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SCC-112, a novel cell cycle-regulated molecule, exhibits reduced expression in human renal carcinomas
Gene 328 (2004) 187 – 196 www.elsevier.com/locate/gene
SCC-112, a novel cell cycle-regulated molecule, exhibits reduced expression in human renal carcinomas Deepak Kumar a, Isamu Sakabe a, Sonal Patel a, Ying Zhang b, Imran Ahmad c, Edmund A. Gehan b, Theresa L. Whiteside d, Usha Kasid a,* a
Department of Radiation Medicine and Biochemistry and Molecular Biology, Lombardi Cancer Center, Georgetown University, E208, Research Building, 3970 Reservoir Road, NW, Washington, DC 20007, USA b Biomathematics and Biostatistics, Lombardi Cancer Center, Georgetown University, Washington, DC 20007, USA c NeoPharm, Inc., Lake Forest, IL 60045, USA d Department of Pathology, The Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA Received 17 July 2003; received in revised form 25 November 2003; accepted 12 December 2003 Received by F. Salvatore
Abstract We report here the identification and an initial characterization of a novel cell cycle-regulated molecule, SCC-112. SCC-112 cDNA (6744 bp) encodes a longest open reading frame (ORF) comprised of 1297 amino acids, representing a f150-kDa nuclear protein. SCC-112 mRNA and protein levels were relatively high during the G2/M phase of the cell cycle in MDA-MB 435 breast cancer cells. Transient expression of SCC-112 cDNA in COS-1 cells led to an increase in the number of cells in sub-G1 phase and enhanced activity of caspase-3, a downstream effector of apoptosis. Stable transfection of SCC-112 cDNA in MDA-MB 231 breast cancer cells also led to an increase in the number of cells in sub-G1 phase (f2 – 3-fold), indicative of apoptosis. The examination of the paired sets of human normal and tumor tissues revealed that the SCC-112 mRNA level was significantly high in normal breast and kidney tissues as compared to the corresponding primary tumor tissues ( P<0.0001; breast, n=50, and kidney, n=20). Consistent with these observations, SCC-112 protein expression (150 kDa) was high in a majority of the normal renal tissues examined as compared to the matched renal tumor tissues (67%, 1.2-fold to > 10-fold, n=18). Taken together, these findings suggest that the SCC-112 gene expression is likely to be associated with normal cell growth and proliferation. D 2004 Published by Elsevier B.V. Keywords: SCC-112; cDNA and predicted amino acid sequences; Subcellular localization; G2/M phase; Cell death; Tumor vs. normal tissue expression
1. Introduction Cells undergo apoptosis or programmed cell death in response to a variety of environmental or stress-related stimuli. Several apoptotic proteins and signaling pathways have been defined (Green, 2004). Cancer cells often exhibit defects in radiation or chemotherapeutic drug-induced apo-
Abbreviations: RCC, renal cell carcinoma; FBS, fetal bovine serum; PMSF, phenylmethylsulphonyl fluoride; DAPI, 4V,6-diamidino-2-phenyindole dilactate; RT-PCR, reverse transcription-polymerase chain reaction; ORF, open reading frame; PEST sequence, a region rich in proline, glutamic acid, serine and threonine. * Corresponding author. Tel.: +1-202-687-2226. E-mail address: [email protected] (U. Kasid). 0378-1119/$ - see front matter D 2004 Published by Elsevier B.V. doi:10.1016/j.gene.2003.12.013
ptosis, implicating an important role of the apoptogenic signals in regulation of normal cell growth and proliferation (Johnstone et al., 2002). The apoptosis inducing molecules are known to play roles in the regulation of damageresponsive cell cycle check points, genomic instability, and DNA repair and recombination (Levine, 1997). Many of these proteins also have an important role in tumor growth suppression, implied, in part, by a reduced level of expression in primary tumors as compared to matched normal adjacent tissues (Soengas et al., 2001). Renal cell carcinomas (RCC) have been the focus of investigation of several candidate tumor suppressor genes including the VHL, the FHIT and the RASSF1A genes (Clifford and Maher, 2001). These and other discoveries have been guided, in part, by a high frequency of loss of
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heterozygosity at chromosome 3p21 – p22 in RCC, and cancers of the lung and breast (Lerman and Minna, 2000). The loss of chromosome 4 has been shown to correlate positively with high tumor stage in clear-cell RCCs (Reutzel et al., 2001), and 4p has been predicted to harbor the tumor suppressor gene(s) (Schouten et al., 1990; Wu et al., 1995; Arribas et al., 1999; Kitamura et al., 2000; Feng et al., 2002). Here, we report the identification of a novel cell cycle-regulated, pro-apoptotic molecule, SCC-112. SCC112 gene is located on chromosome 4p14. Our data suggest a potential correlation between the expression of SCC-112 and normal renal cell growth and proliferation.
2. Materials and methods 2.1. Cell culture Human breast cancer cell lines, MDA-MB 435 and MDA-MB 231, and COS-1 cells were grown in DMEM, supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 25 Ag/ml gentamicin. All culture reagents were purchased from Invitrogen Life Technologies. The cells were grown in 75-cm2 tissue culture flasks in a humidified atmosphere of 5% CO2 and 95% air at 37 jC. 2.2. Cloning and sequence analysis of SCC-112 cDNA A 318-bp novel cDNA fragment (KAS-112) was isolated from a human head and neck squamous carcinoma cell line as described earlier (Patel et al., 1997). Homology search of the National Center for Biotechnological Information (NCBI) GenBank and Human EST databases using the KAS-112 cDNA sequence (GenBank accession no. AF003254) revealed two partially overlapping clones, KIAA0648 (5177 bp, GenBank accession no. AB014548) and an EST sequence (2247 bp, GenBank accession no. AI655954). The KIAA0648 cDNA clone was procured from Kazusa DNA Research Institute, Japan (Ohara et al., 1997), and the EST clone was purchased from Incyte Genomics (Palo Alto, CA). Both strands of the EST and KIAA0648 clones were sequenced using automated DNA sequencer (Applied Biosystems Prism 377) and sequences were assembled using Autoassembler program (Applied Biosystems) (GenBank accession no. AF294791). BLAST sequence database, conserved domain prediction and open reading frame (ORF) searches were performed using the NCBI programs (Altschul et al., 1997). The prediction of the possible nature of the protein including localization based on structural characteristics was performed by pSORT (Nakai and Kanehisa, 1992). 2.3. Generation of anti-SCC-112 antibody A rabbit antiserum was generated against a synthetic peptide representing 20 amino acid residues at the C-
terminus of SCC-112 ORF (amino acids 1278 – 1297, KLQDLAKKAAPAERQIDLQR). The peptide was coupled to keyhole limpet hemocyanin and injected into rabbits. Custom antisera production service was provided by Zymed laboratories (San Francisco, CA). 2.4. Cell synchronization Logarithmically growing MDA-MB 435 cells were treated with 4 Ag/ml aphidicolin (Sigma) for 24 h. The cells were washed thoroughly and medium was replaced with fresh medium and incubation continued for 0, 4 and 8 h, representing G1/S phase, S phase and G2/M phase-enriched cell populations, respectively. Alternatively, cells were treated with 100 ng/ml nocodazole (Sigma) for 16 h to arrest the cells in M phase. The cells were fixed in 70% ethanol, washed with PBS, followed by RNase treatment and propidium iodide staining. The cell cycle distribution profiles were determined by the flow cytometric assay using FACsort (Becton Dickinson). 2.5. Northern blotting and hybridization The total RNA was isolated from MDA-MB 435 cells arrested at various phases of the cell cycle using Trizol reagent according to the manufacturer’s instructions (Invitrogen Life technologies), followed by electrophoresis and transfer to the nylon membrane. Multi-tissue northern blots containing poly A+RNA from human adult and fetal tissues were purchased from Clontech. Northern blots were hybridized using 32P-labeled SCC-112 cDNA insert (5177 bp) as a probe, followed by reprobing with radio-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or h-actin cDNA (Clontech). The autoradiographs were scanned and bands were quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). 2.6. Western blot analysis MDA-MB 435 cells were washed with ice-cold PBS and scrapped in lysis solution containing 20 mM Tris (pH 8.0), 150 mM NaCl, 1% NP-40 (w/v), 10% glycerol (w/v), 1 mM phenylmethylsulphonyl fluoride (PMSF), 20 Ag/ml aprotinin and 20 Ag/ml leupeptin. The cells were lysed for 45 min with vigorous shaking at 4 jC and centrifuged at 12 000g for 15 min at 4 jC. Thirty to fifty micrograms of the total protein was analyzed by Western blotting using 1:2000 dilution of the SCC-S2 antiserum. The frozen human normal and tumor tissues were homogenized in RIPA lysis buffer (150 mM NaCl, pH 7.5; 1% (w/v) sodium deoxycholate; 1% (v/v) Triton X-100; 0.1% (w/v) SDS, 2 mM PMSF, 20 Ag/ml aprotinin and 20 Ag/ ml leupeptin). After incubation on ice for 30 min, the tissue extracts were centrifuged at 12 000g for 15 min at 4 jC and the supernatant was used for Western blot analysis.
