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[Cancer Research 65, 10716-10724, December 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

Identification and Characterization of Survival-Related Gene, a Novel Cell Survival Gene Controlling Apoptosis and Tumorigenesis

Zeng-Rong Yuan1,2, Ruoxiang Wang2, Jennifer Solomon2, Xunyi Luo2, Hong Sun2, Liying Zhang1,2 and Yufang Shi1,2

1 Department of Molecular Genetics, Microbiology and Immunology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey and 2 Department of Immunology, Holland Laboratory of Biomedical Research, American Red Cross, Rockville, Maryland

Requests for reprints: Yufang Shi, Department of Molecular Genetics, Microbiology and Immunology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 661 Hoes Lane, Piscataway, NJ 08854. Phone: 732-235-4501; Fax: 732-235-4505; E-mail: shiyu{at}umdnj.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Apoptosis plays a critical role in cellular homeostasis during development, immune responses, and tumorigenesis. Recent studies have identified a number of genes that control this process. We report here our identification of a novel cell survival-related gene (SRG) from a human expression cDNA library by functional cloning. SRG shows no significant nucleotide sequence homology to any known genes in the Genbank. Our fluorescence in situ hybridization analysis has estimated that SRG is located at 1p36, agreeing with the location at 1p36.22 in the human genome sequence. SRG encodes a putative protein of 172 amino acids, which is mainly located in the perinuclear region. Northern blotting analysis indicates that SRG is highly expressed in many human cancer cell lines although it is low in most tissues except liver and placenta. To investigate the function of SRG in apoptosis, we transfected SRG cDNA into BAF/BO3 and B16/F0 cells and induced apoptosis by cytokine/serum deprivation. We found that SRG-transfected cells are resistant to apoptosis induced by cytokine/serum deprivation. In addition, mice bearing SRG-transfected melanoma had more tumor formation and larger tumor growth. Melanoma transfected with antisense SRG showed significantly less tumor formation and smaller tumor growth. Interestingly, mouse SRG gene was also identified on chromosome 4 and blocking SRG expression with small interfering RNA promoted serum deprivation–induced apoptosis of NIH3T3 cells. Our results show that SRG is a novel cell survival gene that critically controls apoptosis and tumor formation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Apoptosis is considered to be an evolutionarily conserved "suicide" program present in all nucleated metazoan cells (1, 2). It plays a critical role in cellular homeostasis during development, immune responses, and pathophysiologic processes. Apoptosis is now known to be an active cell death process characterized by activation of proteases, autodestruction of chromatin, nuclear condensation, cytoplasmic membrane blebbing, and vesicularization of internal components (3). It is responsible for the widespread and deliberate elimination of excessive cells during various physiologic and pathogenic processes. Apoptosis is strictly controlled by specific genes that are sequentially expressed at different phases of this highly programmed process. Interference with the activity of the genes controlling apoptosis will result in an imbalance between cell death and survival, thus leading to pathologic abnormalities (4–6). Aberrant regulation of apoptosis has been observed to occur in the etiology of some human cancers and the progression of some degenerative diseases.

Among all the factors contributing to cancer formation, increased cell survival and decreased apoptosis are the most critical alterations in cancer cells. In addition, except for surgical procedures, most cancer treatment strategies rely on the induction of apoptosis in cancer cells. Deregulated expression of genes that control cell survival and apoptosis is often seen in cancer cells. Recent investigations have revealed that various oncogenes and tumor suppressor genes exert their tumorigenic effects largely through modulating the apoptosis and cell proliferation processes. In addition, activation of the antiapoptotic signal transduction pathways mediated by autocrine production of cytokines is also believed to contribute to tumorigenesis and the development of drug resistance by cancer cells. Therefore, understanding molecular mechanisms that controls apoptosis in cancer cells is fundamental for understanding tumorigenesis and designing effective therapies (7–11).

