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Carcinogenesis Advance Access originally published online on November 4, 2006
Carcinogenesis 2007 28(4):777-784; doi:10.1093/carcin/bgl211
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Tumor prone phenotype of mice deficient in a novel apoptosis-inducing gene, drs

Yukihiro Tambe1,{dagger}, Atsuko Yoshioka-Yamashita1,11,{dagger}, Ken-ichi Mukaisho2, Seiki Haraguchi1,12, Tokuhiro Chano3, Takahiro Isono4, Takao Kawai5, Yasuhiko Suzuki6, Ryoji Kushima7, Takanori Hattori2, Motohito Goto8, Shuichi Yamada9, Makoto Kiso10, Yumiko Saga10 and Hirokazu Inoue1,*

1 Department of Microbiology, Shiga University of Medical Science Setatsukinowa-cho, Otsu, Shiga 520-2192, Japan
2 Department of Pathology, Shiga University of Medical Science Setatsukinowa-cho, Otsu, Shiga 520-2192, Japan
3 Clinical Laboratory Medicine, Shiga University of Medical Science Setatsukinowa-cho, Otsu, Shiga 520-2192, Japan
4 Central Research Laboratory, Shiga University of Medical Science Setatsukinowa-cho, Otsu, Shiga 520-2192, Japan
5 Department of Infectious Diseases, Osaka Prefectural Institute of Public Health Nakamichi, Higashinari-ku, Osaka, Osaka 537-0025, Japan
6 Department of Global Epidemiology, Research Center for Zoonosis Control, Hokkaido University N18, W9, Kita-ku, Sapporo, Hokkaido 060-0818, Japan
7 Division of Diagnostic Pathology, Shiga University of Medical Science Setatsukinowa-cho, Otsu, Shiga 520-2192, Japan
8 SPOC Inc, Reading Venture Plaza 5F 75-1, Ono-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0046, Japan
9 Animal Research Laboratory, Bioscience Education-Research Center, Akita University 1-1-1 Hondo, Akita, Akita 010-8543, Japan
10 Division of Mammalian Development, National Institute of Genetics Yata 1111, Mishima, Shizuoka 411-8540, Japan
11Present address: Moores UCSD Cancer Center, University of California San Diego, School of Medicine 3855 Health Sciences Drive, La Jolla, CA 92093-0820, USA
12Present address: Chiba Cancer Center Research Institute 666-2 Nitona, Chuoh-ku, Chiba 260-717, Japan

*To whom correspondence should be addressed. Tel: +81 77 548 2177; Fax: +81 77 548 2404; Email: hirokazu{at}belle.shiga-med.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The drs gene was originally isolated as a suppressor of v-src transformation. Expression of drs mRNA is markedly downregulated in a variety of human cancer cell lines and tissues, suggesting the potential role of this gene as a tumor suppressor. Previously, we found that Drs protein associates with ASY/Nogo-B/RTN-xS, an apoptosis-inducing protein in the endoplasmic reticulum, and sequentially activates caspases to induce apoptosis in human cancer cells without involvement of the mitochondria. In this study, we investigated the tumor suppressor function of drs and the correlation between Drs-mediated apoptosis and tumor suppression by generating a gene-knockout (KO) mouse. Between 7 and 12 months after birth, malignant tumors including lymphomas, lung adenocarcinomas and hepatomas were generated in about 30% of the drs KO mice, whereas no tumors were found in any of the wild-type mice during the same period of time. drs KO embryonic fibroblasts also showed enhanced sensitivity to transformation by v-src oncogene. Reintroduction of drs into a tumor cell line derived from the tumor of a drs KO mouse led to the suppression of tumor formation in nude mice, which was accompanied by enhanced apoptosis and the activation of caspase-9 and -3. Furthermore, introduction of drs into this cell line enhanced sensitivity to apoptosis mediated by caspase-3, -9 and -12 under low serum culture conditions. The present results thus indicate that drs contributes to the suppression of malignant tumor formation, and this suppression is closely correlated with drs-mediated apoptosis.

Abbreviations: KO, knockout; ER, endoplasmic reticulum.