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2.7. Immunofluorescence microscopy MDA-MB 435 cells were grown on cover slips. The cells were fixed in chilled methanol for 10 min, followed by rinsing in PBS and permeabilization with 0.01% Triton X100 for 30 s. Non-specific binding was blocked with 1% BSA in PBS (w/v) for 30 min. The cells were incubated with a 1:1000 dilution of the SCC-112 antiserum in 1% BSA/PBS for 1 h, followed by three washings of 10 min each with PBS. Texas red-conjugated goat anti-rabbit secondary antibody (Jackson Immunoresearch) (1:200 dilution in 1% BSA in PBS for 1 h) was used to detect the antigen – antibody complex. After three washings of 10 min each with PBS, the nuclei were stained with 4V,6-diamidino-2-phenyindole dilactate (DAPI; Sigma) for 1 min. The cover slips were rinsed with PBS, mounted on a glass slide with cells facing the slide in fluoromount-G (Southern Biotechnology Associates, Birmingham, AL) and visualized under a fluorescence microscope (Carl Zeiss). 2.8. Expression cloning of SCC-112 cDNA A portion of the full length SCC-112 cDNA (nucleotides 216 – 4125, AF294791) coding for the predicted longest ORF was cloned into a eukaryotic expression vector, pCR3.1 (Invitrogen). Briefly, a 1696-bp fragment was amplified by PCR using the EST clone (AI655954) as a template and primers P1 (5V-GCG-GGC-CCG-GAC-AAGATC-ACC-ACG-GAC-GAG-3V) and P2 (5V-CGG-GCCCGC-GAG-AAA-AGT-GTG-AAG-TAT-GA-3V). In addition, a 2558-bp fragment was amplified by PCR using KIAA0648 clone and primers P3 (5V-GCT-TAT-ATT-ACTTAT-ATG-3V) and P4 (5V-GCA-GAA-GCT-GGA-GCCTGC-3V). The two amplified products were digested with BamHI, and the ligated cDNA was cloned into a TA cloning vector pCR2.1 (Invitrogen). The ligated cDNA (3972 bp) served as a template for a third PCR reaction using primers P5 (5V-ATC-ACC-ACG-GAC-GAG-ATG-3V) and P6 (5VTTA-CCT-TTG-TAA-GTC-AAT-3V). The amplified cDNA fragment (3909 bp) representing nucleotides 216 –4125 of the full-length SCC-112 cDNA was cloned into the pCR3.1 expression vector and the sequence was verified by automated DNA sequencing. To construct the N-terminal FLAG epitope-tagged SCC112 cDNA expression vector, SCC-112 cDNA encoding the open reading frame was amplified by polymerase chain reaction using pCR3.1SCC-112 (described above) as template. The 5V-primer used for amplification was 5VTCC-ACC-ATG-GAT-TAC-AAG-GAT-GAC-GAC-GATAAG-ATG-ATC-AAA-CGC-CTG-AAG-ATG-3V and the 3V-primer was the primer P6 described above. The sequence underlined in the 5V-primer corresponds to the FLAG octapeptide. The amplified product was cloned into the mammalian expression vector pCR3.1 according to the instruction manual (Invitrogen) and verified by automated sequencing.
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2.9. Transient and stable transfections, and expression of SCC-112 cDNA In the transient transfection experiments, COS-1 cells were seeded in six-well plates (1 – 3105 cells/well, in duplicate or triplicate) and transfected with the expression vector pCR3.1 or recombinant vector containing FLAGtagged SCC-112 cDNA (1– 5 Ag/well) using the LipofectAMINE 2000 (Invitrogen). Thirty-six hours after transfection, cells were harvested and lysed at 4 jC for 30 min in lysis buffer (100 mM HEPES, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 Ag/ml each of aprotinin and leupeptin), followed by microcentrifugation for 15 min at 4 jC. Protein concentration was determined using Coomassie G250 protein assay reagent (Pierce). Fifty micrograms of the cell lysate was resolved by 7.5% SDS-PAGE, transferred to Immobilon-P membrane (Millipore) and immunoblotted with 1 Ag/ml mouse monoclonal FLAG-M2 antibody (Sigma). Enhanced chemiluminescence method (Luminol, NEN Life Science Products) was used to detect the signal. The blot was reprobed with mouse monoclonal h-tubulin antibody (Santa Cruz). MDA-MB 231 human breast cancer cells were stably transfected with pCR3.1SCC-112 or pCR3.1 vector. The cells were subcultured in DMEM containing 10% FBS to achieve 70 –80% confluency per T-75 flask at 24 h, washed once with serum-free medium and 4.4 ml of DMEM was added to the flask. Sixty microliters of Fugene 6 reagent (Roche Diagnostics) was diluted with 600 Al of serum-free DMEM. Ten micrograms of plasmid DNA (pCR3.1SCC-112 or pCR3.1 vector) was added to the diluted Fugene 6 reagent and the mixture was incubated for 45 min. The DNA and Fugene 6 mixture was added dropwise to the flask and mixed by gentle swirling. After 48 h, the cells were subcultured (1:6) in DMEM containing 10% FBS, 25 Ag/ml gentamicin, and 800 Ag/ml geniticin (G418, Invitrogen Life Technologies). Selection of stable transfectants in G418-containing medium continued for 2 weeks. At least 100 G418-resistant colonies were pooled and the mass culture was maintained in DMEM supplemented with 10% FBS, 25 Ag/ml gentamicin and 800 Ag/ml of G418. The expression of SCC-112 cDNA in stable transfectants was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) using the Titan one tube RT-PCR kit (Roche Diagnostics) (Kumar et al., 2000). Total RNA was isolated from SCC-112 or vector transfectants and untransfected MDA-MB 231 cells using the Trizol reagent. For the RT-PCR, the 5V-primer used was the T7 primer built into the expression vector pCR3.1 backbone (Invitrogen) and the 3V-primer P7, 5V-GTT-GATCAC-TGA-GAA-GAG-3Vwas designed against a portion of the SCC-112 cDNA (nucleotides 639 –622, AF294791). The reactions in the absence of reverse transcriptase and in the presence of the 18S primers (Ambion) were used as controls. The reaction products were resolved on 1.5% agarose gel and detected by ethidium bromide staining.
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2.10. Apoptosis and caspase-3 activity assays Apoptosis was determined by the flow cytometric analysis of COS-1 and MDA-MB 231 transfectants. At 36 h after the transient transfection of COS-1 cells with various amounts of the FLAG-tagged SCC-112 cDNA or vector, floating cells were pooled with the adherent cells collected by trypsinization and fixed in 2 ml of 75% ethanol for at least 30 min at 4 jC. The percentage of cells containing subG1 DNA content, indicative of apoptosis, was determined by the flow cytometric approach. 1-2105 MDA-MB 231 stable transfectants were seeded per well, in triplicate, in a six-well plate in DMEM containing 10% FBS. After 24 h, cells were switched to serum-free medium for various times. The floating cells were pooled with the adherent cells, followed by the flow cytometric analysis. For measurement of the caspase-3 activity in COS-1 transfectants, the pooled cells were lysed in cell lysis buffer (Clontech) and the caspase-3 activity was determined using the Apoalertk Caspase-3 assay kit (Clontech). 2.11. Hybridization, quantification and statistical analysis of cancer profiling array The Cancer Profiling Array blot (Clontech) spotted with 241 cDNAs pairs from matched normal and tumor tissues from individual patients, representing 13 different tissues, was hybridized using 32P-labeled SCC-112 cDNA insert (5177 bp) as per manufacturer’s user manual (PR11929). After hybridization, the membrane was reprobed with ubiquitin cDNA provided as a loading control. The autoradiographs were scanned and signals were quantified using ImageQuant software (Molecular Dynamics). For statistical significance, the sample sizes were calculated by STPLAN software package (Brown et al., 1996). The ratio of the ubiquitin-normalized SCC-112 mRNA expression values in normal and matched tumor tissue was transformed to natural logarithm. Under the null hypothesis of no difference in gene expression levels between normal and tumor tissues, the average ratio should be one and the average of the natural logs of the ratios should equal 0. To test the null hypothesis for each type of tumor, a one-sample t-test of the natural logs of the ratios was performed and two-sided P values were calculated. 95% confidence intervals were calculated based on the natural log transformations of the data and transformed back to obtain the confidence limits on the ratio scale (Armitage et al., 2001).
3. Results and discussion 3.1. Sequence identification, expression and localization of SCC-112 The complete SCC-112 cDNA sequence deduced from the three partially overlapping clones, KAS-112, KIAA0648
and EST, is 6744 bp in length (Fig. 1A). A small portion of the sequence present at the 3V-ends of the KAS-112 (265 – 318 bp) and EST (1881 – 2247 bp) clones was not found in the KIAA0648 clone. Since this portion of the KAS-112 and EST sequences (hatched boxes, Fig. 1A) is present in the genomic SCC-112 DNA (NCBI Human Genome Data Base, Genbank accession number AC022862), it is likely that KAS-112/EST sequence represents the unspliced, partial SCC-112 mRNA. To further confirm the SCC-112 cDNA sequence at the junction between the KAS-112/EST and KIAA0648 clones, total RNA was isolated from MDA-MB 231 cells and RT-PCR was performed using a EST cDNA sequence-specific 5V-primer (P8, 5V-CCG-TCA-TGC-CACAGC-TTG-3V, at position 956 –973 bp), and a KIAA0648 sequence-specific 3V-primer (P9, 5V-GGC-TTC-TGA-ATCAAT-GTG-CAC-AGG-TGC-G-3V, at position 427– 400 bp). The sequence of the anticipated 1036-bp amplified cDNA product matched the overlapping cDNA sequence obtained from these clones (data not shown). Northern blot analysis using the KIAA0648 cDNA insert as a probe identified two transcripts of f7 kb and f8 kb in normal human adult tissues (Fig. 1B). Similar results were obtained using fetal tissue and cancer cell RNA blots (Clontech) (data not shown). These two transcripts were also seen in MDA-MB 231 cells using a 5V-fragment of the EST sequence as a probe (Fig. 1A, 320-bp EcoRI fragment) (data not shown). The SCC-112 cDNA codes for a novel longest ORF comprised of 1297 amino acids (aa). The poly-adenylation signal sequence could be located at the 3V untranslated region. We did not find a stop codon upstream of the first ATG; however, the antiserum generated against a synthetic C-terminus SCC-112 peptide detected a single f150-kDa band by immunoprecipitation and immunoblotting in lysates from human A549 lung carcinoma cells, corresponding to the predicted size of the protein (146 kDa) (data not shown). The pre-immune serum and mock lysates served as controls in these experiments. In other experiments, COS-1 cells were transiently transfected with expression vector containing the FLAG epitope-tagged SCC-112 cDNA coding for the ORF. SCC-112 expression was detected by immunoblotting with anti-FLAG antibody, followed by a reprobe using the anti-SCC-112 antiserum. The mobility of the FLAG-tagged SCC-112 overlapped with the total SCC112 protein expressed in COS-1 cells (see below, Fig. 4A). A protein homology search indicated a partial homology of SCC-112 to AS3 (69% identity), an androgen-inducible gene located on chromosome 13 in the BRCA2-Rb1 locus, having a role in proliferative shutoff (Geck et al., 1999). SCC-112 aa sequence was similar to SPO76 (22% identity), a chromosome morphogenesis protein in Sorodaria macrospora (Van Heemst et al., 1999), and BimD (22% identity), a Aspergillus nidulans protein involved in mitosis (Van Heemst et al., 2001). SCC-112 ORF also showed a limited homology to yeast Pds5p (23% identity), a chromosome maintenance protein involved in chromosome cohesion
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Fig. 1. Sequence identification, and mRNA and protein expression of SCC-112. (A) Schematic maps of complete SCC-112 cDNA and overlapping partial clones are shown. The gray box represents the coding region of cDNA. The black boxes represent the 5V- and 3V-untranslated regions of cDNA. The hatched box shown in the KAS-112 and EST clones represents the adjacent genomic DNA sequence. E, EcoRI. (B) Expression of SCC-112 mRNA in normal tissues. Normal human adult tissue mRNA blots were probed with radiolabeled SCC-112 cDNA fragment (1568 – 6744 bp). The blots were reprobed with h-actin cDNA. (C) Expression of SCC-112 protein in normal human tissues. Western blot analysis was performed using approximately 30 Ag of total protein in whole cell lysates from various human tissues and the blots were reprobed with anti-actin antibody.
