Alterations of SAG mRNA in human cancer cell lines: requirement for the RING finger domain for apoptosis protection

Yi Sun

Department of Molecular Biology, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Co., 2800 Plymouth Road, Ann Arbor, MI 48105, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
We have recently cloned and characterized a novel zinc RING finger gene, SAG (sensitive to apoptosis gene). SAG has antioxidant activity and protects cells from apoptosis induced by metal ions and redox compounds. During the course of SAG cloning, we identified two forms of deletions of SAG mRNA in colon and testicular carcinoma cell lines. The first form (SAG-MU1) consists of a 7 bp deletion that results in a frameshift and abolishes the RING finger domain. The second (SAG-MU2) consists of an in-frame 48 bp deletion that truncates 16 amino acids in the protein but retains the RING finger domain. Functional studies using stable transfectant lines reveal that, like wild-type SAG, SAG-MU2 retains anti-apoptosis activity, whereas SAG-MU1 shows no such protection. The results indicate that the C-terminal zinc RING finger domain is required for anti-apoptosis activity of SAG.

Abbreviations: OP, 1,10-phenanthroline; SAG, sensitive to apoptosis gene; SAG-MU1, SAG mutant 1; SAG-MU2, SAG mutant 2.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Mutational activation of oncogenes and mutational inactivation of tumor suppressor genes are frequent genetic alterations in human cancers (1,2). Two typical examples are ras oncogene activation and p53 tumor suppressor gene inactivation by point mutations (3,4). Mutated Ras becomes constitutively active, sending proliferative signals to the cell, whereas mutant p53, unlike the wild-type protein, often loses activity in negatively controlling cell proliferation (5,6). The outcome of these mutations is uncontrolled cell growth and tumor development. Point mutations have also been found in the genes encoding the antioxidant enzyme Cu,Zn-superoxidase dismutase. These mutations were associated with a subset of patients with familial amyotrophic lateral sclerosis (7,8).

Apoptosis (programmed cell death) is a genetically programmed active process for maintaining homeostasis under physiological conditions and for responding to various stimuli (9) that is characterized by cell membrane blebbing, cytoplasmic shrinkage, nuclear chromatin condensation and DNA fragmentation (10). Apoptosis can be initiated in various cells by a wide variety of physical, chemical and biological stimuli, including diverse anticancer drugs, oxidative DNA damaging reagents and cytokines (9). Oxidative stress-induced apoptosis is often inhibited by antioxidant proteins or small antioxidant molecules (1114). We have recently cloned a redox-inducible zinc RING finger protein, SAG (sensitive to apoptosis), that acts as an antioxidant to protect cells from apoptosis induced by redox compounds (15). We report here the detection of two SAG deletion mutants in two human cancer cell lines. Biological characterization of these mutants revealed that the RING finger domain is required for the anti-apoptotic activity of SAG.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Cell cultures
The following human tumor cells lines were used: DLD-1 and HT-29 (colon); Saos-2 and U2-OS (bone); H1651, H2009 and H1299 (lung); PC-3, Du145 and LnCap (prostate); 293 and G401 (kidney); Sk-OV3 and A90 (ovarian); Cates-1B (testis); HeLa (cervix). These were obtained from the American Type Culture Collection and cultured as specified. Nasopharyngeal carcinoma lines HONE-T-1 and C15 and human epidermal keratinocytes (Rhek) and the H-ras transformed line (Rhek-ras) were cultured as described (16). Cells were grown to 80% confluency before being harvested for total RNA isolation.

Primary colon cancer tissues
Twelve pairs of primary colon cancer and adjacent normal tissues were collected by National Disease Research Interchange (NDRI, Philadelphia, PA) from cancer patients after surgical removal and snap frozen in liquid nitrogen.