Mitogenic signals generated by the interaction of cytokines with their cognate receptors induce entry into S phase of the cell cycle. Continuous cell proliferation requires coordinated activation of cell cycle genes, inhibition of tumor suppressor genes, and bypass of the programmed cell death pathway. As withdrawal of cytokines, such as interleukin (IL)-2, IL-3, erythropoietin, and nerve growth factor, results in apoptosis in cytokine-dependent cells, it has been suggested that cytokines directly provide antiapoptotic signals. Alternatively, cytokine-promoted cell survival may be exerted indirectly by cooperating with antiapoptotic signals provided by serum or other factors. Based on our observations (12–15), we have proposed a model to account for the balance between cell proliferation and apoptosis. Cell survival signals could be provided by cytokines and other factors in the cellular microenvironment. Without these factors, proliferating cells undergo apoptosis (16, 17). Indeed, we found that when stimulated with IL-3 in the absence of serum, BAF/BO3 cells proliferate and at the same time undergo apoptosis (12). This "abortive cell cycle" and factor withdrawal–induced apoptosis can be distinguished by the phase of the cell cycle at which cells die. As a corollary, cells undergoing apoptosis resulting from abortive cell cycle are actively cycling. In contrast, when cells enter factor withdrawal–induced apoptosis, their growth is arrested (12, 18, 19). This is best represented by IL-3-dependent 32D cells. In this cell, IL-3 withdrawal leads to growth arrest and G1 accumulation followed by apoptosis. Constitutive expression of c-myc inhibits G1 arrest and accelerates apoptosis (20–22).

We have investigated the relationship between cell proliferation and apoptosis by activating cells with mitogenic cytokines and growth factors in the presence or absence of serum. When stimulated with cytokines or growth factors in the absence of serum, several cell types underwent apoptosis. Interestingly, serum induces expression of cell survival genes, such as bcl-2, in this system, whereas mitogenic cytokines induce expression of proapoptotic genes, including bax and c-myc, in the absence of serum. By functional cloning, we have identified a novel cell survival-related gene (SRG). This gene is overexpressed in many cancer cell lines, in the liver, as well as in activated lymphocytes. Significant antiapoptotic activity of the SRG was seen in cells upon serum deprivation. On the other hand, suppression of SRG expression by antisense promoted cell death. Strikingly, blocking SRG expression in mouse melanoma cells results in much smaller or no tumor development in vivo compared with the SRG-transfected or control-transfected melonoma cells. Our results show that the SRG gene is a novel cell survival gene that plays a critical role in cell apoptosis and tumorigenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of cDNA expression library. We constructed a human cDNA library with mRNA isolated from human heart. The cDNA was cloned unidirectionally into the ZAP expression vector (Stratagene, La Jolla, CA) carrying the neomycin selection mark. Briefly, unidirectional cDNA was synthesized with hybrid oligo(dT) linker primer and EcoRI adapter. The cDNA fragment was ligated into ZAP expression vector. The ligation was then packaged to form a primary human ZAP expression library. We amplified the primary library, tested phage titer, and finally constructed a large, stable quantity of a high-titer stock of the library. For gene transfection, pBK-CMV phagemid containing the cloned inserts representing all clones was isolated from the ZAP expression vector by in vivo mass autoexcision.

Functional cloning. The SRG gene was identified by functional cloning. We transfected the human cDNA library into BAF/BO3 cells by electroporation. The transfected cells were cultured in RPMI 1640 supplemented with IL-3 in the absence of serum for 5 days. Surviving cells were further selected by G418 at 1,000 µg/mL for another 5 days. Genomic DNA was extracted from surviving BAF/BO3 cells by the DNeasy mini kit (Qiagen, Valencia, CA). PCR was done to amplify the transfected gene using a pair of primer flanking both arms of the pBK-CMV vector. The PCR fragments were cloned into the pUC18 vector and sequenced using an ABI automatic sequencer. The nucleotide sequence was further confirmed by manual sequencing according to established protocol (23). One sequence was found to contain a complete open reading frame. No homology was found to any known human gene in Genbank, except some sequences of expressed sequence tags (EST). This gene was named SRG.