during mitosis (Panizza et al., 2000). Conserved domain search in NCBI database revealed partial homology of SCC-112 amino acid sequence to domains such as NAdaptin (Kirchhausen et al., 1989) and SKP1 (Stebbins et al., 1999). N-Adaptin domain is found in vesicle associated proteins involved in protein trafficking and clathrin-mediated endocytosis (Kirchhausen, 1999). SKP1 domain has been found in several proteins of SKP1 family required for cell cycle progression (Bai et al., 1996). A search for the known motifs and protein family signature sequences revealed the following main structural features of the SCC-112: RhoGEF domain (2-137 aa), Leucine Zipper pattern (166– 187 aa), N-adaptin domain (127 – 651 aa),
SKP1 domain (249 – 350 aa); three PEST sequence sites (597 –617, 1143 –1663 and 1216 –1227 aa); two tyrosine kinase phosphorylation sites (858 – 865 and 1030 – 1036 aa), a potential caspase 8 cleavage site (672 aa) (IETD#L) (Earnshaw et al., 1999), and six nuclear localization signature sequences (920 – 926, 1225– 1231, 1227 –1230, 1228– 1234, 1232 –1235 and 1251– 1257 aa). Genomic sequence (f120 kb) corresponding to SCC112 cDNA was located in the NCBI Human Genome Data Base (Genbank accession number AC022862) and Celera Human Genome Data Base (clone IDs GA_x2HTBL52FRR and GA_x2HTBL52FKR). Alignment of SCC-112 cDNA and genomic DNA sequences revealed that SCC-112 gene is
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composed of approximately 38 exons. The chromosome localization search at locus link in NCBI database showed that SCC-112/KIAA0648 is localized on chromosome 4p14, a locus previously predicted to harbor the tumor suppressor gene(s) (Schouten et al., 1990; Arribas et al., 1999; Kitamura et al., 2000; Feng et al., 2002). SCC-112 was expressed as a f150-kDa protein in normal human tissues representing colon, lung, ovary, breast and kidney (Fig. 1C). Interestingly, an additional f65-kDa band was detected in 95% of the human normal kidney tissues examined (n=22). The significance of the variant SCC-112 awaits the structure-functional analysis of this molecule in normal and tumor cells (also see below, Fig. 7). The expression of 150-kDa SCC-112 protein was also seen in four human cancer cell lines and their respective tumor xenografts grown in athymic mice (pancreatic, Aspc-1, Colo-357; prostate, PC-3; and breast, MDA-MB 435). Surprisingly, SCC-112 protein was expressed at lower levels in the tumor xenograft tissues as compared to corresponding cells in vitro (D.K. and U.K., unpublished data). The f65-kDa band was not detected in these cancer cells and tissues. The secondary structure prediction of the SCC-112 protein suggested it to be a nuclear protein. In agreement with this, SCC-112 protein was found to be localized to the nucleus in MDA-MB 435 cells (Fig. 2). As noted above, these cells do not express the f65-kDa protein, further confirming that f150-kDa SCC-112 is a nuclear protein. 3.2. Modulation of expression of SCC-112 during cell cycle As shown in Fig. 3A, SCC-112 mRNA levels were elevated in late S/early G2 (f3-fold) and G2 phases of cell cycle (f8-fold) as compared to unsynchronized MDAMB 435 cells. Consistent with this observation, mRNA level was f2-fold higher in nocodazole-treated cells (mitotic phase) as compared to unsynchronized cells (Fig. 3C, upper panel). SCC-112 protein levels were also elevated during S/G2 (f2– 3-fold, Fig. 3B) and G2/M phases
(f5-fold, Fig. 3C, lower panel). These data imply a cell cycle-dependent regulation of the expression of SCC-112 mRNA and protein. 3.3. Correlation between SCC-112 expression and apoptosis Previously, apoptosis has been demonstrated by an increase in the number of cells in sub-G1 phase (Kissil et al., 1999), and increased enzymatic activity of caspase-3, the executioner of apoptosis in most cell types (Thornberry and Lazebnik, 1998). To determine a role of SCC-112 in cell death, COS-1 cells were transiently transfected with the FLAG epitope-tagged SCC-112 cDNA expression vector or the control vector. At 36 h post-transfection, the number of cells in sub-G1 phase was quantified by flow cytometry. A dose-related increase in the number of cells in sub-G1 phase was observed as compared to vector transfectants (1 Ag/ well, f1.8-fold; 5 Ag/well, f2.4-fold) (Fig. 4, panels A and B). Similar observations were made in transiently transfected HeLa cells (data not shown). To further support a role of SCC-112 in apoptosis, caspase-3 activities were measured in transiently transfected COS-1 cells and HeLa cells. We have observed enhanced caspase-3 activity in both cell types. Representative data obtained in COS-1 transfectants are shown in Fig. 4C (increase in caspase-3 activity: 1 Ag/well, f1.6-fold; 5 Ag/well, f3.0-fold). To determine the effect of constitutive overexpression of SCC-112 on apoptosis, MDA-MB 231 breast cancer cells were stably transfected with pCR3.1SCC-112 expression vector or pCR3.1 control vector. Expression of the exogenous SCC-112 mRNA was confirmed by RT-PCR using a 5V-primer designed against a portion of the vector backbone and a 3V-primer representing the SCC-112 cDNA. The anticipated 523-bp cDNA fragment was detected in the SCC-112 transfectants and not in the vector-transfected or untransfected MDA-MB 231 cells (Fig. 5A). Following serum-withdrawal for 72 h, a f2 –3-fold increase in the number of sub-G1 cells in SCC-112 transfectants was observed as compared to vector transfectants (Fig. 5B;
Fig. 2. Nuclear localization of SCC-112. MDA-MB 435 human breast cancer cells were immunostained with anti-SCC-112 antibody, and SCC-112 protein expression was detected using Texas red-conjugated goat anti-rabbit IgG secondary antibody. Nuclei were visualized by DAPI staining procedure as described in Section 2.
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Fig. 3. Cell cycle-dependent modulation of SCC-112 mRNA and protein expression. (A) The total RNA was isolated from MDA-MB 435 cells at indicated times post-aphidicolin treatment and analyzed by Northern blotting using a radiolabeled SCC-112 cDNA as probe, followed by reprobing with a radiolabeled GAPDH cDNA probe. Autoradiographs were scanned and data quantified using the ImageQuant software program. The SCC-112 mRNA levels were normalized against GAPDH expression in the corresponding lanes and the fold changes in normalized expression relative to the unsynchronized cells were plotted. (B) The whole cell lysates of MDA-MB 435 cells were prepared at indicated times post-aphidicolin treatment and analyzed by Western blotting. The blots were reprobed with anti-actin antibody. The SCC-112 and actin levels were quantified as above. The SCC-112 protein levels were normalized against actin expression in the corresponding lanes and the fold changes in normalized expression relative to the unsynchronized cells were plotted. (C) Logarithmically growing MDA-MB 435 cells were treated with 100 ng/ml of nocodazole for 16 h (+) or left untreated (). SCC-112 mRNA (upper) or protein (lower) expression was quantified as in panels A and B, respectively. (D) Cell cycle distribution profiles of MDA-MB 435 cells at various times post-aphidicolin treatment or after treatment with nocodazole for 16 h. US, unsynchronized cells; G1, 50%; S, 35%; G2/M, 15%; post-aphidicolin 0 h; G1, 65%; S, 23%; G2/M, 12%; post-aphidicolin 6 h; G1, 6%; S, 21%; G2/M, 73%; post-aphidicolin 8 h; G1, 3%; S, 17%; G2/M, 80%; nocodazole, 16 h; G1, 2%; S, 18%; G2/M, 80%.
Fig. 4. Correlation between SCC-112 expression, apoptosis and caspase-3 activity in COS-1 cells. (A) Expression of FLAG-tagged SCC-112. COS-1 cells were transiently transfected with FLAG-tagged SCC-112 cDNA, or pCR3.1 vector, followed by immunoblotting with FLAG-M2 antibody (top). The same blot was reprobed with anti-h-tubulin antibody and anti-SCC-112 antiserum (bottom panels). (B) SCC-112 expression induces apoptosis. Flow cytometric analysis was performed at 36 h after transfection of COS-1 cells with the FLAG epitope-tagged SCC-112 cDNA or pCR3.1 vector DNA using indicated amount of the plasmid DNA per well, as explained in Section 2. (C) SCC-112 expression correlates with enhanced caspase-3 activity. The caspase-3 activity was measured in COS-1 transfectants as described in Section 2.