RNA isolation and RT–PCR analysis
Total RNA was isolated from human cancer cell lines or colon cancer tissues using RNAzol solution (Tel-Test, Friendswood, TX) according to the manufacturer's instructions. RT–PCR analysis was performed as described previously (17). Briefly, 5–10 µg of total RNA were mixed with 1 µg of oligo(dT) primer (Pharmacia, Piscataway, NJ), 60 U of avian myeloblastosis virus reverse transcriptase (Seikagaku American, Gaithersburg, MD), 40 U of RNasin (Promega, Madison, WI) and reaction buffer in a total volume of 25 µl. The reaction mixture was incubated at 42°C for 90 min. The RNA was then hydrolyzed in 0.5 N NaOH at 70°C for 30 min. The synthesized first strand cDNA was precipitated with ethanol, washed, dried and dissolved in 50–75 µl of TE buffer. Five microliters of cDNA were used in the PCR reaction. The primers used for PCR cloning of cDNA flanking the entire SAG open reading frame were SagP.01 [5'-CGGGATCCATGGCCGACGTGGAAG-3' (upstream)] and SagT.02 [5'-CGGGATCCTCATTTGCCGATTC TTTGGAC-3' (downstream)]. The resulting PCR fragment of 358 bp was digested with BamHI and subcloned into predigested pcDNA3 vector (Invitrogen, San Diego, CA). The resultant clones were sequenced manually using a DNA sequencing kit (Sequenase v.2.0; Amersham, Arlington Heights, IL) to confirm both sense and antisense orientation and freedom from PCR-generated mutations. DNA sequencing revealed a wild-type SAG clone as well as two deletion mutants, SAG mutant 1 (SAG-MU1), with a 7 bp deletion, and SAG mutant 2 (SAG-MU2), with a 48 bp deletion, in DLD-1 tumor cells. To determine SAG deletions in other human tumor cell lines and primary colon cancer tissues, RT–PCR analysis was conducted in the presence of [35S]dATP (Amersham), as detailed previously (18). The primers used were SagT.02 and hSAG.M1 (5'-GCCATCTGCAGGGTCCAG-3', starting at nucleotide 151 of hSAG cDNA). The resulting fragment was 200 bp for wild-type SAG. The PCR products were resolved in 6% denaturing sequencing gels. The bands corresponding to wild-type SAG and the SAG deletion mutants were confirmed by excising them from the gel and PCR amplification with the same set of primers, followed by TA cloning (Invitrogen) and DNA sequencing.

DNA transfection and stable transfectant selection
DLD-1 cells were transfected with the plasmids neo control pcDNA-3 (Invitrogen), pcDNA-SAG, pcDNA-SAG-MU1 and pcDNA-SAG-MU2. The SAG mutant constructs were generated by RT–PCR as described above. Neomycin-resistant colonies were selected by G418 selection (600 µg/ml) and stable clones were ring-isolated and expanded in culture (15). Expression of SAG was measured by northern analysis as described (19).

DNA fragmentation
Subconfluent DLD-1 transfectant cells were treated with 150 µM 1,10-phenanthroline (OP) for 24 h. Both detached and attached cells in 2x100 mm dishes were harvested and subjected to DNA fragmentation analysis as described (20). Briefly, cells were collected by centrifugation and lysed with lysis buffer (5 mM Tris–HCl, pH 8, 20 mM EDTA, 0.5% Triton X-100) on ice for 45 min. Fragmented DNA in the supernatant of a 14 000 r.p.m. centrifugation (45 min at 4°C) was extracted twice with phenol/chloroform and once with chloroform and precipitated with ethanol and salt. The DNA pellet was washed once with 70% ethanol and resuspended in TE buffer with 100 µg/ml RNase at 37°C for 2 h. The fragmented DNA was separated by 1.8% agarose gel electrophoresis, stained with ethidium bromide and visualized under UV light.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Identification of two deletion mutants of human SAG in the DLD-1 colon carcinoma cell line
SAG was cloned by differential display as a redox-inducible gene during OP-induced apoptosis (15,20). SAG consists of 113 amino acids with a zinc RING finger domain at the C-terminus. To study SAG function, we cloned SAG cDNA flanking the entire open reading frame into the pcDNA3 eukaryotic expression vector (Invitrogen) by RT–PCR. Total RNA isolated from DLD-1 colon carcinoma cells was used as the template. During sequence verification of the resulting clones, we found that in addition to wild-type sequence, several clones contained either a 7 or 48 bp deletion at nucleotide 170 or 177, respectively, assigning the first A of the start codon as nucleotide 1 (Figure 1Go). Both deletion mutants contain the first 56 amino acids unchanged. SAG-MU1 contains a 7 bp frameshift deletion that encodes a protein of 90 amino acids without the zinc RING finger domain. SAG-MU2 contains an in-frame 48 bp deletion that encodes a protein of 97 amino acids with the zinc RING finger domain (Figure 2Go).