Gene mapping. Fluorescence in situ hybridization (FISH) was done to map the chromosomal location of the SRG gene. This was done by SeeDNA Biotech, Inc. (Windsor, ON, Canada). Briefly, phytohemagglutinin (PHA)-activated human T cells were used for chromosome preparation. SRG cDNA was used as a probe according to established protocols. 4',6-Diamidino-2-phenylindole (DAPI) banding was used to identify the specific chromosome. The assignment between signals from the probe and the short arm of chromosome 1 was obtained. The detailed position was further determined based on the summary from 10 photos.

Northern blotting analysis and densitometric analysis. Human tissue RNA membrane, which included total RNA of 12 different human tissues, was purchased from OriGene Technologies, Inc. (Rockville, MD). Human cancer RNA samples were also isolated from cancer cell lines. Human endothelial cells were used as a control. Total RNA was extracted by Tripure Reagent (Roche, Indianapolis, IN). Twenty micrograms of total RNA of each sample were separated on 1% agarose/2.2 mol/L formaldehyde denaturing gel and transferred onto a Nytran membrane (Schleicher & Schuell, Inc., Keene, NH). SRG cDNA was used as a probe after labeling with [32P]dCTP by random priming (Roche) according to the instructions of the manufacturer. Prehybridization and hybridization were done at 42°C in a solution containing 5x SSC (10x SSC is 1.5 mol/L NaCl and 0.15 mol/L sodium citrate), 2.5 mmol/L EDTA, 0.1% SDS, 5x Denhardt's solution, 2 mmol/L sodium PPi, 50 mmol/L sodium phosphate, and 50% formamide. Membranes were washed with 0.2x SSC, 0.1% SDS at 56°C for 1 hour, and hybridization signals were detected by autoradiography. The same membranes were rehybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal quantitative control.

Densitometric analysis was done by using Kodak Image Station 440 with the Kodak 1D 3.5 software (Kodak, Rochester, NY). The density of SRG band was determined by the value of SRG divided by the density of corresponding GAPDH. The relative density of SRG was calculated by using the expression level in the brain tissue or endothelial cells, respectively, as 1.0; the fold increase in various samples was assigned accordingly.

Immunochemical staining. Human breast carcinoma cell line, MCF-7, and human breast epithelial cell line, MCF-10A, were cultured in MEM supplemented with 1 mmol/L sodium pyruvate, 0.01 mg/mL bovine insulin, and 10% fetal bovine serum (FBS) in Nunc Lab-Tak chambers precoated with human fibronectin (50 µg/mL). When grown to semiconfluence, cells were fixed with fresh 4% paraformaldehyde in PBS for 10 minutes then treated with 0.5% Triton X-100 in PBS for 5 minutes at room temperature. The cell-coated slide was stained with primary preimmune serum or rabbit anti-SRG polyclonal antibody (made in our laboratory) followed by secondary FITC-conjugated goat anti-rabbit IgG antibody (BD Science, San Diego, CA) in blocking buffer composed of 5% goat serum in PBS for 1 hour in a humid atmosphere. After washing twice with PBS, the slide was mounted with ProLong antifade reagent (Molecular Probes, Eugene, OR) and covered with a glass coverslip. Localization of SRG protein was detected using a Nikon inverted fluorescence microscope (Eclipse TE 2000-S, Nikon, Cedar Knolls, NJ).