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3.4. Reduced SCC-112 mRNA expression in human breast and kidney tumor tissues
Fig. 5. Induction of apoptosis in MDA-MB 231 cells stably transfected with SCC-112 cDNA. (A) MDA-MB 231 cells were stably transfected with pCR3.1SCC-112 cDNA (SCC-112) or pCR3.1 vector (Vector). Expression of the exogenous SCC-112 mRNA was confirmed by RT-PCR amplication of a portion of the SCC-112 cDNA (523 bp). 18S primers (Ambion) were used as a positive control. (B) For apoptosis assay, stable transfectant cultures (SCC-112 or Vector) were switched to serum-free medium for 72 h, followed by flow cytometric measurement of the fraction of cells in sub-G1 as described in Section 2. Three independent experiments were performed. Data from a representative experiment are shown.
P=0.0001, n=3). Together, these data demonstrate that expression of the exogenous SCC-112 gene correlates with enhanced apoptosis in different cell types.
Pro-apoptotic molecules may possess potential tumor suppressor activity correlating, in part, with a relatively low expression in tumor tissues as compared to normal tissues. To this end, we examined the mRNA expression profile of SCC-112 in normal and tumor tissues from various cancer patients. Quantification data showing foldchanges in SCC-112 mRNA expression in normal versus tumor tissue of individual patients are shown in Fig. 6. A majority of normal breast and kidney tissues had a higher steady state level of SCC-112 mRNA as compared to corresponding tumor tissues (breast, 76%, n=50; kidney, 90%, n=20). As shown in Table 1, the average ratio of the mRNA expression in normal to tumor tissue for breast and kidney were significantly greater than one ( P<0.0001), and the 95% confidence intervals for the ratios were substantially above one. On the same blot, SCC-112 mRNA expression was found to be lower in the three metastatic breast tumor tissues as compared to corresponding normal tissues ( P<0.05) (data not shown). For uterus, the average ratio of normal to tumor tissue was 0.90, and the 95% confidence intervals for the ratios were below one. The average ratios of SCC-112 expression for colon and stomach were not statistically significantly different from one at 5%
Fig. 6. Reduced SCC-112 mRNA expression in human breast and kidney tumor tissues. A Cancer Profiling Array blot (Clontech) containing cDNAs from normal, tumor and metastatic tissues from 241 patients was hybridized with 32P-labeled SCC-112 cDNA probe, followed by reprobing with ubiquitin cDNA. The SCC-112 mRNA expression levels observed in normal (N) and matched tumor (T) tissues were normalized with the corresponding ubiquitin mRNA expression levels and fold change in normalized SCC-112 mRNA expression in individual patients (N/T ratio) was plotted as shown. n, total number of patients.
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Table 1 Statistical analysis of the fold change in SCC-112 mRNA expression level in tumor tissue versus corresponding normal tissue from individual cancer patients Tumor type
Number of patients (n)
Mean N/Ta (log ratio)
Mean N/Ta (ratio)
Standard error (log ratio)
95% Confidence interval (log ratio)
95% Confidence interval (ratio)
P-value (two-sided)
Kidney Breast Rectum Thyroid Ovary Lung Colon Stomach Uterus
20 50 18 6 14 21 35 27 42
0.20 0.20 0.11 0.10 0.05 0.05 0.06 0.08 0.10
1.22 1.22 1.11 1.10 1.05 0.95 0.94 0.92 0.90
0.03 0.05 0.07 0.10 0.10 0.07 0.05 0.07 0.04
(0.13, 0.26) (0.10, 0.29) (0.04, 0.25) (0.15, 0.35) (0.16, 0.26) (0.19, 0.10) (0.16, 0.04) (0.22, 0.06) (0.19, 0.01)
(1.14, (1.11, (0.96, (0.86, (0.85, (0.83, (0.86, (0.81, (0.82,
<0.0001 <0.0001 0.13 0.35 0.63 0.50 0.25 0.25 0.02
a
1.30) 1.34) 0.25) 1.42) 1.30) 1.10) 1.04) 1.06) 0.99)
N/T 3 SCC-112 mRNA.
level. Transcriptional silencing of the tumor suppressor genes has been attributed, in part, to the promoter methylation (Baylin and Herman, 2000). Whether SCC-112 mRNA level is elevated in tumor cells following treatment of cells with a DNA methylation inhibitor remains to be seen. These data indicate a notable decrease in the mRNA expression of SCC-112 in a majority of the human breast and kidney tumor tissues examined. 3.5. Reduced SCC-112 protein expression in human renal tumor specimens We examined SCC-112 protein expression in a group of renal tumor and normal adjacent kidney tissues. Renal tissues were chosen based on the SCC-112 mRNA expression data (Fig. 6 and Table 1), and the availability of the frozen tumor tissues and matched normal adjacent tissues. Among the eighteen available independent paired sets of renal tumor and matched normal adjacent tissues analyzed, twelve pairs showed increased expression of f150-kDa SCC-112 protein in the normal renal tissue as compared to matched renal tumor tissue (1.2 – 10-fold, n=6; >10-fold, n=6). Representative data are shown in Fig. 7 (paired samples from patient #7, #5 and #1). Interestingly, a higher
Fig. 7. Reduced SCC-112 protein expression in human renal tumor tissues. The tissues were homogenized and whole cell lysates were analysed by western blotting using anti-SCC-112 antibody, followed by reprobing with anti-actin antibody, as explained in Section 2. Representative data are shown. Pt. I. D., patient identification number; T, renal cell carcinoma; N, normal adjacent tissue.
level of the f150-kDa protein was seen in some tumor tissues as compared to matched normal adjacent tissues (for example, patient #6, Fig. 7). In addition to f150-kDa protein, a prominent f65-kDa band was present in 17 out of the 18 normal renal tissues examined (94%) (Fig. 7). In contrast, only 3 of the 18 matched renal tumor tissues showed expression of the variant f65-kDa SCC-112 (17%) (data not shown). Remarkably, in three sets of paired samples (#5, #19 and #21), both protein bands were undetectable in the renal tumor tissues and were present in the normal adjacent tissues (see paired samples from patient #5 shown in Fig. 7). The appearance of a smaller molecular weight SCC-112 (f65 kDa) in a majority of the normal kidney tissues is intriguing at this time. The 65-kDa protein does not appear to be a non-related cross-reactive protein because its expression is undetectable in a number cell types, in all of which the f150-kDa SCC-112 protein is detectable, e.g., COS-1, and human cancer cell lines (breast, MDA-MB 231, MDA-MB 435; prostate, PC-3, LNCap; renal, ACHN, 769-P; pancreatic, Aspc-1, Colo 357; and Hela cells, D.K. and U.K. unpublished data).
4. Conclusions This study is the first report of SCC-112, a novel cell cycle-regulated molecule. SCC-112 is an inducer of cell death, and it may also be involved in the regulation of cell growth and proliferation based on the following observations: (1) expression of exogenous SCC-112 stimulates apoptosis in COS-1 cells and human breast cancer cells; (2) expression of exogenous SCC-112 enhances caspase-3 activity in COS-1 cells; (3) SCC-112 mRNA expression is significantly high in human normal breast and renal tissues as compared to corresponding tumor specimens; and (4) SCC-112 protein expression is high in a majority of the available human normal renal tissues as compared to the matched tumor specimens. Interestingly, chromosomal location of SCC-112 at 4p14 is in agreement with a recently reported linkage of a subset of hereditary nasopharyngeal carcinoma to a major susceptibility locus D4S405 on chromosome 4p12– p15 (Feng et al., 2002). Future inves-
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tigations will determine the role of SCC-112 in normal cells, and potential diagnostic and therapeutic usefulness of SCC112 in human cancers.
Acknowledgements The sequences reported in this study have been deposited in the GenBank database (KAS-112, accession no. AF003254 and SCC-112, accession no. AF294791). Histologically confirmed, frozen human renal tumor and matched normal adjacent tissues were kindly provided by the Cooperative Human Tissue Network (CHTN) resource of the National Cancer Institute, National Institutes of Health. The statistical analysis was performed at the Biostatistics Shared Resource; the human breast tumor cell lines and COS-1 cells were obtained from the Tissue Culture Shared Resource; and automated DNA sequencing was performed at the Macromolecular Synthesis and Sequencing Shared Resource of the Lombardi Cancer Center, supported by National Institutes of Health Grant P30-CA51008. This work was supported by National Institutes of Health grants CA68322 and CA74175, and by NeoPharm. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389 – 3402. Armitage, P., Berry, G., 2001. Statistical Methods in Medical Research, 3rd ed. Blackwell, Malden, MA. Arribas, R., Ribas, M., Risques, R.A., et al., 1999. Prospective assessment of allelic losses at 4p14 – 16 in colorectal cancer: two mutational patterns and a locus associated with poorer survival. Clin. Cancer Res. 11, 3454 – 3459. Bai, C., Sen, P., Hofmann, K., Ma, L., Goebl, M., Harper, J.W., Elledge, S.J., 1996. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86, 263 – 274. Baylin, S.B., Herman, J.G., 2000. DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet. 16, 168 – 174. Brown, B., et al., 1996. The University of Texas M.D. Anderson Cancer Center, Department of Biomathematics. Clifford, S.C., Maher, E.R., 2001. Von Hippel-Lindau disease: clinical and molecular perspectives. Adv. Cancer Res. 82, 85 – 105. Earnshaw, W.C., Martins, L.M., Kaufmann, S.H., 1999. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 68, 383 – 424. Feng, B.J., Huang, W., Shugart, Y.Y., et al., 2002. Genome-wide scan for familial nasopharyngeal carcinoma reveals evidence of linkage to chromosome 4. Nat. Genet. 31, 395 – 399. Geck, P., Szelei, J., Jimenez, J., Sonnenschein, C., Soto, A.M., 1999. Early gene expression during androgen-induced inhibition of proliferation of prostate cancer cells: a new suppressor candidate on chromosome 13, in the BRCA2-Rb1 locus. J. Steroid Biochem. Mol. Biol. 68, 41 – 50. Green, D.R., Knight, R.A., Melino, G., Finazzi-AgroV , A., Orrenius, S., 2004. Ten years of publication in cell death. Cell Death Differ. 11, 2 – 3.