   Introduction
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 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Apoptosis is closely associated with the development of cancer. Alterations in apoptosis-related genes, including p53, Bcl-2 and Akt, are frequently detected in cancer cells (1–4) and these alterations often make cells resistant to apoptosis. This inhibition of apoptosis may be indispensable for the development of cancer. The drs gene was originally isolated as a novel suppressor gene of v-src transformation (5,6). Although the drs gene is expressed in normal rat fibroblasts, it is completely downregulated in cells transformed by viral oncogenes such as v-src, v-fps, v-K-ras, v-mos and v-abl. The drs cDNA has an open reading frame encoding for a 464 amino acid protein. This protein has one transmembrane domain, a short intracellular domain in the C-terminus and three consensus repeats (CRs) designated as sushi motifs that are conserved in the extracellular domain of the selectin family of adhesion molecules and complement-binding proteins (7–10). The C-terminal region containing the transmembrane domain of drs shares sequence similarity with three repetitive elements of the putative tumor suppressor gene, DRO1, the mRNA expression of which is downregulated by some known oncogenes including ß-catenin, activated H-ras and c-myc (11). DRO1 has been shown to sensitize cells to anoikis and CD95-induced apoptosis. In previous studies, we demonstrated that the expression of drs mRNA is markedly downregulated in a variety of human cancer cell lines and malignant tumor tissues, including those of the colon, bladder, ovary, lung and prostate (12–16). Introduction of the drs cDNA into these cancer cell lines using a retroviral vector was shown to suppress anchorage-independent growth (12). These previous findings suggest that the drs gene acts as a tumor suppressor in various types of human cancer. In addition, we previously found that ectopic expression of the Drs protein induced apoptosis associated with the activation of caspase-12 (like), -9 and -3 in various human cancer cell lines (17). The release of cytochrome c from the mitochondria into the cytoplasm was not observed in the apoptosis induced by drs. Instead, the Drs protein interacted with ASY/Nogo-B/RTN-xS, an apoptosis-inducing protein localized in the endoplasmic reticulum (ER) (18–20). Co-expression of these genes was shown to increase the efficiency of apoptosis. These findings indicate that Drs induces apoptosis via a novel ER-mediated pathway and suggest that this pathway might contribute to the suppression of tumor formation. In order to gain a better understanding of the relationship between Drs-mediated apoptosis and tumor suppression, we generated drs-knockout (KO) mice and analyzed the tumor spectrum of these mice.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Construction of targeting vector and homologous recombination
BAC clones for the mouse drs gene were isolated from a mouse 129/Sv BAC library using the mouse drs cDNA probe. Targeting strategies are shown in Figure 1A. A 5 kb fragment of the 5'-flanking region (long arm) and a 1.5 kb fragment of the 3'-flanking region (short arm) were subcloned into the targeting vector, pgk-neo-pgk DT-A cassette (pgk, phosphoglycerokinase promoter; neo, neomycine-resistant gene; DT-A, diphtheria toxin fragment A). The neo gene and the DT-A gene are driven by the pgk promoter. This vector also contains the lox P sequences at the 5'- and 3'-sides of the neo gene allowing the neo gene to be excised after construction of the KO mouse. The targeting vector was linearized at the NotI site and introduced into R1 ES cells (21) by electroporation. Among the 1203 G418-resistant ES clones examined, four clones, including #349, were detected as homologous recombinants by PCR using the primers p1-2-21 (the 3'-side of the short arm: 5'-GGAAGTGCTCCTGCAAAGCTT-3') and pGK-R (inside the neo-resistant gene: 5'-CTAAAGCGCATGCTCCTCCAGACT-3'). These clones were further verified by Southern blot analysis.


Figure 1
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  		Fig. 1 Targeting strategy and analysis of drs in mice embryo. (A) Schematic representation of targeting strategy. CR, consensus repeat; TM, transmembrane domain; AAA, poly A signal; H, HindIII; Neor, neomycine-resistant gene; DT-A, diphtheria toxin fragment A. (B) RT–PCR-Southern blot analysis of expression of drs mRNA in developing wild-type mouse embryos. Embryo of a drs KO mouse was used as a negative control (NC). (C) Southern blot analysis of drs-deficient mice embryos. Genomic DNA was digested with HindIII, and hybridized with the 5'-probe indicated in A. The upper band represents a 9 kb wild-type (WT) allele, and the lower band represents a neo-deleted KO allele. (D) Northern blot analysis of drs mRNA expression in drs-deficient mice embryos.

 
Generation of drs-deficient mice
The microinjection method was used to generate chimeric mice. A germ-line chimera was obtained using ES clone #349. The chimera mice were crossed with CAG-Cre mice in order to excise the neo gene (22), and these founder mice were bred with C57BL/6 mice. All analyses were performed in this genetic background unless otherwise indicated. Genotyping was performed routinely by PCR using pLA5-1 (3'-region of the long arm): 5'-AGGGAGGCCACAGAAGGGTCC-3' (for the targeted allele), or pmDrsU2 (5'-side of the initiation codon ATG in the first exon): 5'-TTAAGTGAGCTGTGCAGCCT-3' (for the normal allele) and p1-2-21 (3'-side of the short arm): 5'-GGAAGTGCTCCTGAAAGCTT-3'. The CAG-Cre mouse was kindly provided by Dr J. Miyazaki (Osaka University).