Apoptosis induction and survival-related gene overexpression. BAF/BO3 cells, from a mouse B cell line, were transfected with sense SRG in pcDNA3.1 or the empty vector by electroporation. Stably transfected cell clones were selected by G418 cloning followed by serial dilution and PCR analysis. The vector and SRG-transfected BAF/BO3 cells were cultured in 24-well plate at 2 x 105 cells per well in RPMI 1640 in the presence or absence of IL-3 at 200 units/mL and 10% FBS as indicated. After 24 and 48 hours, the cells were harvested and washed twice with PBS. Cell pellets were resuspended in staining buffer (PBS with 20 µg/mL propidium iodide, 0.2% saponin, and 50 µg/mL RNase) and incubated at room temperature for 30 minutes. DNA content was analyzed by flow cytometry using a FACScan (Becton Dickinson, San Diego, CA) with fluorescence signals collected using a linear scale. The percentage of cells with sub-G0-G1 DNA content was enumerated as apoptotic cells.

B16/F0 cells, mouse melanoma cell line, were separately transfected with sense or antisense SGR or pcDNA3.1 vector alone using SuperFect transfection reagent following the protocol of the manufacturer (Qiagen). Transfected cells were selected using G418. Surviving cells were cloned and further cultured in complete DMEM medium with G418 then subjected to serum deprivation for 1 week. Apoptosis was analyzed by morphologic alteration under light microscopy and by DNA content analysis.

Mouse model for tumor formation. B16/F0 clones transfected with SRG cDNA in different orientations were expanded in complete DMEM medium. Each mouse was injected with 1 x 106 cells in 100 µL saline solution. In one study, mice were injected i.v. through tail vein. In the other study, mice were injected the B16/F0 and the vector-transfected B16/F0 i.m. into the outside of each thigh in one group or injected the SRG sense– and antisense–transfected B16/F0, respectively. The mice were observed daily. Mice were euthanized when tumor began to significantly affect the mobility. For pathohistologic analysis, the mice were perfused with 10% formamide in PBS. The melanoma tumors were examined by morphologic and pathohistologic analyses.

Small interfering RNA. Small interfering RNA corresponding to mouse SRG homologue on chromosome 4 was selected using the following sequence: CTCCAGTTTAGATGGTCCTGG. The whole siRNA cassette consists of human U6 promoter and terminator sequence flanking the insert encoding a hairpin of mouse SRG siRNA. Mouse fibroblast cell line, NIH3T3, was transfected with SRG siRNA by SuperFect reagent following the protocol of the manufacturer (Qiagen). The cells were then subjected to serum deprivation. Apoptosis was analyzed by light microscopy and DNA content.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Functional cloning of survival-related gene. To functionally clone genes that are capable of regulating cell survival, we constructed a human heart cDNA expression library by unidirectional cloning into the ZAP expression vector carrying the neomycin selection marker. Phagemid DNA representing all the clones in the library was isolated by autoexcision, then transfected into the murine prepro-B cell line, BAF/BO3. The BAF/BO3 cells are IL-3-dependent and quickly undergo apoptosis upon deprivation of IL-3. These cells also undergo IL-3-driven apoptosis when serum is not present in the culture medium, a phenomenon we called cytokine-promoted apoptosis. Thus, the transfected cells were stimulated with IL-3 under cytokine-promoted apoptosis condition for 5 days. After further selection with G418, surviving cells were cloned and their transfected genes were isolated from genomic DNA by PCR with primers derived from sequences flanking the phagemid arms. One of the isolated clones, a 1,390 bp sequence with a poly(A) tail, was named SRG. It contains one open reading frame encoding a protein of 172 amino acids (Fig. 1A). To verify the presence of this mRNA species in human cells, we used SRG cDNA to screen two human libraries (Stratagene) and found several clones. Sequence analysis of these clones further confirmed the presence of these unrecognized genes. We did extensive searches against known genes and ESTs in Genbank. Although we found no homologous genes, we did find several EST sequences identical to SRG, further confirming the existence of SRG as a transcribed gene. It was only recently that a predicted open reading frame (accession no. XM_514368) of the chimpanzee (Pan troglodytes) genome was identified with 97% homology to the human SRG sequence.



