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Molecular Cancer Research 4:539-547 (2006)
© 2006 American Association for Cancer Research

Cancer Genes and Genomics

Cyclooxygenase-2 Is Involved in S100A2-Mediated Tumor Suppression in Squamous Cell Carcinoma

Wan-Chi Tsai1, Sen-Tien Tsai5,6, Ying-Tai Jin2,7 and Li-Wha Wu3,4

Institutes of 1 Basic Medical Sciences, 2 Oral Medicine, 3 Molecular Medicine, and 4 Cardiovascular Research Center College of Medicine, National Cheng Kung University; and Departments of 5 Otolaryngology, 6 Radiation Oncology, and 7 Pathology, National Cheng Kung University Hospital, Tainan, Taiwan, Republic of China

Requests for reprints: Li-Wha Wu, Institute of Molecular Medicine, College of Medicine, National Cheng Kung University, 1 University Road, Tainan 70101, Taiwan, Republic of China. Phone: 886-6-2353535, ext. 3618; Fax: 886-6-2095845. E-mail: liwhawu{at}mail.ncku.edu.tw


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
S100A2 is considered a putative tumor suppressor due to its loss or down-regulation in several cancer types. However, no mechanism has been described for the tumor suppressor role of S100A2. In this study, ectopic expression of S100A2 in the human malignant squamous cell carcinoma cell line KB resulted in a significant inhibition of proliferation, migration, and invasion. Moreover, S100A2 significantly reduced the number of colonies (≥0.5 mm) formed in semisolid agar and decreased tumor growth and burden in nude mice. cDNA microarray analysis was used to compare mRNA expression profiles of vector- and S100A2-expressing isogenic cells. Among the genes deregulated by S100A2, the expression of cyclooxygenase-2 (COX-2) mRNA was significantly suppressed by S100A2 (2.4-fold). Western blot analysis confirmed that S100A2 reduced the expression of COX-2 protein in stably and transiently transfected KB and RPMI-2650 cells. COX-2 is frequently overexpressed in various types of cancer and plays an important role in tumor progression. Partial restoration of COX-2 expression attenuated the antitumor effect of S100A2 both in vitro and in vivo. Although the interplay between S100A2 and COX-2 remains to be clarified, these findings first showed a potent antitumor role of S100A2 in squamous cell carcinoma partly via reduced expression of COX-2. (Mol Cancer Res 2006;4(8):539–47)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
S100 family (the largest superfamily of EF-hand calcium-binding proteins, composed of at least 20 members) has multiple functions in various cell types and tissues (1, 2). Many cellular processes, including cell proliferation, differentiation, motility, secretion, membrane permeability, protein synthesis, and extracellular signal transduction, are regulated by members of this family (3). Sixteen of the 20 members are tightly clustered on human chromosome 1q21. In this chromosome region, S100A2 together with 13 other S100 members are located within the epidermal differentiation complex. This region encodes many other genes that are expressed in epidermal keratinocytes (4). Among these genes, expression of involucrin is highly correlated with epidermal differentiation (5). The location of S100 genes in epidermal differentiation complex has heightened interest in their role in differentiation of the epidermis.

Expression levels of S100 proteins vary considerably in different tumors and with respect to the progression of malignancy (2, 6). Multiple sequence alignments clearly show that S100 proteins can be divided into four subgroups (7). S100A2, S100A3, S100A4, S100A5, and S100A6 are in the same subgroup, indicating that these proteins are evolutionarily related. Although these proteins display high levels of sequence similarity, their characteristics are distinct. S100A4 is overexpressed in many types of tumors and is considered a well-established marker for tumor progression, invasion, metastasis, and poor survival prognosis (8). S100A6 is markedly up-regulated in many types of tumor cells, including melanoma, adenocarcinoma, and neuroblastoma (9). Unlike the relationship of other related S100 members to carcinogenesis, S100A2 is the first putative tumor suppressor protein.