Northern blot analysis
Total RNA was isolated from the cells using the SV Total RNA Isolation System (Promega). Samples of 20 µg of total RNA were subjected to electrophoresis on 1% agarose gels containing 1.8% formaldehyde, and then transferred to nylon filters. Filters were then hybridized with 32P-labeled probes at 41°C overnight in hybridizing buffer (50% formamide, 0.6 M sodium chloride, 60 mM sodium citrate, 0.2% SDS, 0.1% bovine serum albumin (BSA), 0.1% Ficoll, 0.1% polyvinylpyrrolidone and 50 µg of herring sperm DNA/ml). The hybridized filters were washed with 15 mM sodium chloride, 1.5 mM sodium citrate and 0.1% SDS at 50°C, and then autoradiographed. The full-length cDNA of mouse drs was used as a probe.

Southern blot analysis
DNA from each sample was digested with restriction enzymes, separated by 1% agarose gel electrophoresis and transferred to a nylon filter. Hybridization and washing were performed as described above for the northern blot analysis.

RT–PCR
Reverse transcription was carried out using total RNAs (2 µg) from normal mouse embryos. Oligo(dT)-adaptor primers (RNA LA PCR kit, Version 1.1, Takara Shuzo Co., Ltd) were used for the reverse transcription reaction. The reverse transcription products were amplified by 22 or 30 PCR cycles using degenerate primers (0.4 µM), TaKaRa LA Taq polymerase (2.5 U), and reverse transcription products in a TaKaRa PCR Model MP Thermal cycler. The primer sets used for PCR in order to detect mouse drs mRNA were 5'-TTAAGTGAGCTGTGCAGCCT-3' for the forward primer (pmDrsU2), and 5'-TAACAGCACATCAGACGTTGC-3' for the reverse primer (pmDrsSR3, this is located in the 3' downstream of the CR 2 of mouse drs). Amplicons were separated on 1.5% agarose gel.

Immunohistochemistry
Mice were sacrificed using an overdose of diethyl ether. Resected tumors and tissues were fixed in 10% formaldehyde in PBS (–) for 4 h, and the tissues were embedded in paraffin. Serial sections (4 µm) were sliced, and the sections were stained with hematoxylin and eosin. Immunohistochemical methods were used to classify the lymphomas that developed in the drs KO mice. Dewaxed sections of the lymphomas were examined using antibodies against anti-mouse CD3 (T cell marker) or anti-mouse CD79a (B cell marker) using the streptavidin–biotin–peroxidase method (Histofine MAX-POMULTI, Nichirei, Tokyo).

Cell cultures
The cell lines used in this study were 293T (a human embryonic kidney cell line expressing the E1 gene of adenovirus type 5 and the T antigen of SV40) and LC-T1 (a mouse lung cancer cell line derived from a lung adenocarcinoma generated in a drs-deficient mouse). MEFs lacking drs (–/Y), or normal MEFs (+/Y) were prepared from male embryos resulting from the mating of drs heterozygous (+/–) female and wild-type (+/Y) male mice. WT-LT and KO-LT cells were established by transfection of the large T gene of SV40 (pSV-Large T) to immortalize wild-type MEFs and drs KO MEFs, respectively. All cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS).

Construction of recombinant retrovirus and virus infection
Mouse drs cDNA (mDRS-1) or v-src cDNA was cloned into a pCXbsr retroviral vector containing murine retrovirus LTRs and the blasticidin-resistant gene, used as a selection marker (23,24). The recombinant plasmids were prepared using a Qiagen column (Qiagen). The pCXbsr vector carrying the mouse drs cDNA was transfected into 293T cells with the helper plasmid pCL-Ampho (25) using Lipofectamine-Plus reagent (Invitrogen). In the case of the pCXbsr retrovirus carrying the v-src gene, the recombinant plasmid was transfected into 293T cells with the helper plasmid pCL-Eco (25). The amphotropic and ecotropic viruses in the culture medium were collected 48 h after transfection, and the virus titer was assayed via its introduction into F2408 (a rat fibroblast cell line) and then by selection with blasticidin. For the viral infection, 2 x 105 cells were plated onto 60 mm dishes and were cultured overnight at 37°C. After polybrene treatment (2 µg/ml) for 30 min, the viruses were added to the cultures, which were then incubated for 1 h at 37°C. After 7 days of incubation in selection medium (blasticidin 2 µg/ml), the blasticidin-resistant colonies were pooled and used for assays.