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  		Figure 1. Complete sequence of human SRG and its chromosomal localization. A, the SRG gene was identified by functional cloning. BAF/B03 cells were transfected with a human expression cDNA library and subjected to IL-3-induced apoptosis in the absence of serum. Cells were further selected by G418. The expressed transfected cDNA was isolated. The complete cDNA SRG contains 1,390 nucleotides with a poly(A) tail. Its open reading frame encodes a putative protein of 172 amino acids. B, SRG gene chromosomal localization was determined by FISH. PHA-activated human T cells were used for chromosome preparation. SRG cDNA was used as a probe. DAPI banding was used to identify the specific chromosome. The detailed position was further determined based on the summary from 10 photos. There is a single location at 1p36.

 
The chromosome localization of SRG was determined on PHA-activated human lymphocytes by FISH with the probe of SRG cDNA. Among 100 mitotic figures, 45 of them showed hybridization signals on one pair of chromosomes. The assignment between signals from the probe and the short arm of chromosome 1 was obtained by DAPI banding analysis. The detailed position was further determined based on the summary from 10 photos (Fig. 1B). The gene was mapped to chromosome1p36. Blast of the human genome database further confirmed that the SRG gene is located on chromosome1p36.22, a chromosome site believed to be associated with cancer development (24, 25).

Expression of survival-related gene in human tissues and human cancer cell lines. We studied SRG expression in human tissues and human cancer cell lines. The SRG transcript was detected by Northern blot analysis. Among 12 human tissues analyzed, only liver and placenta showed high SRG expression; the expression of SRG was very low in other human tissues (Fig. 2A). Increased SRG expression was also observed in human peripheral lymphocytes upon activation. In addition, many cancer cells generally have high SRG expression: Among 18 cancer cell lines, 15 of them were highly positive (Fig. 2B). However, SRG expression was low in prostate cancer cells and HeLa cell (epithelial carcinoma cell). These results showed that SRG is expressed mainly in cancer and actively proliferating cells, suggesting a role for cancer development.



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  		Figure 2. Expression of SRG in human normal tissues and cancer cell lines. SRG expression was detected in normal tissues (A) and human cancer cell lines (B) by Northern blot analysis. GAPDH expression was used as a loading control. The level of expression was determined by densitometric analysis. The density of SRG band was determined by the value of SRG divided by the density of corresponding GAPDH. The relative density of SRG was calculated by using the expression level in the brain tissue or endothelial cells, respectively, as 1.0; the fold increase in various samples was assigned accordingly. C, SRG distribution in normal breast epithelial and cancer cell lines. Preimmune serum and polyclonal anti-SRG antibody were used to verify SRG localization by immunohistochemical staining.

 
To determine the expression of SRG protein, we generated a rabbit polyclonal antibody against SRG protein. This polyclonal antibody was used to verify the cellular presence of SRG by immunochemical staining. Using cell lines derived from human breast epithelial cells and breast cancer cells, we found that SRG protein is expressed highly in the perinuclear region of cancer cells, a pattern that is not apparent in normal breast cells (Fig. 2C). Therefore, SRG is a cytoplasmic protein that is present in the perinuclear regions of cancer cells.

Survival-related gene inhibits apoptosis in vitro. To identify the role of SRG in cell survival, BAF/BO3 cells were transfected with sense SRG or empty vector. Stably transfected BAF/B03 cells were cloned separately and were verified by PCR analysis (data not shown). The cells were subjected to IL-3 and/or serum deprivation. Apoptosis was detected by DNA content analysis and trypan blue staining (Life Technologies, Rockville, MD). As shown in Fig. 3A, SRG-transfected cells exhibited a much higher cell survival ratio than vector-transfected cells when cultured under IL-3 deprivation or IL-3 plus serum deprivation condition. Furthermore, nontransfected cells or cells transfected with empty vector showed almost complete cell death by day 4, whereas three SRG-transfected clones were >50% alive and still had viable cells even after 12 days (Fig. 3B). Our data clearly showed that SRG is involved in cell survival.