The S100A2 (formerly called CaN19 or S100L) gene was first identified via subtractive hybridization screening (10). Markedly reduced level of S100A2 has been shown in breast tumor biopsies (11), melanomas, and esophageal, lung, and other cancer types (12-14) and related to the prognosis of certain cancers (15, 16). Reduced nuclear staining of S100A2 in early-stage oral cancers correlates with shorter disease-free survival (17). Therefore, expression of S100A2 was proposed as a valuable prognostic marker. By contrast, S100A2 overexpression was recently found to correlate with prognosis in ovarian, gastric, and lung cancers (18-20). Taken together, the role of S100A2 in carcinogenesis remains controversial.

To clarify this role, we employed stable clones of squamous cell carcinoma (SCC) ectopically expressing S100A2. The effects of S100A2 on cell growth, motility, invasion, colony-forming ability, and tumorigenesis in vivo were examined. We show that S100A2 could exert its antitumor activity by reducing expression of cyclooxygenase-2 (COX-2).


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Decreased Expression of S100A2 mRNA in Oral Cancer Lines
We previously showed down-regulation of S100A2 mRNA in oral cancer cells using cDNA microarray analysis (17). The expression of S100A2 mRNA in primary normal oral keratinocytes (NOK) and various cell lines (one precancerous dysplastic oral keratinocyte line DOK and nine established oral SCC lines) was further examined using semiquantitative reverse transcription-PCR and found to be significantly down-regulated in precancerous and nine cancer cell lines compared with NOK (Fig. 1 ). KB cells expressed the least amount of S100A2 mRNA (Fig. 1, top) and were used to establish stable clones for the following studies.


Figure 1
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  		FIGURE 1. Diminished expression of S100A2 mRNA in oral cancer cell lines. Subconfluent cells of NOK and various cell lines were harvested for preparation of total RNA for semiquantitative reverse transcription-PCR. A. Expression level of S100A2 mRNA in NOK was higher than that in one precancerous DOK cell and nine oral cancer cell lines. B. S100A2/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio of NOK in the indicated cell lines. Columns, mean of three independent experiments; bars, SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus NOK.

 
Ectopically Expressed S100A2 Attenuated Cellular Growth via Delayed Entry into G2-M Phase
We first established two myc-tagged S100A2-expressing clones (S100A2-1 and S100A2-2) and a pool of vector control clones. Western blot analysis confirmed the presence of S100A2 proteins in S100A2-1 and S100A2-2 clones (Fig. 2A ). The protein level of S100A2 in both stable clones was at least 4-fold higher than the endogenous level of S100A2 protein in NOK. No detectable S100A2 protein was detected in vector control cells.


Figure 2
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  		FIGURE 2. Levels of S100A2 protein in stable clones and their effects on cellular growth and cell cycle progression. A. Western blot analysis of S100A2 protein in NOK and stable clones using anti-S100A2 antibodies. Ectopically expressed myc-tagged S100A2 resulted in increased molecular weight of S100A2 compared with the endogenous S100A2. Level of myc-tagged S100A2 protein in S100A2-1 and S100A2-2 was higher than that of endogenous S100A2 in NOK. No myc-tagged S100A2 could be detected in vector control cells. Actin served as an internal control for protein loading. B. Growth rate of S100A2-1 and S100A2-2 was significantly decreased when compared with the parental and vector control cells. ***, P < 0.001 versus vector control cells. C. Distribution of G1, S, and G2-M for vector and S100A2-2 cells during cell cycle progression after double thymidine block. Following the release, cells were harvested at 0, 4, 8, 12, and 22 hours. DNA histograms for vector and S100A2-2 cells: histogram with percentages of cycling cells in G1, S, and G2-M shows one representative experiment of four independent repeats with similar data.

 
To unravel the role of S100A2 overexpression in the malignant phenotype of KB cells, growth rate (direct enumeration of cells at the indicated time points) was examined. Four days after plating, growth rates were significantly slower in S100A2-expressing clones than in both parental KB and vector control cells (P < 0.001; Fig. 2B). The growth rates of parental KB and vector control cells were not altered by transfection of vector alone. Fluorescence-activated cell sorting analysis of the phase distribution of cells synchronized in G1-S by double thymidine treatment and harvested at 0, 4, 8, 12, and 22 hours showed no difference at 0, 4, and 8 hours in the distribution of cycling cells between vector and S100A2-2 clones (Fig. 2C). By 12 hours, 28% and 39% of the vector control and 43% and 48% of the S100A2-2 cells were in G1 and S phases, respectively, and >30% of vector control cells and only 9% of S100A2-2 cells were in G2-M phase. These data indicate that overexpression of S100A2 stalls cells in G1 and S phases and delays their entry into G2-M.