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SCC-112, a novel cell cycle-regulated molecule, exhibits reduced expression in human renal carcinomas Deepak Kumar a, Isamu Sakabe a, Sonal Patel a, Ying Zhang b, Imran Ahmad c, Edmund A. Gehan b, Theresa L. Whiteside d, Usha Kasid a,* a
Department of Radiation Medicine and Biochemistry and Molecular Biology, Lombardi Cancer Center, Georgetown University, E208, Research Building, 3970 Reservoir Road, NW, Washington, DC 20007, USA b Biomathematics and Biostatistics, Lombardi Cancer Center, Georgetown University, Washington, DC 20007, USA c NeoPharm, Inc., Lake Forest, IL 60045, USA d Department of Pathology, The Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA Received 17 July 2003; received in revised form 25 November 2003; accepted 12 December 2003 Received by F. Salvatore
Abstract We report here the identification and an initial characterization of a novel cell cycle-regulated molecule, SCC-112. SCC-112 cDNA (6744 bp) encodes a longest open reading frame (ORF) comprised of 1297 amino acids, representing a f150-kDa nuclear protein. SCC-112 mRNA and protein levels were relatively high during the G2/M phase of the cell cycle in MDA-MB 435 breast cancer cells. Transient expression of SCC-112 cDNA in COS-1 cells led to an increase in the number of cells in sub-G1 phase and enhanced activity of caspase-3, a downstream effector of apoptosis. Stable transfection of SCC-112 cDNA in MDA-MB 231 breast cancer cells also led to an increase in the number of cells in sub-G1 phase (f2 – 3-fold), indicative of apoptosis. The examination of the paired sets of human normal and tumor tissues revealed that the SCC-112 mRNA level was significantly high in normal breast and kidney tissues as compared to the corresponding primary tumor tissues ( P<0.0001; breast, n=50, and kidney, n=20). Consistent with these observations, SCC-112 protein expression (150 kDa) was high in a majority of the normal renal tissues examined as compared to the matched renal tumor tissues (67%, 1.2-fold to > 10-fold, n=18). Taken together, these findings suggest that the SCC-112 gene expression is likely to be associated with normal cell growth and proliferation. D 2004 Published by Elsevier B.V. Keywords: SCC-112; cDNA and predicted amino acid sequences; Subcellular localization; G2/M phase; Cell death; Tumor vs. normal tissue expression
1. Introduction Cells undergo apoptosis or programmed cell death in response to a variety of environmental or stress-related stimuli. Several apoptotic proteins and signaling pathways have been defined (Green, 2004). Cancer cells often exhibit defects in radiation or chemotherapeutic drug-induced apo-
Abbreviations: RCC, renal cell carcinoma; FBS, fetal bovine serum; PMSF, phenylmethylsulphonyl fluoride; DAPI, 4V,6-diamidino-2-phenyindole dilactate; RT-PCR, reverse transcription-polymerase chain reaction; ORF, open reading frame; PEST sequence, a region rich in proline, glutamic acid, serine and threonine. * Corresponding author. Tel.: +1-202-687-2226. E-mail address: [email protected] (U. Kasid). 0378-1119/$ - see front matter D 2004 Published by Elsevier B.V. doi:10.1016/j.gene.2003.12.013
ptosis, implicating an important role of the apoptogenic signals in regulation of normal cell growth and proliferation (Johnstone et al., 2002). The apoptosis inducing molecules are known to play roles in the regulation of damageresponsive cell cycle check points, genomic instability, and DNA repair and recombination (Levine, 1997). Many of these proteins also have an important role in tumor growth suppression, implied, in part, by a reduced level of expression in primary tumors as compared to matched normal adjacent tissues (Soengas et al., 2001). Renal cell carcinomas (RCC) have been the focus of investigation of several candidate tumor suppressor genes including the VHL, the FHIT and the RASSF1A genes (Clifford and Maher, 2001). These and other discoveries have been guided, in part, by a high frequency of loss of
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heterozygosity at chromosome 3p21 – p22 in RCC, and cancers of the lung and breast (Lerman and Minna, 2000). The loss of chromosome 4 has been shown to correlate positively with high tumor stage in clear-cell RCCs (Reutzel et al., 2001), and 4p has been predicted to harbor the tumor suppressor gene(s) (Schouten et al., 1990; Wu et al., 1995; Arribas et al., 1999; Kitamura et al., 2000; Feng et al., 2002). Here, we report the identification of a novel cell cycle-regulated, pro-apoptotic molecule, SCC-112. SCC112 gene is located on chromosome 4p14. Our data suggest a potential correlation between the expression of SCC-112 and normal renal cell growth and proliferation.
2. Materials and methods 2.1. Cell culture Human breast cancer cell lines, MDA-MB 435 and MDA-MB 231, and COS-1 cells were grown in DMEM, supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 25 Ag/ml gentamicin. All culture reagents were purchased from Invitrogen Life Technologies. The cells were grown in 75-cm2 tissue culture flasks in a humidified atmosphere of 5% CO2 and 95% air at 37 jC. 2.2. Cloning and sequence analysis of SCC-112 cDNA A 318-bp novel cDNA fragment (KAS-112) was isolated from a human head and neck squamous carcinoma cell line as described earlier (Patel et al., 1997). Homology search of the National Center for Biotechnological Information (NCBI) GenBank and Human EST databases using the KAS-112 cDNA sequence (GenBank accession no. AF003254) revealed two partially overlapping clones, KIAA0648 (5177 bp, GenBank accession no. AB014548) and an EST sequence (2247 bp, GenBank accession no. AI655954). The KIAA0648 cDNA clone was procured from Kazusa DNA Research Institute, Japan (Ohara et al., 1997), and the EST clone was purchased from Incyte Genomics (Palo Alto, CA). Both strands of the EST and KIAA0648 clones were sequenced using automated DNA sequencer (Applied Biosystems Prism 377) and sequences were assembled using Autoassembler program (Applied Biosystems) (GenBank accession no. AF294791). BLAST sequence database, conserved domain prediction and open reading frame (ORF) searches were performed using the NCBI programs (Altschul et al., 1997). The prediction of the possible nature of the protein including localization based on structural characteristics was performed by pSORT (Nakai and Kanehisa, 1992). 2.3. Generation of anti-SCC-112 antibody A rabbit antiserum was generated against a synthetic peptide representing 20 amino acid residues at the C-
terminus of SCC-112 ORF (amino acids 1278 – 1297, KLQDLAKKAAPAERQIDLQR). The peptide was coupled to keyhole limpet hemocyanin and injected into rabbits. Custom antisera production service was provided by Zymed laboratories (San Francisco, CA). 2.4. Cell synchronization Logarithmically growing MDA-MB 435 cells were treated with 4 Ag/ml aphidicolin (Sigma) for 24 h. The cells were washed thoroughly and medium was replaced with fresh medium and incubation continued for 0, 4 and 8 h, representing G1/S phase, S phase and G2/M phase-enriched cell populations, respectively. Alternatively, cells were treated with 100 ng/ml nocodazole (Sigma) for 16 h to arrest the cells in M phase. The cells were fixed in 70% ethanol, washed with PBS, followed by RNase treatment and propidium iodide staining. The cell cycle distribution profiles were determined by the flow cytometric assay using FACsort (Becton Dickinson). 2.5. Northern blotting and hybridization The total RNA was isolated from MDA-MB 435 cells arrested at various phases of the cell cycle using Trizol reagent according to the manufacturer’s instructions (Invitrogen Life technologies), followed by electrophoresis and transfer to the nylon membrane. Multi-tissue northern blots containing poly A+RNA from human adult and fetal tissues were purchased from Clontech. Northern blots were hybridized using 32P-labeled SCC-112 cDNA insert (5177 bp) as a probe, followed by reprobing with radio-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or h-actin cDNA (Clontech). The autoradiographs were scanned and bands were quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). 2.6. Western blot analysis MDA-MB 435 cells were washed with ice-cold PBS and scrapped in lysis solution containing 20 mM Tris (pH 8.0), 150 mM NaCl, 1% NP-40 (w/v), 10% glycerol (w/v), 1 mM phenylmethylsulphonyl fluoride (PMSF), 20 Ag/ml aprotinin and 20 Ag/ml leupeptin. The cells were lysed for 45 min with vigorous shaking at 4 jC and centrifuged at 12 000g for 15 min at 4 jC. Thirty to fifty micrograms of the total protein was analyzed by Western blotting using 1:2000 dilution of the SCC-S2 antiserum. The frozen human normal and tumor tissues were homogenized in RIPA lysis buffer (150 mM NaCl, pH 7.5; 1% (w/v) sodium deoxycholate; 1% (v/v) Triton X-100; 0.1% (w/v) SDS, 2 mM PMSF, 20 Ag/ml aprotinin and 20 Ag/ ml leupeptin). After incubation on ice for 30 min, the tissue extracts were centrifuged at 12 000g for 15 min at 4 jC and the supernatant was used for Western blot analysis.