Soft agar assay
To assess the colony formation in soft agar, 1 x 104 cells were inoculated into 0.5% agar containing DMEM supplemented with 10% FCS in a 60 mm dish. After 2 weeks of incubation, the number of large colonies (over 0.125 mm in diameter) in each plate was scored. Each cell line was tested in duplicate dishes.

Tumorigenicity assay
The tumorigenicity of the cancer cells was determined by subcutaneously injecting 5 x 106 cells in a 0.2 ml volume into 5-week-old BALB/c-nu/nu female nude mice. The tumorigenic potential was evaluated at 4 weeks post-injection. The tumors were fixed in 10% formaldehyde in PBS (–) for 4 h, and the tissues were embedded in paraffin.

Apoptosis assay
The characterization and quantification of apoptosis was performed using the Annexin-PI assay. Cells were stained with the MEBCYTO-Apoptosis kit (MBL) and were analyzed with FACScan (BD) following the manufacturer's protocol. For the detection of apoptotic cells in tumor tissues, terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assays were performed using the Apoptosis in situ Detection Kit (Wako pure chemical industries) according to the manufacturer's protocol.

Immunoblotting
Cells were lysed in Laemli-SDS buffer containing 62.5 mM Tris–HCl (pH 6.8), 10% glycerol, 5% 2-mercaptoethanol, 2% SDS, 0.01% bromophenol blue, 5 mM EDTA and 1 mM orthovanadate. Cell lysates were subjected to SDS–PAGE, and the separated proteins were transferred to PVDF membranes (Immobilon-P, Millipore). Membranes were blocked with TBS-T (10 mM Tris–HCl, pH 7.6, 150 mM sodium chloride, 0.1% Tween-20) containing 5% BSA, and then incubated with primary antibody for up to 14 h in TBS-T containing 2% BSA. The membranes were then washed in TBS-T and incubated for 1 h in horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibody (Amersham International) diluted 1:20 000 in TBS-T containing 2% BSA. After several washes with TBS-T, the immunoreactivity was detected by the ECL system (Amersham International) using the procedures recommended by the manufacturer.

Antibodies
Anti-caspase-3 (#9622, polyclonal), anti-caspase-9 (#9504, polyclonal) and anti-cleaved caspase-9 (#9505, polyclonal) antibodies were purchased from Cell Signaling Technology. Anti-caspase-12 polyclonal antibody was purchased from Medical and Biological Laboratories (MBL). Anti-cleaved caspase-3 (Ab-4, monoclonal) antibody was purchased from Oncogene Research. Anti-{alpha}-tubulin (DM1A, monoclonal) antibody was purchased from SIGMA. Anti-Ki67 antibody was purchased from Novocastle Laboratories. Anti-SV40 large T antigen monoclonal antibody (clone PAb101) was purchased from BD Bioscience. Anti-avian Src monoclonal antibody (clone EC10) was purchased from Upstate Biotechnology.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Tumor formation in drs-deficient mice
To investigate the role of the drs gene in the induction of apoptosis and to determine its role as tumor suppressor in vivo, we isolated a genomic clone of mouse drs, including the first exon, and generated drs KO mice. We have previously isolated two types of mouse drs cDNA, mDRS-1 and mDRS-2; the former is the full-length form containing three CRs, and the latter is a splicing variant lacking CR1 (Figure 1A) (23). The mDRS-1 cDNA has the ability to suppress anchorage-independent growth of human cancer cell lines whereas mDRS-2 does not, suggesting that mouse drs is also able to function as a tumor suppressor and that the presence of CR1 is critical for the suppression of the malignant phenotype of cancer cells by drs. Both types of mRNA were found to be expressed in most normal mouse tissues including heart, lung, stomach, intestine, kidney, ovary, uterus, testis, spleen, muscle, brain and liver. However, the amount of mDRS-2 mRNA was shown to be very low compared to that of mDRS-1 mRNA in these tissues (23). With regards to the development of the mouse embryo, the expression of drs mRNA was detected on day 10.5, and levels of expression were found to increase gradually during development (Figure 1B). As shown in Figure 1A, we constructed a targeting vector in order to disrupt the first exon of the mouse drs gene, which in turn abolished the expression of both types of drs mRNA and thereby generated drs KO mice. The genotypes of the drs KO mice were determined by PCR and verified by Southern blot analysis (Figure 1C). As the drs gene is located on the X chromosome in both humans and mice, wild-type (+/Y) and KO (–/Y) mice were generated in males, while in females, wild-type (+/+), heterozygous (+/–) and KO (–/–) mice were generated. As shown in Figure 1D, the expression of drs mRNA was completely lost in KO mice (–/Y and –/–), whereas the expression level of drs mRNA in heterozygous mice (+/–) was about half that of wild-type mice (+/Y and +/+). All genotypes of mice were born according to Mendelian genetics. While drs-deficient mice were viable and showed no significant abnormality six months after birth, ~30% (14/46) of these mice developed malignant tumors (six lymphomas, four lung adenocarcinomas, three hepatomas and one sarcoma) between 7 and 12 months (Table I and Figure 2A, I, K and M). Lymphomas were also detected in 10% (2/20) of the heterozygous mice during the same period of time. No tumors were found in any of the 23 wild-type mice examined up to 12 months after birth. In the six drs-deficient mice with lymphomas, tumors of the mesenteric lymph nodes were found in the intestine (Figure 2A) and the spleen was enlarged in comparison to those of age-matched wild-type mice (Figure 2E). In addition, the growth of atypical lymphocytes was detected in the mesenteric lymph node (Figure 2B), kidneys (Figure 2C), liver (Figure 2D) and spleen (Figure 2F). Immunostaining with antibodies for markers of T cells (CD3) and B cells (CD79a) showed that these lymphomas were derived from T cells (Figure 2G and H). Pictures representative of the histology of other tumor types (lung adenocarcinoma, hepatoma and sarcoma) generated in drs-deficient mice are shown in Figure 2I–N. These results indicate that the loss of the drs gene promotes the genesis of malignant tumors in vivo.