Ectopic Expression of S100A2 Reduced Cell Motility and Invasive Ability
Time-lapse video microscopy with ImageTool software was used to measure the motility of sparsely seeded vector, S100A2-1, and S100A2-2 clones. Compared with vector cells, individual S100A2-1 and S100A2-2 cells showed significantly decreased (2-fold less) motility over the 6 hours of recording (Fig. 3A ). The Matrigel invasion ability of S100A2-expressing cells (relative to that of vector cells) was also significantly decreased (Fig. 3B). The inhibitory effect of S100A2 on invasion was more dramatic than that on migration.


Figure 3
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  		FIGURE 3. Attenuation of the migration and invasion ability of KB cells on S100A2 overexpression. A. After cell seeding in duplicates onto culture dishes for 16 to 24 hours, the distance of individual cell migration was traced for 6 hours. Pixels represent migration distance. The distance of S100A2-expressing clones was significantly decreased by at least 2-fold compared with that of control cells. B. Cells invading Matrigel after 24 hours were detected as blue stained cells on the opposite side of membrane. Top, representative photomicrographs of vector, S100A2-1, and S100A2-2 cells; bottom, quantitative result of invasion ability. ***, P < 0.001 versus vector control cells. The ability of S100A2-expressing cells to invade Matrigel was significantly attenuated compared with vector control cells. Representative of two independent experiments.

 
S100A2 Reduces Colony Size In vitro and Tumorigenesis In vivo
Soft agar assay was used to measure the anchorage-independent growth for 21 days of S100A2-expressing cells. Although the size of colonies formed by vector control was much larger than those of S100A2-expressing clones, there was no significant variation in the total number of colonies individually formed by these three clones (Fig. 4A ). However, the number of colony size ≥0.5 mm was significantly reduced by S100A2 (85-90% reduction; Fig. 4A). Consistent with a negative role of S100A2 in cell cycle progression, increased S100A2 expression reduced the size but not the number of colonies formed in soft agar.


Figure 4
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  		FIGURE 4. Decreased in vitro and in vivo tumorigenicity by ectopically expressed S100A2. A. Numbers of colonies formed by control vector, S100A2-1, and S100A2-2 clones appearing on soft agar after 21 days of growth were enumerated. Top, representative photomicrographs of colonies formed by vector, S100A2-1, and S100A2-2 cells; bottom, quantitative data of colony-forming ability. Gray columns, total number of colonies; black columns, number of colonies (≥0.5 mm). **, P < 0.01 versus control vector; N.S., not significant versus control vector. S100A2 significantly reduced the number of colonies (≥0.5 mm) but not the total number of colonies on soft agar. B. Size of subcutaneous tumors formed by control vector and S100A2-2 clones was measured for 3 weeks by a caliper every 2 to 3 days. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus vector control. Increased expression of S100A2 diminished tumor formation in nude mice. C. Tumor sizes (top) and weights (bottom) derived from S100A2-2 clones were significantly reduced compared with those derived from vector control. D. Western blot analysis using anti-S100A2 antibodies confirmed the presence of S100A2 protein in all five tumors isolated from S100A2-2 tumor-bearing nude mice but not those from their matched control mice. Representative of two independent experiments.

 
An in vivo tumorigenesis assay in tumor-bearing nude mice showed that significantly smaller tumors developed in mice injected with the S100A2-2 clone than in those injected with the vector clone at 28 days after injection (Fig. 4B). S100A2-overexpressing tumors weighed significantly less (reduced by 50-70%) than vector control tumors (Fig. 4C). Western blot analysis confirmed that the expression of human S100A2 protein was maintained in all S100A2-overexpressing tumors but not in vector clone tumors (Fig. 4D). Together, S100A2 attenuates the tumorigenic ability of malignant KB cells in vitro and in vivo.