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2.7. Immunofluorescence microscopy MDA-MB 435 cells were grown on cover slips. The cells were fixed in chilled methanol for 10 min, followed by rinsing in PBS and permeabilization with 0.01% Triton X100 for 30 s. Non-specific binding was blocked with 1% BSA in PBS (w/v) for 30 min. The cells were incubated with a 1:1000 dilution of the SCC-112 antiserum in 1% BSA/PBS for 1 h, followed by three washings of 10 min each with PBS. Texas red-conjugated goat anti-rabbit secondary antibody (Jackson Immunoresearch) (1:200 dilution in 1% BSA in PBS for 1 h) was used to detect the antigen – antibody complex. After three washings of 10 min each with PBS, the nuclei were stained with 4V,6-diamidino-2-phenyindole dilactate (DAPI; Sigma) for 1 min. The cover slips were rinsed with PBS, mounted on a glass slide with cells facing the slide in fluoromount-G (Southern Biotechnology Associates, Birmingham, AL) and visualized under a fluorescence microscope (Carl Zeiss). 2.8. Expression cloning of SCC-112 cDNA A portion of the full length SCC-112 cDNA (nucleotides 216 – 4125, AF294791) coding for the predicted longest ORF was cloned into a eukaryotic expression vector, pCR3.1 (Invitrogen). Briefly, a 1696-bp fragment was amplified by PCR using the EST clone (AI655954) as a template and primers P1 (5V-GCG-GGC-CCG-GAC-AAGATC-ACC-ACG-GAC-GAG-3V) and P2 (5V-CGG-GCCCGC-GAG-AAA-AGT-GTG-AAG-TAT-GA-3V). In addition, a 2558-bp fragment was amplified by PCR using KIAA0648 clone and primers P3 (5V-GCT-TAT-ATT-ACTTAT-ATG-3V) and P4 (5V-GCA-GAA-GCT-GGA-GCCTGC-3V). The two amplified products were digested with BamHI, and the ligated cDNA was cloned into a TA cloning vector pCR2.1 (Invitrogen). The ligated cDNA (3972 bp) served as a template for a third PCR reaction using primers P5 (5V-ATC-ACC-ACG-GAC-GAG-ATG-3V) and P6 (5VTTA-CCT-TTG-TAA-GTC-AAT-3V). The amplified cDNA fragment (3909 bp) representing nucleotides 216 –4125 of the full-length SCC-112 cDNA was cloned into the pCR3.1 expression vector and the sequence was verified by automated DNA sequencing. To construct the N-terminal FLAG epitope-tagged SCC112 cDNA expression vector, SCC-112 cDNA encoding the open reading frame was amplified by polymerase chain reaction using pCR3.1SCC-112 (described above) as template. The 5V-primer used for amplification was 5VTCC-ACC-ATG-GAT-TAC-AAG-GAT-GAC-GAC-GATAAG-ATG-ATC-AAA-CGC-CTG-AAG-ATG-3V and the 3V-primer was the primer P6 described above. The sequence underlined in the 5V-primer corresponds to the FLAG octapeptide. The amplified product was cloned into the mammalian expression vector pCR3.1 according to the instruction manual (Invitrogen) and verified by automated sequencing.
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2.9. Transient and stable transfections, and expression of SCC-112 cDNA In the transient transfection experiments, COS-1 cells were seeded in six-well plates (1 – 3105 cells/well, in duplicate or triplicate) and transfected with the expression vector pCR3.1 or recombinant vector containing FLAGtagged SCC-112 cDNA (1– 5 Ag/well) using the LipofectAMINE 2000 (Invitrogen). Thirty-six hours after transfection, cells were harvested and lysed at 4 jC for 30 min in lysis buffer (100 mM HEPES, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 Ag/ml each of aprotinin and leupeptin), followed by microcentrifugation for 15 min at 4 jC. Protein concentration was determined using Coomassie G250 protein assay reagent (Pierce). Fifty micrograms of the cell lysate was resolved by 7.5% SDS-PAGE, transferred to Immobilon-P membrane (Millipore) and immunoblotted with 1 Ag/ml mouse monoclonal FLAG-M2 antibody (Sigma). Enhanced chemiluminescence method (Luminol, NEN Life Science Products) was used to detect the signal. The blot was reprobed with mouse monoclonal h-tubulin antibody (Santa Cruz). MDA-MB 231 human breast cancer cells were stably transfected with pCR3.1SCC-112 or pCR3.1 vector. The cells were subcultured in DMEM containing 10% FBS to achieve 70 –80% confluency per T-75 flask at 24 h, washed once with serum-free medium and 4.4 ml of DMEM was added to the flask. Sixty microliters of Fugene 6 reagent (Roche Diagnostics) was diluted with 600 Al of serum-free DMEM. Ten micrograms of plasmid DNA (pCR3.1SCC-112 or pCR3.1 vector) was added to the diluted Fugene 6 reagent and the mixture was incubated for 45 min. The DNA and Fugene 6 mixture was added dropwise to the flask and mixed by gentle swirling. After 48 h, the cells were subcultured (1:6) in DMEM containing 10% FBS, 25 Ag/ml gentamicin, and 800 Ag/ml geniticin (G418, Invitrogen Life Technologies). Selection of stable transfectants in G418-containing medium continued for 2 weeks. At least 100 G418-resistant colonies were pooled and the mass culture was maintained in DMEM supplemented with 10% FBS, 25 Ag/ml gentamicin and 800 Ag/ml of G418. The expression of SCC-112 cDNA in stable transfectants was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) using the Titan one tube RT-PCR kit (Roche Diagnostics) (Kumar et al., 2000). Total RNA was isolated from SCC-112 or vector transfectants and untransfected MDA-MB 231 cells using the Trizol reagent. For the RT-PCR, the 5V-primer used was the T7 primer built into the expression vector pCR3.1 backbone (Invitrogen) and the 3V-primer P7, 5V-GTT-GATCAC-TGA-GAA-GAG-3Vwas designed against a portion of the SCC-112 cDNA (nucleotides 639 –622, AF294791). The reactions in the absence of reverse transcriptase and in the presence of the 18S primers (Ambion) were used as controls. The reaction products were resolved on 1.5% agarose gel and detected by ethidium bromide staining.
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2.10. Apoptosis and caspase-3 activity assays Apoptosis was determined by the flow cytometric analysis of COS-1 and MDA-MB 231 transfectants. At 36 h after the transient transfection of COS-1 cells with various amounts of the FLAG-tagged SCC-112 cDNA or vector, floating cells were pooled with the adherent cells collected by trypsinization and fixed in 2 ml of 75% ethanol for at least 30 min at 4 jC. The percentage of cells containing subG1 DNA content, indicative of apoptosis, was determined by the flow cytometric approach. 1-2105 MDA-MB 231 stable transfectants were seeded per well, in triplicate, in a six-well plate in DMEM containing 10% FBS. After 24 h, cells were switched to serum-free medium for various times. The floating cells were pooled with the adherent cells, followed by the flow cytometric analysis. For measurement of the caspase-3 activity in COS-1 transfectants, the pooled cells were lysed in cell lysis buffer (Clontech) and the caspase-3 activity was determined using the Apoalertk Caspase-3 assay kit (Clontech). 2.11. Hybridization, quantification and statistical analysis of cancer profiling array The Cancer Profiling Array blot (Clontech) spotted with 241 cDNAs pairs from matched normal and tumor tissues from individual patients, representing 13 different tissues, was hybridized using 32P-labeled SCC-112 cDNA insert (5177 bp) as per manufacturer’s user manual (PR11929). After hybridization, the membrane was reprobed with ubiquitin cDNA provided as a loading control. The autoradiographs were scanned and signals were quantified using ImageQuant software (Molecular Dynamics). For statistical significance, the sample sizes were calculated by STPLAN software package (Brown et al., 1996). The ratio of the ubiquitin-normalized SCC-112 mRNA expression values in normal and matched tumor tissue was transformed to natural logarithm. Under the null hypothesis of no difference in gene expression levels between normal and tumor tissues, the average ratio should be one and the average of the natural logs of the ratios should equal 0. To test the null hypothesis for each type of tumor, a one-sample t-test of the natural logs of the ratios was performed and two-sided P values were calculated. 95% confidence intervals were calculated based on the natural log transformations of the data and transformed back to obtain the confidence limits on the ratio scale (Armitage et al., 2001).
3. Results and discussion 3.1. Sequence identification, expression and localization of SCC-112 The complete SCC-112 cDNA sequence deduced from the three partially overlapping clones, KAS-112, KIAA0648
and EST, is 6744 bp in length (Fig. 1A). A small portion of the sequence present at the 3V-ends of the KAS-112 (265 – 318 bp) and EST (1881 – 2247 bp) clones was not found in the KIAA0648 clone. Since this portion of the KAS-112 and EST sequences (hatched boxes, Fig. 1A) is present in the genomic SCC-112 DNA (NCBI Human Genome Data Base, Genbank accession number AC022862), it is likely that KAS-112/EST sequence represents the unspliced, partial SCC-112 mRNA. To further confirm the SCC-112 cDNA sequence at the junction between the KAS-112/EST and KIAA0648 clones, total RNA was isolated from MDA-MB 231 cells and RT-PCR was performed using a EST cDNA sequence-specific 5V-primer (P8, 5V-CCG-TCA-TGC-CACAGC-TTG-3V, at position 956 –973 bp), and a KIAA0648 sequence-specific 3V-primer (P9, 5V-GGC-TTC-TGA-ATCAAT-GTG-CAC-AGG-TGC-G-3V, at position 427– 400 bp). The sequence of the anticipated 1036-bp amplified cDNA product matched the overlapping cDNA sequence obtained from these clones (data not shown). Northern blot analysis using the KIAA0648 cDNA insert as a probe identified two transcripts of f7 kb and f8 kb in normal human adult tissues (Fig. 1B). Similar results were obtained using fetal tissue and cancer cell RNA blots (Clontech) (data not shown). These two transcripts were also seen in MDA-MB 231 cells using a 5V-fragment of the EST sequence as a probe (Fig. 1A, 320-bp EcoRI fragment) (data not shown). The SCC-112 cDNA codes for a novel longest ORF comprised of 1297 amino acids (aa). The poly-adenylation signal sequence could be located at the 3V untranslated region. We did not find a stop codon upstream of the first ATG; however, the antiserum generated against a synthetic C-terminus SCC-112 peptide detected a single f150-kDa band by immunoprecipitation and immunoblotting in lysates from human A549 lung carcinoma cells, corresponding to the predicted size of the protein (146 kDa) (data not shown). The pre-immune serum and mock lysates served as controls in these experiments. In other experiments, COS-1 cells were transiently transfected with expression vector containing the FLAG epitope-tagged SCC-112 cDNA coding for the ORF. SCC-112 expression was detected by immunoblotting with anti-FLAG antibody, followed by a reprobe using the anti-SCC-112 antiserum. The mobility of the FLAG-tagged SCC-112 overlapped with the total SCC112 protein expressed in COS-1 cells (see below, Fig. 4A). A protein homology search indicated a partial homology of SCC-112 to AS3 (69% identity), an androgen-inducible gene located on chromosome 13 in the BRCA2-Rb1 locus, having a role in proliferative shutoff (Geck et al., 1999). SCC-112 aa sequence was similar to SPO76 (22% identity), a chromosome morphogenesis protein in Sorodaria macrospora (Van Heemst et al., 1999), and BimD (22% identity), a Aspergillus nidulans protein involved in mitosis (Van Heemst et al., 2001). SCC-112 ORF also showed a limited homology to yeast Pds5p (23% identity), a chromosome maintenance protein involved in chromosome cohesion
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Fig. 1. Sequence identification, and mRNA and protein expression of SCC-112. (A) Schematic maps of complete SCC-112 cDNA and overlapping partial clones are shown. The gray box represents the coding region of cDNA. The black boxes represent the 5V- and 3V-untranslated regions of cDNA. The hatched box shown in the KAS-112 and EST clones represents the adjacent genomic DNA sequence. E, EcoRI. (B) Expression of SCC-112 mRNA in normal tissues. Normal human adult tissue mRNA blots were probed with radiolabeled SCC-112 cDNA fragment (1568 – 6744 bp). The blots were reprobed with h-actin cDNA. (C) Expression of SCC-112 protein in normal human tissues. Western blot analysis was performed using approximately 30 Ag of total protein in whole cell lysates from various human tissues and the blots were reprobed with anti-actin antibody.