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  		Table I Tumor formation in drs-deficient mice

 


Figure 2
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  		Fig. 2 Malignant tumor generation in drs-deficient mice. Representative photographs (A, E, I, K and M), histological section (HE staining; B–D, F, J, L and N), and immunohistochemical staining for CD3 antibody (T cell marker, G) and CD79a antibody (B cell marker, H). Metastatic lymphoma in mesenteric lymph nodes (A and B), kidney (C), liver (D) and spleen (E–H). Lung adenocarcinoma (I and J). Hepatoma (K and L). Sarcoma (M and N). Note that the arrowheads indicate the tumor tissues (A, I, K and M). White scale bars, 10 mm; black scale bars, 50 µm.

 
Enhanced sensitivity of drs KO embryonic fibroblasts to transformation by viral oncogenes
To investigate whether the loss of drs gene increases the sensitivity to in vitro transformation by viral oncogenes, we established the immortalized cells, WT-LT and KO-LT, by introducing the large T antigen of SV40 into wild-type and drs KO embryonic fibroblasts, respectively. We then tested the anchorage-independent growth of these cells after infection with a retrovirus containing the v-src oncogene. As shown in Figure 3A and B, KO-LT cells produced a small number of colonies (0.5%) in soft agar, whereas WT-LT cells hardly produced any colonies (0.03%). Primary embryonic fibroblasts from WT and drs KO mice did not produce any colonies in soft agar (data not shown). In addition, KO-LT cells infected with a virus expressing v-src (KO-LT/Src) also produced more colonies in soft agar (8.45%) than WT-LT cells infected with v-src (3.63%). The colonies formed by KO-LT/Src cells in soft agar were also markedly larger than those formed by WT-LT/Src cells (Figure 3A). Immunoblot analyses indicated that the v-src protein was expressed similarly in both WT-LT/Src and KO-LT/Src cells and that T antigen was expressed similarly in all cells infected with T antigen regardless of src expression (Figure 3C). These results indicate that drs-deficient cells are more sensitive to transformation by viral oncogenes, suggesting that the drs gene acts to suppress transformation.


Figure 3
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  		Fig. 3 The sensitivity of drs KO embryonic fibroblasts to transformation by viral oncogenes. (A) Pictures of colonies in soft agar. (B) The colony forming efficiency of WT-LT/pCX, WT-LT/Src, KO-LT/pCX and KO-LT/Src cells in soft agar. *P < 0.05, **P < 0.01 by Welch's t-test. (C) Immunoblotting for the large T antigen of SV40 and v-Src protein in WT-LT/pCX, WT-LT/Src, KO-LT/pCX and KO-LT/Src cells.