S100A2 Reduced the mRNA and Protein Expression of COX-2
Affymetrix GeneChip Human Genome U133A arrays (with filtering criteria of ≥1.8-fold change in expression and all "present calls" in at least one group) were used to compare the mRNA expression profile in vector and S100A2-expressing isogenic cells. Two hundred seventeen genes were differentially regulated by S100A2 (data not shown). These genes were further categorized into at least nine functional groups based on their annotation (Fig. 5 ). The mRNA expression of two up-regulated genes (S100A2 and thrombomodulin [TM]) and two down-regulated genes (COX-2 and cyclin D1) were validated with semiquantitative reverse transcription-PCR (Fig. 6A ), indicating a robustness of the microarray analysis used in this study. Western blot analysis further confirmed that the difference in COX-2 mRNA levels translated into a significant suppression of COX-2 protein (≥10-fold) in the S100A2-expressing cells (Fig. 6B). This decreased expression of COX-2 protein was also recapitulated in both KB and RPMI-2650 cells transiently transfected with S100A2-expressing vectors (Fig. 6C and D). Taken together, these data suggest that COX-2 could be one of the downstream targets of S100A2.


Figure 5
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  		FIGURE 5. Functional categories of genes that are significantly altered by S100A2. Gene annotations were obtained from Affymetrix and PubMed databases. X axis, names of functional categories; Y axis, numbers of genes in each functional category. Note that the annotation is constantly evolving.

 

Figure 6
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  		FIGURE 6. Reduced expression of COX-2 in S100A2-expressing stable clones. Western blot analysis of protein lysates isolated from vector, S100A2-1, and S100A2-2 clones using the indicated antibodies. Numbers in parentheses, average fold increase (+) or decrease (–) of mRNA expression in microarray data analysis. ß-Actin or {alpha}-tubulin served as a loading control. A. RT-PCR analysis indicated that the expression of TM was increased, whereas expression of cyclin D1 was repressed by S100A2. B. Western blot analysis indicates that COX-2 protein was suppressed in both S100A2-expressing clones, S100A2-1 and S100A2-2. C and D. Increased expression of S100A2 is accompanied with the reduced expression of COX-2 in transiently transfected KB and RPMI-2650 cells, respectively.

 
COX-2 Caused Partial Loss of S100A2-Mediated Antitumor Effects
Stable clones, S100A2-2-control and S100A2-2-COX-2, were established from S100A2-2 cells transfected with either empty vector or a COX-2 cDNA expression construct to measure the effects of COX-2 restoration on cellular growth, invasion, and colony-forming ability in semisolid agar. Western blot analysis confirmed the ectopic expression of COX-2 protein in the S100A2-2-COX2 stable clone. Although expression of COX-2 was still lower than that in the KB vector clone, the level of COX-2 protein in S100A2-2-COX-2 was at least thrice higher than that in the S100A2-2-control clone (Fig. 7A ). Using a growth curve assay, the growth rate of COX-2-expressing S100A2-2-COX-2 cells was significantly increased compared with S100A2-2-control cells (P < 0.01; Fig. 7B). Reintroduction of a COX-2 cDNA expression construct also significantly restored the ability of S100A2-2 cells to invade Matrigel (Fig. 7C). The abilities of S100A2-2-COX-2 to grow and to invade Matrigel were, however, not as high as those of control vector cells. Furthermore, the S100A2-2-COX-2 clone manifested a significant increase in the number of colonies (P < 0.05; Fig. 7D).