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Fig. 1. Deletions of SAG mRNA in DLD-1 colon carcinoma cells. Total RNA was isolated from cells and subjected to analysis by RT–PCR, subcloning and DNA sequencing. DNA sequencing ladders from three individual clones are shown. WT, wild-type; MU1, SAG mutant 1; MU2, SAG mutant 2.

 


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Fig. 2. Comparative alignments of the coding sequence of wild-type as well as two SAG deletion mutants. (A) Primary amino acid sequence of wild-type SAG and the two SAG mutants. Identity is shaded black. (B) Consensus sequence of the zinc RING finger domain and RING finger sequence found in wild-type SAG and SAG-MU2. C3H2C3 residues are in bold. The numbers shown above the sequences are the codon numbers in the proteins.

 
Infrequent SAG deletions in human cancer cell lines
To examine whether SAG deletions can also be detected in other cancer cell lines, RT–PCR in the presence of [35S]dATP followed by sequencing gel separation was conducted in 20 human tumor and transformed lines (see Materials and methods). To detect whether deletions were also found at the DNA level, genomic DNA was isolated from these cell lines and subjected to the same analysis. The primers used flank the C-terminal half of the SAG molecule, including both deletion regions. Within this region there is no intron (unpublished observation) so that both genomic DNA and cDNA should yield the same size band of 200 bp as wild-type SAG. If there are deletions, SAG-MU1 should give a band of 193 bp in size, whereas SAG-MU2 should yield a band of 152 bp. As shown in Figure 3Go, three bands, corresponding to the wild-type as well as the two deletion mutants (which have been confirmed by DNA sequencing; data not shown), were detected in an RNA sample from Cates-1B cells, an embryonal testicular carcinoma line (lane 3). The DNA sample from the same cells contained the wild-type band only (lane 4). As a control, HONE-1 cells, a nasopharyngeal carcinoma line (16), was shown to contain the wild-type band only (lane 2). All other human cell lines tested contained only the wild-type band in both DNA and RNA samples (data not shown). These results indicate that the SAG deletions occur very rarely in human cancer cell lines. Several lines of evidence suggest that the deletions described here are unlikely to be RT–PCR artifacts or a result of alternative splicing of SAG mRNA. First, multiple cell lines were examined and mutations were detected in only two. Second, the deletions are not in a GC-rich region and they involve multiple bases (7 and 48 bp, respectively). Third, there is no intron within this region. Detection of the deletions in RNA but not in genomic DNA may reflect alterations in RNA editing of SAG mRNA in these tumor cells (21,22).



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Fig. 3. Detection of SAG deletion mutants in Cates-1B testicular carcinoma cells. Total RNA and genomic DNA were isolated from multiple human tumor cell lines and subjected to RT–PCR or PCR analysis in the presence of [35S]dATP, respectively. The radio-labelled PCR products were resolved in 6% sequencing gels. The gel was dried and exposed to X-ray film. Wild-type SAG and the two SAG mutants are indicated.

 
Lack of SAG deletions in primary human colon carcinomas
We have detected two SAG mutants in colon carcinoma DLD-1 cells and testicular carcinoma Cates-1B cells. To determine whether the same deletions also occur in primary cancer tissues, we performed the same RT–PCR analysis of 12 colon cancer tissues, snap frozen after surgical removal from cancer patients. All the samples yielded the 200 bp wild-type band and none of them contained either SAG-MU1 or SAG-MU2 (data not shown). We were unable to perform the same analysis in primary testicular carcinoma tissues due to the difficulties in sample collection. Thus, SAG deletions, occurring in a few cancer cell lines at the RNA level, may arise from cell culture selection.