 
Reintroduction of drs suppresses tumorigenesis and enhances caspase-mediated apoptosis in drs-KO lung cancer cells
To confirm that drs can function as a tumor suppressor in vivo, we established a tumor cell line, LC-T1, from an adenocarcinoma of a drs-deficient mouse and then reintroduced the wild-type drs gene (mDRS-1) into this cell line using a retroviral vector. The expression of drs mRNA from the retrovirus containing drs was confirmed by northern blot analysis (Figure 4A). Immunofluorescent experiments using a retrovirus expressing Flag-tagged Drs indicated that the Drs protein is primarily localized in the ER of LC-T1 cells, as the staining from an antibody to the Flag epitope merges with an antibody to BiP (an ER marker protein) (Figure 4B). Under normal serum (10%) culture conditions, the in vitro proliferation of LC-T1 cells expressing wild-type drs (LC-T1 mDrs) was almost identical to that of LC-T1 cells infected with the empty viral vector (LC-T1 pCX) (Figure 4C). However, under low serum (0.2%) culture conditions, the number of apoptotic cells increased among LC-T1 mDrs cells, as compared to LC-T1 pCX cells (Figure 4D). The amount of the cleaved forms of caspase-3 (18 kD), -9 (39 kD) and -12 (20 kD) also increased in LC-T1 mDrs cells under 0.2% serum culture conditions (Figure 4E). The intermediate of caspase-12 (45 kD) prominently increased at 24 h, probably because the activation of caspase-12 precedes to the processing of caspase-9 and -3. Treatment with a pan-caspase inhibitor, z-VAD-fmk, led to the suppression of this type of apoptosis (Figure 4D), indicating that the apoptosis of LC-T1 cells under low-concentration serum conditions is mediated by the activation of these caspases. These results suggest that drs has the ability to confer sensitivity to this ER-mediated apoptosis induced by low concentrations of serum in this drs-deficient tumor cell line.


Figure 4
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  		Fig. 4 Effects of the reintroduction of the drs gene on the growth and apoptotic properties of a lung cancer cell line (LC-T1) derived from a lung adenocarcinoma of a drs-deficient mouse. (A) Reintroduction of the drs gene in LC-T1 cells. Northern blot analysis of endogenous and viral drs mRNA. The expression of G3PDH mRNA served as an internal control. (B) The expression and subcellular localization of the Drs protein. Flag-tagged Drs (left) and BiP (middle, an ER marker) are colocalized in the ER (right, merged). (C) Growth curves of LC-T1 pCX and LC-T1 mDrs cells in 10, 1 and 0.2% FCS medium. *P < 0.05 by Welch's t-test. (D) Apoptosis of LC-T1 pCX and LC-T1 mDrs cells cultured under normal (10%) or low (0.2%) FCS conditions. Annexin-PI assay was conducted at 48 h after the inoculation of each cell type under each culture condition. A pan-inhibitor of caspases (z-VAD-fmk, 2 µM) was added to medium containing 0.2% FCS when the culture conditions were altered. ***P < 0.001 by Welch's t-test. (E) Immunoblotting for activated caspase-3, -9 and -12 in LC-T1 pCX and LC-T1 mDrs cells during apoptosis induced by low (0.2%) FCS conditions. The expression of {alpha}-tubulin served as an internal control.

 
To investigate whether the drs gene is responsible for the suppression of malignant tumor formation in vivo, we injected LC-T1 pCX and LC-T1 mDrs cells subcutaneously into nude mice and compared the growth of tumors. As shown in Figure 5A, the growth of LC-T1 tumors was significantly suppressed by the reintroduction of the wild-type drs gene. Histological analysis of these tumors indicated that apoptotic cells containing piknotic chromatin condensation, nuclear blebbing or the presence of apoptotic bodies, which could be distinguished from mitotic cells, were significantly increased in LC-T1 mDrs tumors when compared to LC-T1 pCX tumors (Figure 5B and D). TUNEL analysis also indicated that the number of apoptotic cells significantly increased in LC-T1 mDrs tumors (Figure 5B and D). Furthermore, activation of caspase-3 and -9 was evaluated by immunohistochemistry to define the apoptotic cells in the tumor tissues (Figure 5C). The number of cells with active caspase-3 and -9 was markedly increased in the LC-T1 mDrs tumors compared to LC-T1 pCX tumors (Figure 5D). Immunostaining for Ki-67 also indicated that cell proliferation in vivo was moderately decreased in LC-T1 mDrs tumors when compared to LC-T1 pCX tumors (Figure 5C and D). These results indicate that drs is able to suppress in vivo tumor growth and stimulate apoptosis mediated by the activation of caspase-3 and -9. Taken together, these results suggest that the suppression of malignant tumor formation in drs-deficient mice is closely correlated with drs-mediated apoptosis.