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  		Figure 3. SRG inhibits apoptosis of BAF/BO3 cells in vitro. BAF/BO3 cells with or without constitutive expression of SRG were subjected to IL-3 and/or serum deprivation. A, apoptosis was detected by DNA content analysis after 48 hours. B, trypan blue staining was used to measure long-term survival of BAF/BO3 cells transfected with SRG upon IL-3 deprivation in the presence of serum. The control and empty vector–transfected cells showed almost complete cell death by day 4, whereas live cells can be detected in the three SRG-transfected cell clones even after 12 days.

 
Because cancer cells usually have high SRG expression, we examined the function of SRG in tumor cells. Murine melanoma B16/F0 cells were transfected with sense or antisense SRG, or empty vector and cultured in parallel with untransfected cells. When cultured under the serum deprivation condition for 5 to 7 days, cell viability was significantly different in the four cell groups. Both untransfected and empty vector–transfected B16/F0 cells showed significant apoptosis. In contrast, the sense SRG–transfected B16/F0 cells still exhibited healthy growth, although cell density was somewhat less than control cells cultured in complete medium in control. More interestingly, blocking SRG expression by transfecting antisense SRG into cells dramatically accelerated B16/F0 cell death (Fig. 4). This result showed that SRG critically controls cancer cell growth in vitro by promoting cell survival.



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  		Figure 4. Blocking SRG expression accelerates cell death in melanoma cells. Murine melanoma B16/F0 cells transfected with sense SRG (S.SRG), antisense SRG (As.SRG), or empty vector were cultured under the serum deprivation condition for 7 days. A, apoptosis was analyzed by light microscopy for morphology and flow cytometry for DNA content. B, the transcript SRG was detected by RT-PCR with a pair of primer derived from SRG and the transfection vector. SRG protein level was detected by dot blot assay with ß-actin as a quantitative control.

 
Survival-related gene promotes melanoma formation in vivo. Tumorigenesis in the body is a complicated pathologic process that involves deregulation of many genes. To determine the role of SRG in tumor formation in vivo, we used the established C57BL/N mice as an experimental model for tumor development in vivo with B16/F0 melanoma cells. Different B16/F0 cell clones were prepared as described above. By injecting i.v. with these cell clones, we assayed the ability of these cells to form tumor. We found that the tumor cells migrated to liver where multiple black tumor colonies developed. Comparing tumor colony number and size resulting in each experimental group, the sense SRG–transfected cells developed more and bigger tumor colonies than the untransfected cells or the antisense SRG–transfected cells (Fig. 5A). This finding indicated that SRG plays a critical role in tumorigenesis. To examine the function of SRG in regulating tumor growth, cells of two different B16/F0 transfectants were injected i.m., one into the outside of each thigh of the same mouse. As shown in Fig. 5B, sense SRG–transfected cells in the left thigh grew into much bigger tumors (>10 mm in diameter) than those arising from antisense SRG–transfected cells in the right thigh (<2 mm in diameter or undetectable). In contrast, tumors grown from untransfected and empty vector–transfected cells were medium in size (5-6 mm in diameter). The tumor volume was also calculated and statistical analysis was done. The tumor derived from sense SRG–transfected cells is significantly bigger, whereas the tumor from antisense SRG–transfected cells is significantly smaller (P < 0.0001; Fig. 5C). Thus, our results clearly showed that SRG promotes growth and survival of tumor cells. Histologic analysis of tumors showed significant pathologic difference among the experimentally generated tumors (Fig. 5D). In comparison with the control melanoma cells, those transfected with sense SRG reveal a tighter and more even cellular arrangement, indicating that the sense SRG–transfected melanoma cells grew quickly and survived better. In contract, antisense SRG–transfected melanoma cells were arranged relatively loosely and irregularly. Various necrotic cells and cell fragments were observed, suggesting that blockade of SRG allowed tumor cells to undergo cell death and massive apoptotic cells accumulate and become necrotic. In addition, because the sense and antisense SRG–transfected cells were injected into the same mouse, it is unlikely that the effect of SRG is exerted through production of a soluble cytokine or factor.



