2001). Recent evidence suggests that overexpression of Cyclin D1 is an early, causative event in hepatocarcinogenesis (Joo et al. 2001). A constitutive enhancement of cyclin D1 expression observed in tob−/− mice may lead to development of cancers. In addition, increased susceptibility to the alkylating agent DEN of tob−/− mice and tob−/− MEF strongly suggests that Tob contributes to genome stability in vivo. Because tob expression is often decreased in human cancers (Table 2; M. Komoda, M. Suganuma, K. Iwanaga, N. Sueoka, E. Sueoka, T. Suzuki, Y. Yoshida, and T. Yamamoto, unpubl.), depletion and/or epigenetic suppression of tob may contribute to development of human cancers.

Materials and methods

DEN treatment of mice

Mice were maintained under standard specific-pathogen-free conditions. On day 15 after birth, offspring received a single intraperitoneal injection of DEN (20 μg/g body weight) in PBS. DEN-treated and untreated mice were sacrificed at 6 or 9 mo, and were analyzed for macroscopically visible tumors. Experiments with animals were carried out following guidelines for animal use issued by the Committee of Animal Experiments, Institute of Medical Science, University of Tokyo.

Generation of tob−/−p53−/− mice

To generate tob−/−p53−/− mice, tob+/−p53+/− mice were crossed. Wild-type, p53−/−, and tob−/− mice of the same background were used for comparisons. These mice were maintained in a hybrid C57BL/6J/129 SV background (75%/25%, respectively).

Cells and culture

MEFs were obtained from 14.5-day-old embryos by an established procedure (Todaro and Green 1963). MEFs, HeLa cells, and 293T cells were maintained in DMEM containing 10% FBS, 50 μM β-mercaptoethanol, and antibiotics. Growth rates of the cells at the twentieth passage were determined by plating triplicate cultures of 1 × 105 cells in 60-mm dishes. DEN (1 mg/mL) treatment was performed for 48 h after the third passage. The DEN-treated cells were washed and then cultured for another 12 h in fresh medium without DEN for the cytogenetic examination. EBC1 cells of a human lung cancer cell line (American Type Culture Collection) were cultured in RPMI 1640, supplemented with 10% FBS and antibiotics.

Chromosome analysis

MEFs, after four passages, were exposed to colcemid (0.02 μg/mL) for 2 h. Mitotic chromosome spreads were prepared by standard procedures and stained with 4‘,6‘-diamidino-2-phenylindole. At least 300 metaphase spreads were subjected to the analysis.

Northern blot analysis

Total RNAs were isolated with ISOGEN (Nippon Gene) per the manufacturer’s instructions. Total RNAs were prepared from mouse livers before and 3.5 h after partial hepatectomy and from 293T cells transfected with pME18S (Ikematsu et al. 1999) or pMETob-Flag (Tob; Yoshida et al. 2000). RNA samples (20 μg) were subjected to Northern blot hybridization using cyclin D1 cDNA labeled with [α-32P]dCTP by random priming as described previously (Ikematsu et al. 1999).

Transient transfection and reporter gene assays

HeLa cells were transfected with various combinations of the following plasmids by lipofection: pME18S, pMETob-Flag, pRL-TK, and –1745-CD1-Luc (Matsumura et al. 1999). Forty-eight hours after transfection, cell extracts were analyzed for luciferase activity with a Dual-Luciferase Reporter System (Promega). Transfection efficiency was standardized with an internal control plasmid, pRL-TK.

Immunoprecipitation and immunoblotting

Immunoprecipitation and immunoblotting were performed as described previously (Yoshida et al. 2000). Antibodies used for blotting were anti-Myc monoclonal antibody (Santa Cruz Biotechnology), anti-Flag monoclonal antibody (Sigma), and anti-Tob antibodies (Matsuda et al. 1996).

DNA microarray analysis

Recombinant adenovirus vectors, Ad-Tob and Ad-LacZ, were constructed using homologous recombination between the expression cosmid and the parental virus genome as described (Miyake et al. 1996). EBC1 cells, which show a low level of Tob expression (Yanagie et al. 1998), were infected with the adenovirus vectors (5–30 × 109 pfu/mL) at MOI = 30 for 1 h. RNAs were prepared from the infected cells and from wild-type and tob−/− MEFs. The RNAs were subjected to DNA microarray analysis using compact-sized DNA array filter (Okuno et al. 2001; GeneticLab, Sapporo).

HDAC assay

HDAC activity was measured as described (Nomura et al. 1999). Lysates were prepared from 293T cells transfected with pMETob-Flag or empty pME18S vector and were immunoprecipitated with anti-Flag antibody. Immunocomplexes were incubated for 5 h at 37°C with 1500 cpm of acid-soluble 3H-labeled histones.

ChIP assay

HeLa cells stably transfected with pMETob-Flag and parental cells were fixed with 1% HCHO. After fixation, chromatins were prepared from the cell lysates and subjected to ChIP assay (Upstate Biotechnology). The average size of the DNA fragments was ∼300 bp. The same amount of chromatin was used for immunoprecipitations with specific antibodies. The presence of the cyclin D1 promoter was analyzed by quantitative PCR with the promoter specific primer pair 5′-GGCGATTTGCATTTCTATGA-3′ (forward) and 5′-CAAAACTCCCCTGTAGTCCGT-3′ (reverse).

RT–PCR

RNAs were prepared from human lung cancers and subjected to semiquantitative RT–PCR. Briefly, the cDNAs were amplified by PCR using specific primers for tob (F, 5′-CACAGGATCTTAGTGTTTGGATCGA-3′; R, 5′-TTCTTCATTTTGGTAGAGCCGAACT-3′) for 24 cycles and for actin (F, 5′-CAAGAGATGGCCACGGCTGCT-3′; R, 5′-TCCTTCT GCATCCTGTCGGCA-3′) for 19 cycles at 94°C for 30 sec, 55°C for 30 sec, and 72°C for 60 sec in the presence of [α-32P]dCTP. PCR products were analyzed by 5% polyacrylamide gel electrophoresis. Radioactivity of PCR product was determined by BAS 2000 Bioimage Analyzer (Fuji Photo Film). Expression of actin mRNA was used as a control. Informed consent was obtained from all patients. Experiments with human materials were carried out following guidelines issued by the Human Science Ethical Committee, Institute of Medical Science, University of Tokyo.

Acknowledgments

We thank R. Ajima, M. Watanabe, N. Ikematsu, H. Nishimura, H. Toki, M Ohsugi, T. Tezuka, Y. Yamanashi, S. Matsuda, J. Inoue, T. Nomura, and C. Sherr for critical discussions; I. Matsumura and N. Kanakura for providing us with the cyclin D1 promoter constructs; M. Katsuki and K Nakao for providing us with p53−/− mice; and K. Nomura for technical advice. We also thank H. Fujiki for discussion and encouragement. This work was supported by a grant for Advanced Cancer Research from the Ministry of Education, Science, Sports and Culture of Japan, and grants from the Organization for Pharmaceutical Safety and Research of Japan, and from the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.

Footnotes

E-MAIL tyamamot@ims.u-tokyo.ac.jp; FAX 81-3-5449-5413.

Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1088003.

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