Figure 5
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  		Fig. 5 Effect of drs on the growth of LC-T1 cell tumors in nude mice. (A) (Upper) Representative photographs of tumors produced by LC-T1 pCX and LC-T1 mDrs cells. Tumors were dissected from nude mice at 4 weeks after the injection of malignant cells. The scale bar indicates 10 mm. (Lower) Average weights of five tumors induced by LC-T1 pCX and LC-T1 mDrs. ***P < 0.001 by Welch's t-test. (B) HE staining and TUNEL assay of dissected LC-T1 pCX and LC-T1 mDrs tumors. Apoptotic nuclei (black) and mitotic nuclei (white) are indicated by arrowheads. The scale bar indicates 0.1 mm. (C) Comparison of immunostaining with an antibody for Ki67 (left), an antibody for the cleaved active form of caspase-3 (middle), and an antibody for the cleaved active form of caspase-9 (right) in LC-T1 pCX and LC-T1 mDrs tumors. The scale bar indicates 0.1 mm. (D) Percentage of apoptotic nuclei observed with HE staining, TUNEL-positive cells, Ki67-positive cells, cleaved caspase-3-positive cells and cleaved caspase-9-positive cells in the image of each of the tumors. *P < 0.05, **P < 0.01 by Welch's t-test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this study, we generated Drs KO mice to demonstrate for the first time that the drs gene plays a role as a tumor suppressor. Using a tumor cell line, LC-T1, derived from a lung adenocarcinoma of a drs KO mouse, we also demonstrated that the reintroduction of the drs gene suppresses the tumorigenicity and enhances caspase-mediated apoptosis of this type of tumor cell in nude mice. Furthermore, retroviral expression of drs sensitizes drs-deficient tumor cells to caspase-mediated apoptosis under low serum culture conditions. These findings strongly suggest that drs-mediated apoptosis is closely correlated with the suppression of malignant tumor formation. The malfunction of apoptosis is well known to be closely associated with the development of cancer (26) and alterations of apoptosis-related genes are frequently detected in cancer cells. p53, an apoptosis-inducing gene, has been shown to be inactivated in a variety of cancers (1). Bcl-2, an anti-apoptotic gene, has been found to be overexpressed in B cell follicular lymphoma and other malignancies (2). Akt, another anti-apoptotic gene, was first identified as a transforming gene of retroviruses (3), and perturbations of the Akt signaling pathway have been reported in a variety of human cancers (4). Expression of drs mRNA has also been shown to be downregulated in rat cells transformed by various viral oncogenes such as v-src and v-K-ras (5,6) and a variety of human cancer cell lines and malignant tissue types (12–16). Experiments using a temperature-sensitive mutant of the v-src gene indicated that downregulation of drs mRNA was dependent on the expression of a functional v-Src kinase (6). Furthermore, serum stimulation of starved cells and other retroviral oncogenes such as v-abl, v-fps, v-mos and v-sis also induced downregulation of drs mRNA (5,6). These findings suggest that mitogenic signals are necessary to downregulate expression of drs. Neither gross deletion nor rearrangement of the drs gene was detected by Southern blot analyses in any of the human cancer cell lines and tissues with downregulated drs (12,13,15,16). Downregulation of drs mRNA in human cancers might be caused by epigenetic mechanisms including DNA methylation, or effect of the activation of cellular oncogenes, such as c-src and c-ras. However, we cannot completely exclude the possibility that minor mutations in the drs gene cause the downregulation of drs.

About 30% of drs KO mice developed malignant tumors between 7 and 12 months after birth. Oncogenesis involves a series of genetic alterations, including the activation of dominant oncogenes and the inactivation of tumor suppressor genes. Inactivation of drs in human cancers by downregulation of drs mRNA can be considered to be one of many alterations that can lead to tumorigenesis. The genetic loss of the drs gene in drs KO mice may not cause malignant tumors on its own, but may increase the sensitivity to tumor formation caused by inactivation of other tumor suppressor genes or activation of other oncogenes. In the 70% of drs KO mice, that did not develop tumors during the 12 months after birth, the accumulation of other genetic changes might not be enough to produce malignant tumors. Experiments with drs KO embryonic fibroblasts also support this hypothesis. As shown in Figure 3, drs KO embryonic fibroblasts showed enhanced sensitivity to transformation by viral oncogenes, whereas primary drs KO embryonic fibroblasts did not show any transformed phenotype. The increased anchorage independent growth of KO-LT/Src might be due to the loss of anti-mitogenic activity in addition to pro-apoptotic activity of drs. We have previously reported that retroviral introduction of drs into human cells suppressed the anchorage independent growth (12). The reduced Ki-67 staining in LC-T1 mDrs cells (Figure 5) may be also due to anti-mitogenic effect of drs in the tumor tissue, in which the anchorage was lost.