The zinc RING domain is required for SAG apoptosis-protective activity
Although the lack of SAG deletions in primary cancers suggests that SAG may not be actively involved in human cancer development, the two deletion mutants offered an opportunity to study structure–function relationships of SAG protein. As shown in Figure 2Go, wild-type SAG, SAG-MU1 and SAG-MU2 share 100% identity in the first 56 amino acids. Due to a frameshift deletion, SAG-MU1 contains no zinc RING finger domain; this is retained, however, in the in-frame deletion mutant SAG-MU2. We have previously shown that wild-type SAG can protect cells from apoptosis induced by OP (15). Using these two mutants, we determined whether the zinc RING finger is required for this anti-apoptotic activity. Stable transefectants expressing wild-type SAG, SAG-MU1 or SAG-MU2, along with the vector control, were generated by DNA transfection and neo resistance selection. SAG expression was monitored by northern analysis. As shown in Figure 4Go, the vector controls D1-3 and D1-6 express very low levels of endogenous SAG mRNA. Expression was remarkably increased in SAG transfectants: wild-type SAG D12-1 and D12-8; SAG-MU1 D3-3 and D3-4; SAG-MU2 D4-2 and D4-5. To determine their potential apoptosis-protective activity, DNA fragmentation, a hallmark of apoptosis, was analyzed in these stable transfectants upon exposure to OP. Cells were seeded at 3.5x106 per 100 mm dish and exposed after 16–24 h to 150 µM OP for 24 h. Both detached and attached cells in 2x100 mm dishes were harvested and subjected to the assay. As shown in Figure 5Go, OP induces DNA fragmentation in the vector control cells (D1-6 and D1-3) as well as SAG-MU1 cells (D3-4 and D3-3). Much less DNA fragmentation was observed in wild-type SAG-transfected cells (D12-1 and D12-8) and RING-containing SAG-MU2 cells (D4-5 and D4-2). These results indicate that ectopic expression of SAG protects cells against OP-induced apoptosis and that the zinc RING finger domain is required for this protection.



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Fig. 4. Expression of SAG in SAG stable transfectants. Constructs expressing wild-type SAG, SAG-MU1 or SAG-MU2 and the vector control were transfected into DLD-1 cells, followed by G418 selection and stable line cloning. Total RNA was isolated from these stable clones and subjected to northern analysis for ecotopic expression of exogenous SAG. Two independent clones from each construct transfection are shown. Ethidium bromide staining of 28S and 18S rRNA as equal loading controls is shown at the bottom.

 


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Fig. 5. DNA fragmentation of SAG transfectants treated with OP. Stable transfectants were seeded in 2x100 mm dishes and exposed to OP (150 µM) for 24 h. Both detached and attached cells were harvested and subjected to DNA fragmentation assay as described in Materials and methods. A molecular marker (100 bp) is included on the left. The transfectants are: the vector controls D1-6 and D1-3 (lanes 1 and 5); wild-type SAG D12-1 and D12-8 (lanes 2 and 6); SAG-MU1 D3-4 and D3-3 (lanes 3 and 7); SAG-MU2 D4-5 and D4-2 (lanes 4 and 8).

 
Through differential display we identified an OP-inducible, apoptosis-protective gene, SAG, that encodes a novel zinc RING finger protein (15). The zinc RING finger group is a new gene family with a rapidly increasing membership (23). The members of this gene family contain a C3HC4 or C3H2C3 motif and are involved in DNA binding, RNA binding and protein–protein interactions (2427). Biologically, the zinc RING finger proteins are involved in many processes, including oncogenesis, signal transduction and development, among others (23). Some of the RING finger proteins have been found to inhibit apoptosis, including baculovirus protein, p35 and mammalian homologs of baculovirus inhibitors of apoptosis (28,29). The RING finger domain is required for apoptosis inhibition in some cases, but not in others (28,29). Here we show the detection of two deletion mutants of a RING finger protein, SAG, in two tumor cell lines. Co-existence of the wild-type and two mutants in mRNA, but not in DNA, from these tumor cells may reflect abnormal RNA editing in some of the SAG messenger (21,22). Through analysis of these two SAG mutants it appears that the RING finger domain (codons 61–102) of SAG is required for protection of cells from apoptosis induced by the redox-sensitive compound OP. Elucidation of the structure–function relationship would facilitate the study of SAG function(s) and its mechanism of action.

While this manuscript was under review, there were four publications reporting the isolation and characterization of Rbx-1, a VHL tumor suppressor binding protein (3032), and ROC-1, a cullin-binding protein (33,34). Rbx/ROC was identified to be a component of ubiquitin ligase that catalyzes the ubiquitination of I{kappa}B{alpha}, an inhibitor of NF-{kappa}B (33,34). SAG, reported here and previously (15), has been identified to be Rbx-2/ROC-2, with a similar function to ROC-1 (33). Thus, SAG may protect cells from apoptosis through either its metal ion-binding/ROS scavenging activity (15) or its promotion of I{kappa}B degradation, leading to activation of NF-{kappa}B that functions as an apoptosis protector (35,36).


    Acknowledgments
 
The colon cancer tissues used in this study were provided by the National Disease Research Interchange.


    Notes
 
Email: yi.sun{at}wl.com


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

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Received February 26, 1999; revised May 7, 1999; accepted May 19, 1999.





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