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  		Figure 5. A critical role of SRG in melanoma formation in mouse. A, B16/F0-derived tumor was assayed in C57BL/N mice. B16/F0 cells with or without the expression of SRG in sense or antisense orientation were injected i.v. through tail vein. Tumor formation, as indicated by multiple black tumor colonies (arrows) in the liver, was assayed. B, C57BL/N mice were injected i.m. with B16/F0 cell. For comparison, each thigh received different cell lines. In one group, sense SRG–transfected cells were injected into the left thigh, whereas antisense SRG–transfected cells were injected in the right thigh. In the other group, untransfected cells were injected into the left side, whereas control vector–transfected cells were injected into the right side. C, the tumor volume was calculated and the differences were analyzed statistically by t test (P > 0.0001). D, pathohistologic analysis of melanoma tumors. Melanoma tumors from perfused mice were extracted. The paraffin sections were stained with H&E. Micrographs of representative areas are taken by light microscopy.

 
Survival-related gene small interfering RNA reduces cell survival. Our findings have shown the cell survival function of human SRG in the mouse both in vitro and in vivo. To discover a possible mouse homologue to human SRG, we did Genbank blast analysis and found a similar sequence in mouse chromosome 4. The existence of this sequence as a transcript was confirmed by reverse transcription-PCR (RT-PCR). The amino acid sequence has 85.7% identity with the human protein (data not shown). Therefore, we examined the function of mouse SRG in apoptosis by blocking its expression with siRNA in mouse NIH 3T3 cells. SRG siRNA was designed as a cassette with antisense oligonucleotide of mouse SRG, which has strong homology (81%) with human SRG. When transfected, NIH3T3 cells were induced to undergo apoptosis by serum deprivation, whereas siRNA transfectants showed more apoptosis (Fig. 6). Thus, down-regulation of SRG by siRNA decreases cell viability under the serum deprivation condition.



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  		Figure 6. Mouse siRNA SRG reduces cell survival. NIH3T3 cells transfected with siRNA corresponding to mouse SRG were cloned. A, cells were cultured in the absence of serum for 7 days and assayed by morphology and DNA content analysis. B, the expression of SRG was tested by RT-PCR with a pair of primer derived from SRG. The level of SRG protein was detected by dot blot.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cell differentiation requires coordinated activation of cell cycle genes, inhibition of tumor suppressor genes, and bypassing apoptosis. As withdrawal of cytokines, such as IL-2, IL-3, erythropoietin, and nerve growth factor, results in apoptosis in cytokine-dependent cells, it has been suggested that cytokines directly provide antiapoptotic signals (26, 27). Based on our previous studies on the ability of cytokines to drive cells to undergo apoptosis in absence of serum (12, 15), we hypothesize that when cells proliferate, they require a higher threshold of cell survival signals, without which cells undergo apoptosis as a result of an "abortive cell cycle." This cytokine-promoted apoptosis was also observed in IL-2-responsive human T-cell blasts in response to IL-2 (27) and in Rat-1 fibroblasts in response to epidermal growth factor and fibroblast growth factor (28). It has been shown that constitutive expression of c-myc promotes both cell cycle entry and death of fibroblasts under low serum conditions, whereas parental cells without altered c-myc expression do not undergo apoptosis under such conditions (29–31). Therefore, mitogenic signals from c-myc could induce apoptosis in fibroblasts due to an abortive cell cycle in the absence of serum cell survival signals. Recent studies have shown that IL-2 is required for activation-induced apoptosis in primary activated mature T cells (32, 33), indicating that IL-2, which induces c-myc expression, contributes to apoptosis as well as to cell proliferation. On the surface, this would seem to be incompatible with the notion that IL-2 withdrawal could induce apoptosis in IL-2-dependent cell lines. However, we suggest that signals generated from the interaction of cytokines with their receptors can promote cell proliferation, cell survival, or cell death depending on differentiation stages and the milieu of the responding cells. Based on this scenario, we device a novel system to functionally clone genes that are involved in the regulation of cell survival. We identified a novel gene, SRG. Because the function of SRG clearly protects cells from apoptosis induced by cytokine/serum deprivation, we believe that this gene is involved in cell survival. We also found that most cancer cells show high SRG expression and blocking its expression in melanoma cancer cells also significantly inhibited their ability to grow into tumor. Therefore, this gene is likely to be involved in cancer development.