The main pathway of p53-induced apoptosis involves the efflux of cytochrome c from the mitochondria into the cytoplasm, which results in the activation of caspase-9 and -3 (27). Lymphomas are also frequently generated in p53 KO mice (28–33). In contrast to p53, drs associates with another apoptosis-inducing protein, ASY/Nogo-B/RTN-xS (18–20), which is localized in the ER and activates caspase-12, -9 and -3 to induce apoptosis without the involvement of p53 and the mitochondrial pathway, as reported in a previous paper (17). This mechanism is consistent with the localization of the Drs protein to the ER and the activation of caspase-12, -9 and -3 in LC-T1 cells infected with a retrovirus containing drs, as shown in Figure 4. p53-mediated apoptosis is thought to be involved in the exclusion of precancerous cells, the DNA of which has been damaged by ultraviolet light, ionized radiation, and DNA-damaging reagents (34), although the p53-mediated responses by DNA damage is not necessarily correlated with tumor suppression (35,36). Drs-mediated apoptosis differs from mitochondria-mediated apoptosis by p53. Drs may thus exclude emerging malignant tumor cells in the process of the progression from benign tumors to malignant tumors. This idea is consistent with findings showing that the downregulation of the drs gene occurs primarily at later stages of tumorigenesis (13–16).

Retroviral reintroduction of drs into a drs KO tumor cell line enhanced sensitivity to apoptosis accompanied with the activation of caspase-12, -9 and -3 under low serum culture conditions and suppressed in vivo tumorigenicity in nude mice. In tumors from LC-T1 mDrs cells, the number of apoptotic cells that were associated with the activation of caspase-9 and -3 was significantly increased, in comparison with those from LC-T1 cells containing vector, although we could not examine the activation of caspase-12 because the antibody specific for a cleaved form of caspase-12 was not available. When tumors grow in vivo, the nutrients, growth factors and oxygen that are required to develop large solid tumors become limited in supply. This causes many tumor cells to die by apoptosis induced by hypoxia and the depletion of growth factors and nutrients. Drs may play a role in the induction of apoptosis due to such environmental stresses. One of the functions of the ER is to sense cellular physiological changes. We consider that drs induces apoptosis in response to such stresses. The increased apoptosis seen in LC-T1 mDrs cells under low serum conditions in vitro might reflect the apoptotic cell death caused by stresses such as growth factor depletion in vivo. Rescuing tumor cells from apoptosis caused by such stresses is considered to be a critical step in the development of malignant tumors.

The Drs-binding protein, ASY/Nogo-B/RTN-xS, was shown to interact with both Bcl-xL and Bcl-2 in the ER and to reduce their respective anti-apoptotic activities (20), suggesting that Drs induces apoptosis by interacting with these proteins in the ER. Recent studies have shown that pro-apoptotic and anti-apoptotic Bcl-2 family proteins can localize on the ER as well as in the mitochondria, and that they are able to regulate apoptosis induced by ER stress or by alterations in Ca2+ homeostasis (37–41). Further investigations will be necessary to clarify the physiological role(s) played by drs in the induction of apoptosis in the ER and in the upstream signaling pathways leading to apoptosis. Drs KO mice and drs KO cells are expected to provide potent tools for the resolution of such issues. Our present findings suggest that a drs-mediated apoptosis pathway that differs from the known mitochondrial pathway is involved in the suppression of malignant tumor development. Elucidation of an ER-mediated pathway of drs-induced apoptosis will provide new insights into the mechanisms of carcinogenesis and contribute toward developing a new anticancer therapy.


   Footnotes
 
{dagger}These authors contributed equally to this work. Back


   Acknowledgments
 
We would like to thank Hiroko Kita (Department of Microbiology, Shiga University of Medical Science) and Takefumi Yamamoto (Central Research Laboratory, Shiga University of Medical Science) for their technical assistance. We are also grateful to Scott Stuart, Michelle V. Wagner and Dr Naohisa Yoshioka (University of California San Diego) for their helpful comments on the manuscript and Dr Hideki Kato (Hamamatsu University School of Medicine), Dr Hiroki Kokubo (National Institute of Genetics), Dr Masahito Ikawa (Osaka University) and Dr Masaru Okabe (Osaka University) for their technical advice. This project was supported by a Grant-in Aid for Scientific Research (C) (Grant No. 15590335 and No. 17590341), a Grant-in-Aid for Scientific Research on Priority Areas (C) (Grant No. 16021222) from the Ministry of Education, Science, Sports, and Culture of Japan (H.I.), and a grant from Japan Society for the Promotion of Science Research Fellowships for Young Scientists (A.Y.). This work was also supported by a grant from the NOVARTIS Foundation (Japan) for the Promotion of Science (H.I.) and a grant from the Charitable Trust Oosaka Cancer Researcher-Fund (A.Y.).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 24, 2006; revised October 21, 2006; accepted October 24, 2006.


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