during mitosis (Panizza et al., 2000). Conserved domain search in NCBI database revealed partial homology of SCC-112 amino acid sequence to domains such as NAdaptin (Kirchhausen et al., 1989) and SKP1 (Stebbins et al., 1999). N-Adaptin domain is found in vesicle associated proteins involved in protein trafficking and clathrin-mediated endocytosis (Kirchhausen, 1999). SKP1 domain has been found in several proteins of SKP1 family required for cell cycle progression (Bai et al., 1996). A search for the known motifs and protein family signature sequences revealed the following main structural features of the SCC-112: RhoGEF domain (2-137 aa), Leucine Zipper pattern (166– 187 aa), N-adaptin domain (127 – 651 aa),
SKP1 domain (249 – 350 aa); three PEST sequence sites (597 –617, 1143 –1663 and 1216 –1227 aa); two tyrosine kinase phosphorylation sites (858 – 865 and 1030 – 1036 aa), a potential caspase 8 cleavage site (672 aa) (IETD#L) (Earnshaw et al., 1999), and six nuclear localization signature sequences (920 – 926, 1225– 1231, 1227 –1230, 1228– 1234, 1232 –1235 and 1251– 1257 aa). Genomic sequence (f120 kb) corresponding to SCC112 cDNA was located in the NCBI Human Genome Data Base (Genbank accession number AC022862) and Celera Human Genome Data Base (clone IDs GA_x2HTBL52FRR and GA_x2HTBL52FKR). Alignment of SCC-112 cDNA and genomic DNA sequences revealed that SCC-112 gene is
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composed of approximately 38 exons. The chromosome localization search at locus link in NCBI database showed that SCC-112/KIAA0648 is localized on chromosome 4p14, a locus previously predicted to harbor the tumor suppressor gene(s) (Schouten et al., 1990; Arribas et al., 1999; Kitamura et al., 2000; Feng et al., 2002). SCC-112 was expressed as a f150-kDa protein in normal human tissues representing colon, lung, ovary, breast and kidney (Fig. 1C). Interestingly, an additional f65-kDa band was detected in 95% of the human normal kidney tissues examined (n=22). The significance of the variant SCC-112 awaits the structure-functional analysis of this molecule in normal and tumor cells (also see below, Fig. 7). The expression of 150-kDa SCC-112 protein was also seen in four human cancer cell lines and their respective tumor xenografts grown in athymic mice (pancreatic, Aspc-1, Colo-357; prostate, PC-3; and breast, MDA-MB 435). Surprisingly, SCC-112 protein was expressed at lower levels in the tumor xenograft tissues as compared to corresponding cells in vitro (D.K. and U.K., unpublished data). The f65-kDa band was not detected in these cancer cells and tissues. The secondary structure prediction of the SCC-112 protein suggested it to be a nuclear protein. In agreement with this, SCC-112 protein was found to be localized to the nucleus in MDA-MB 435 cells (Fig. 2). As noted above, these cells do not express the f65-kDa protein, further confirming that f150-kDa SCC-112 is a nuclear protein. 3.2. Modulation of expression of SCC-112 during cell cycle As shown in Fig. 3A, SCC-112 mRNA levels were elevated in late S/early G2 (f3-fold) and G2 phases of cell cycle (f8-fold) as compared to unsynchronized MDAMB 435 cells. Consistent with this observation, mRNA level was f2-fold higher in nocodazole-treated cells (mitotic phase) as compared to unsynchronized cells (Fig. 3C, upper panel). SCC-112 protein levels were also elevated during S/G2 (f2– 3-fold, Fig. 3B) and G2/M phases
(f5-fold, Fig. 3C, lower panel). These data imply a cell cycle-dependent regulation of the expression of SCC-112 mRNA and protein. 3.3. Correlation between SCC-112 expression and apoptosis Previously, apoptosis has been demonstrated by an increase in the number of cells in sub-G1 phase (Kissil et al., 1999), and increased enzymatic activity of caspase-3, the executioner of apoptosis in most cell types (Thornberry and Lazebnik, 1998). To determine a role of SCC-112 in cell death, COS-1 cells were transiently transfected with the FLAG epitope-tagged SCC-112 cDNA expression vector or the control vector. At 36 h post-transfection, the number of cells in sub-G1 phase was quantified by flow cytometry. A dose-related increase in the number of cells in sub-G1 phase was observed as compared to vector transfectants (1 Ag/ well, f1.8-fold; 5 Ag/well, f2.4-fold) (Fig. 4, panels A and B). Similar observations were made in transiently transfected HeLa cells (data not shown). To further support a role of SCC-112 in apoptosis, caspase-3 activities were measured in transiently transfected COS-1 cells and HeLa cells. We have observed enhanced caspase-3 activity in both cell types. Representative data obtained in COS-1 transfectants are shown in Fig. 4C (increase in caspase-3 activity: 1 Ag/well, f1.6-fold; 5 Ag/well, f3.0-fold). To determine the effect of constitutive overexpression of SCC-112 on apoptosis, MDA-MB 231 breast cancer cells were stably transfected with pCR3.1SCC-112 expression vector or pCR3.1 control vector. Expression of the exogenous SCC-112 mRNA was confirmed by RT-PCR using a 5V-primer designed against a portion of the vector backbone and a 3V-primer representing the SCC-112 cDNA. The anticipated 523-bp cDNA fragment was detected in the SCC-112 transfectants and not in the vector-transfected or untransfected MDA-MB 231 cells (Fig. 5A). Following serum-withdrawal for 72 h, a f2 –3-fold increase in the number of sub-G1 cells in SCC-112 transfectants was observed as compared to vector transfectants (Fig. 5B;
Fig. 2. Nuclear localization of SCC-112. MDA-MB 435 human breast cancer cells were immunostained with anti-SCC-112 antibody, and SCC-112 protein expression was detected using Texas red-conjugated goat anti-rabbit IgG secondary antibody. Nuclei were visualized by DAPI staining procedure as described in Section 2.
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Fig. 3. Cell cycle-dependent modulation of SCC-112 mRNA and protein expression. (A) The total RNA was isolated from MDA-MB 435 cells at indicated times post-aphidicolin treatment and analyzed by Northern blotting using a radiolabeled SCC-112 cDNA as probe, followed by reprobing with a radiolabeled GAPDH cDNA probe. Autoradiographs were scanned and data quantified using the ImageQuant software program. The SCC-112 mRNA levels were normalized against GAPDH expression in the corresponding lanes and the fold changes in normalized expression relative to the unsynchronized cells were plotted. (B) The whole cell lysates of MDA-MB 435 cells were prepared at indicated times post-aphidicolin treatment and analyzed by Western blotting. The blots were reprobed with anti-actin antibody. The SCC-112 and actin levels were quantified as above. The SCC-112 protein levels were normalized against actin expression in the corresponding lanes and the fold changes in normalized expression relative to the unsynchronized cells were plotted. (C) Logarithmically growing MDA-MB 435 cells were treated with 100 ng/ml of nocodazole for 16 h (+) or left untreated (). SCC-112 mRNA (upper) or protein (lower) expression was quantified as in panels A and B, respectively. (D) Cell cycle distribution profiles of MDA-MB 435 cells at various times post-aphidicolin treatment or after treatment with nocodazole for 16 h. US, unsynchronized cells; G1, 50%; S, 35%; G2/M, 15%; post-aphidicolin 0 h; G1, 65%; S, 23%; G2/M, 12%; post-aphidicolin 6 h; G1, 6%; S, 21%; G2/M, 73%; post-aphidicolin 8 h; G1, 3%; S, 17%; G2/M, 80%; nocodazole, 16 h; G1, 2%; S, 18%; G2/M, 80%.
Fig. 4. Correlation between SCC-112 expression, apoptosis and caspase-3 activity in COS-1 cells. (A) Expression of FLAG-tagged SCC-112. COS-1 cells were transiently transfected with FLAG-tagged SCC-112 cDNA, or pCR3.1 vector, followed by immunoblotting with FLAG-M2 antibody (top). The same blot was reprobed with anti-h-tubulin antibody and anti-SCC-112 antiserum (bottom panels). (B) SCC-112 expression induces apoptosis. Flow cytometric analysis was performed at 36 h after transfection of COS-1 cells with the FLAG epitope-tagged SCC-112 cDNA or pCR3.1 vector DNA using indicated amount of the plasmid DNA per well, as explained in Section 2. (C) SCC-112 expression correlates with enhanced caspase-3 activity. The caspase-3 activity was measured in COS-1 transfectants as described in Section 2.