Apoptosis is considered to be a key factor for cancer development (34, 35). Disruption of several apoptotic genes has been shown in malignant tumor of many model systems (4, 36, 37). The bcl-2 family includes both proapoptotic and antiapoptotic factors and the relative expression of the various bcl-2 members is a central determinant of apoptosis (38, 39). Many studies have shown overexpression of bcl-2, a cell survival gene, in a variety of human cancers, including solid tumors and leukemias (40–42). The overexpression of bcl-2 prevents the cell death from apoptosis, allowing the continual division of mutated cells, which eventually develop into tumors. Several studies have indicated that bcl-2 and bcl-xL localize mainly to the mitochondrial membrane and regulate a series of mitochondrial alterations associated with apoptosis (43–45). They are able to maintain mitochondrial membrane potential function and prevent the release of cytochrome c from the intermembrane space.

Our results showed that SRG functions much like bcl-2 in its antiapoptotic effect. We analyzed the expression of bcl-2, bcl-1, and bim in the sense and antisense SRG–transfected cells by real-time RT-PCR. Interestingly, the expression levels of these genes were not changed by the expression level of SRG. Therefore, we believe that antiapoptotic mechanism of SRG is likely by a pathway independent of these members of the bcl-2 family. The precise mechanism of SRG function still needs further study. Because bcl-2 is a pivotal regulator of apoptotic cell death and it is always overexpressed in many cancers, targeting bcl-2 by specific antisense oligonucleotides or small molecular bcl-2 inhibitors have shown promise as an immunotherapy for a broad range of cancer (46–50). Undoubtedly, as the molecular mechanism through which SRG regulates tumorigenesis is being elucidated, SRG will be an attractive target for the development of new strategies to treat cancer.

In conclusion, we have discovered a novel gene, SRG, which plays an important role in control cell survival and tumorigenesis. It likely functions to coordinate mutagenic signals and survival signals. The perinuclear localization of SRG suggests that it may function through the mitochondrial apoptosis pathway. We believe that further investigation of mechanisms through which SRG regulates cell survival will lead to a better understanding of the mechanisms by which cells are stimulated to undergo apoptosis or proliferate in normal growth, aging, and development, in the abnormal regulatory mechanisms that lead to aberrant apoptosis and growth in transformed cells.


   Acknowledgments
 
Grant support: USPHS grants CA76492, AI43384, and AI50222.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Arthur Roberts for his critical review of the manuscript and Dr. Jim Xiang (Department of Oncology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada) for the B16 cells and the advice on the mouse melanoma cells.


   Footnotes
 
Note: R. Wang is currently in Molecular Urology and Therapeutics Program, Department of Urology, Emory University School of Medicine, Atlanta, GA 30322. J. Solomon is currently in Miltenyi Biotec, Inc., Auburn, CA 95602. X. Luo is currently in Campbell Family-Institute for Breast Cancer Research, Princess Margaret Hospital, Toronto, ON, Canada M5G 2C1. H. Sun is currently in The Obstetrics and Gynecology Hospital, Medical Center of Fudan University, Shanghai 200011, People's Republic of China.

Received 6/21/05. Revised 8/25/05. Accepted 9/26/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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