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3.4. Reduced SCC-112 mRNA expression in human breast and kidney tumor tissues
Fig. 5. Induction of apoptosis in MDA-MB 231 cells stably transfected with SCC-112 cDNA. (A) MDA-MB 231 cells were stably transfected with pCR3.1SCC-112 cDNA (SCC-112) or pCR3.1 vector (Vector). Expression of the exogenous SCC-112 mRNA was confirmed by RT-PCR amplication of a portion of the SCC-112 cDNA (523 bp). 18S primers (Ambion) were used as a positive control. (B) For apoptosis assay, stable transfectant cultures (SCC-112 or Vector) were switched to serum-free medium for 72 h, followed by flow cytometric measurement of the fraction of cells in sub-G1 as described in Section 2. Three independent experiments were performed. Data from a representative experiment are shown.
P=0.0001, n=3). Together, these data demonstrate that expression of the exogenous SCC-112 gene correlates with enhanced apoptosis in different cell types.
Pro-apoptotic molecules may possess potential tumor suppressor activity correlating, in part, with a relatively low expression in tumor tissues as compared to normal tissues. To this end, we examined the mRNA expression profile of SCC-112 in normal and tumor tissues from various cancer patients. Quantification data showing foldchanges in SCC-112 mRNA expression in normal versus tumor tissue of individual patients are shown in Fig. 6. A majority of normal breast and kidney tissues had a higher steady state level of SCC-112 mRNA as compared to corresponding tumor tissues (breast, 76%, n=50; kidney, 90%, n=20). As shown in Table 1, the average ratio of the mRNA expression in normal to tumor tissue for breast and kidney were significantly greater than one ( P<0.0001), and the 95% confidence intervals for the ratios were substantially above one. On the same blot, SCC-112 mRNA expression was found to be lower in the three metastatic breast tumor tissues as compared to corresponding normal tissues ( P<0.05) (data not shown). For uterus, the average ratio of normal to tumor tissue was 0.90, and the 95% confidence intervals for the ratios were below one. The average ratios of SCC-112 expression for colon and stomach were not statistically significantly different from one at 5%
Fig. 6. Reduced SCC-112 mRNA expression in human breast and kidney tumor tissues. A Cancer Profiling Array blot (Clontech) containing cDNAs from normal, tumor and metastatic tissues from 241 patients was hybridized with 32P-labeled SCC-112 cDNA probe, followed by reprobing with ubiquitin cDNA. The SCC-112 mRNA expression levels observed in normal (N) and matched tumor (T) tissues were normalized with the corresponding ubiquitin mRNA expression levels and fold change in normalized SCC-112 mRNA expression in individual patients (N/T ratio) was plotted as shown. n, total number of patients.
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Table 1 Statistical analysis of the fold change in SCC-112 mRNA expression level in tumor tissue versus corresponding normal tissue from individual cancer patients Tumor type
Number of patients (n)
Mean N/Ta (log ratio)
Mean N/Ta (ratio)
Standard error (log ratio)
95% Confidence interval (log ratio)
95% Confidence interval (ratio)
P-value (two-sided)
Kidney Breast Rectum Thyroid Ovary Lung Colon Stomach Uterus
20 50 18 6 14 21 35 27 42
0.20 0.20 0.11 0.10 0.05 0.05 0.06 0.08 0.10
1.22 1.22 1.11 1.10 1.05 0.95 0.94 0.92 0.90
0.03 0.05 0.07 0.10 0.10 0.07 0.05 0.07 0.04
(0.13, 0.26) (0.10, 0.29) (0.04, 0.25) (0.15, 0.35) (0.16, 0.26) (0.19, 0.10) (0.16, 0.04) (0.22, 0.06) (0.19, 0.01)
(1.14, (1.11, (0.96, (0.86, (0.85, (0.83, (0.86, (0.81, (0.82,
<0.0001 <0.0001 0.13 0.35 0.63 0.50 0.25 0.25 0.02
a
1.30) 1.34) 0.25) 1.42) 1.30) 1.10) 1.04) 1.06) 0.99)
N/T 3 SCC-112 mRNA.
level. Transcriptional silencing of the tumor suppressor genes has been attributed, in part, to the promoter methylation (Baylin and Herman, 2000). Whether SCC-112 mRNA level is elevated in tumor cells following treatment of cells with a DNA methylation inhibitor remains to be seen. These data indicate a notable decrease in the mRNA expression of SCC-112 in a majority of the human breast and kidney tumor tissues examined. 3.5. Reduced SCC-112 protein expression in human renal tumor specimens We examined SCC-112 protein expression in a group of renal tumor and normal adjacent kidney tissues. Renal tissues were chosen based on the SCC-112 mRNA expression data (Fig. 6 and Table 1), and the availability of the frozen tumor tissues and matched normal adjacent tissues. Among the eighteen available independent paired sets of renal tumor and matched normal adjacent tissues analyzed, twelve pairs showed increased expression of f150-kDa SCC-112 protein in the normal renal tissue as compared to matched renal tumor tissue (1.2 – 10-fold, n=6; >10-fold, n=6). Representative data are shown in Fig. 7 (paired samples from patient #7, #5 and #1). Interestingly, a higher
Fig. 7. Reduced SCC-112 protein expression in human renal tumor tissues. The tissues were homogenized and whole cell lysates were analysed by western blotting using anti-SCC-112 antibody, followed by reprobing with anti-actin antibody, as explained in Section 2. Representative data are shown. Pt. I. D., patient identification number; T, renal cell carcinoma; N, normal adjacent tissue.
level of the f150-kDa protein was seen in some tumor tissues as compared to matched normal adjacent tissues (for example, patient #6, Fig. 7). In addition to f150-kDa protein, a prominent f65-kDa band was present in 17 out of the 18 normal renal tissues examined (94%) (Fig. 7). In contrast, only 3 of the 18 matched renal tumor tissues showed expression of the variant f65-kDa SCC-112 (17%) (data not shown). Remarkably, in three sets of paired samples (#5, #19 and #21), both protein bands were undetectable in the renal tumor tissues and were present in the normal adjacent tissues (see paired samples from patient #5 shown in Fig. 7). The appearance of a smaller molecular weight SCC-112 (f65 kDa) in a majority of the normal kidney tissues is intriguing at this time. The 65-kDa protein does not appear to be a non-related cross-reactive protein because its expression is undetectable in a number cell types, in all of which the f150-kDa SCC-112 protein is detectable, e.g., COS-1, and human cancer cell lines (breast, MDA-MB 231, MDA-MB 435; prostate, PC-3, LNCap; renal, ACHN, 769-P; pancreatic, Aspc-1, Colo 357; and Hela cells, D.K. and U.K. unpublished data).
4. Conclusions This study is the first report of SCC-112, a novel cell cycle-regulated molecule. SCC-112 is an inducer of cell death, and it may also be involved in the regulation of cell growth and proliferation based on the following observations: (1) expression of exogenous SCC-112 stimulates apoptosis in COS-1 cells and human breast cancer cells; (2) expression of exogenous SCC-112 enhances caspase-3 activity in COS-1 cells; (3) SCC-112 mRNA expression is significantly high in human normal breast and renal tissues as compared to corresponding tumor specimens; and (4) SCC-112 protein expression is high in a majority of the available human normal renal tissues as compared to the matched tumor specimens. Interestingly, chromosomal location of SCC-112 at 4p14 is in agreement with a recently reported linkage of a subset of hereditary nasopharyngeal carcinoma to a major susceptibility locus D4S405 on chromosome 4p12– p15 (Feng et al., 2002). Future inves-
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tigations will determine the role of SCC-112 in normal cells, and potential diagnostic and therapeutic usefulness of SCC112 in human cancers.
Acknowledgements The sequences reported in this study have been deposited in the GenBank database (KAS-112, accession no. AF003254 and SCC-112, accession no. AF294791). Histologically confirmed, frozen human renal tumor and matched normal adjacent tissues were kindly provided by the Cooperative Human Tissue Network (CHTN) resource of the National Cancer Institute, National Institutes of Health. The statistical analysis was performed at the Biostatistics Shared Resource; the human breast tumor cell lines and COS-1 cells were obtained from the Tissue Culture Shared Resource; and automated DNA sequencing was performed at the Macromolecular Synthesis and Sequencing Shared Resource of the Lombardi Cancer Center, supported by National Institutes of Health Grant P30-CA51008. This work was supported by National Institutes of Health grants CA68322 and CA74175, and by NeoPharm. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389 – 3402. Armitage, P., Berry, G., 2001. Statistical Methods in Medical Research, 3rd ed. Blackwell, Malden, MA. Arribas, R., Ribas, M., Risques, R.A., et al., 1999. Prospective assessment of allelic losses at 4p14 – 16 in colorectal cancer: two mutational patterns and a locus associated with poorer survival. Clin. Cancer Res. 11, 3454 – 3459. Bai, C., Sen, P., Hofmann, K., Ma, L., Goebl, M., Harper, J.W., Elledge, S.J., 1996. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86, 263 – 274. Baylin, S.B., Herman, J.G., 2000. DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet. 16, 168 – 174. Brown, B., et al., 1996. The University of Texas M.D. Anderson Cancer Center, Department of Biomathematics. Clifford, S.C., Maher, E.R., 2001. Von Hippel-Lindau disease: clinical and molecular perspectives. Adv. Cancer Res. 82, 85 – 105. Earnshaw, W.C., Martins, L.M., Kaufmann, S.H., 1999. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 68, 383 – 424. Feng, B.J., Huang, W., Shugart, Y.Y., et al., 2002. Genome-wide scan for familial nasopharyngeal carcinoma reveals evidence of linkage to chromosome 4. Nat. Genet. 31, 395 – 399. Geck, P., Szelei, J., Jimenez, J., Sonnenschein, C., Soto, A.M., 1999. Early gene expression during androgen-induced inhibition of proliferation of prostate cancer cells: a new suppressor candidate on chromosome 13, in the BRCA2-Rb1 locus. J. Steroid Biochem. Mol. Biol. 68, 41 – 50. Green, D.R., Knight, R.A., Melino, G., Finazzi-AgroV , A., Orrenius, S., 2004. Ten years of publication in cell death. Cell Death Differ. 11, 2 – 3.
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