Figure 7
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  		FIGURE 7. Partial restoration of malignant behaviors of S100A2-expressing cells by reintroduction of COX-2 cDNA S100A2-2 cells containing empty vector (S100A2-control) and the same cells expressing ectopic COX-2 (S100A2-2-COX-2) were established. KB cells with vector only (Vector) served as a negative control for S100A2-expressing cells. A. Western blot analysis confirmed increased expression of COX-2 protein in S100A2-2-COX2 compared with that in S100A2-2-control cells. B. Direct enumeration of vector, S100A2-2-control, and S100A2-2-COX2 during 4 days of growth after seeding. Increased expression of COX-2 significantly increased the growth rate of S100A2-2-COX-2 cells compared with the S100A2-2-control cells. **, P < 0.01 versus S100A2-2-control. C. Invasion assay indicates the ectopically expressed COX-2 significantly restored the ability of cells to invade Matrigel compared with their control cells. ***, P < 0.001 versus S100A2-2-control. D. S100A2-2-COX-2 manifested a higher ability to form colonies (≥0.5 mm) in soft agar compared with the control cells. *, P < 0.05 versus S100A2-2-control. Representative of three independent experiments with similar data. E and F. S100A2-2-COX-2 cells not only formed tumors in nude mice at a higher growth rate but also induced larger tumors than S100A2-2-control cells. *, P < 0.05. Consistent with the previous finding, S100A2 manifested a significant antitumor effect compared with KB-vector cells. #, P < 0.05. Representative of three independent experiments with similar data.

 
To confirm the positive role of COX-2 in S100A2-expressing cells in vivo, we inject nude mice, respectively, with S100A2 transfectants ectopically expressing vector control and COX-2. KB cells expressing vector serves as a negative control. As shown in Fig. 7E and F, COX-2 significantly attenuated the antitumor effect of S100A2 by slowing down tumor growth and burden (P < 0.05).


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Although many articles suggest that S100A2 may act as a tumor suppressor, none provide direct evidence that S100A2 is such a gene. In this report, the human SCC cell line KB was used as a model to show a tumor suppressor role for S100A2. Overexpression of S100A2 in KB cells not only diminished the ability of these cells to grow, migrate, and invade Matrigel but also suppressed their tumorigenicity in vitro and in vivo. Comparison between mRNA expression profiles of vector control and S100A2-expressing stable clones indicated that 217 genes were differentially regulated by S100A2. Thus, genes involved in a wide spectrum of cell behaviors (e.g., growth, apoptosis, migration, invasion, metabolism, and inflammation) participate in the antitumor effect of S100A2.

Of the >20 S100 family members, only S100A2 was predicted to be a tumor suppressor gene for several reasons. First, expression of S100A2 is down-regulated in many types of cancer tissues, including breast, lung, and esophagus (12-14). Second, treatment of mammary carcinoma cells with the inhibitor of DNA methylation 5-aza-2'-deoxycytidine resulted in the enhanced expression of S100A2 mRNA, suggesting that promoter inactivation by methylation is involved in decreased expression of S100A2 (10). Third, S100A2 is located within the epidermal differentiation complex on chromosome 1q21 and this region is frequently rearranged in cancers (21). Fourth, loss of heterozygosity in chromosome 1q is also frequently observed (22). These observations are consistent with our finding that ectopically expressed S100A2 possesses antitumor effects in vitro and in vivo.

The notion that S100A2 is a tumor suppressor gene has also been challenged. Xia et al. reported that (a) S100A is expressed at high levels in hyperplastic perilesional skin and (b) its expression together with that of keratin 6 is induced by calcium, arguing for a role of S100A2 in regenerative differentiation (23). S100A2 was overexpressed in tumor tissues of stomach, lung, and ovary and increased expression positively correlated with poor prognosis (18-20). The tissue-specific effect of S100A2 thus cannot be ruled out. The extensive in vitro and in vivo approaches used in this study support the view that S100A2 behaves as an antitumor gene in human SCC. Although no S100A2 knockout mice have been produced, generation of S100A2 knockout animals in a tissue-specific fashion may be helpful in teasing apart the exact role of S100A2 in carcinogenesis.

Overexpression of S100A2 significantly slowed cellular growth both in vitro and in vivo. Because any alteration on the ability of cells to attach substratum, proliferate, or induce apoptosis is likely to affect cellular growth, we also conducted cell adhesion assay and apoptotic index measurement following serum withdrawal in addition to growth curve and cell cycle analyses. There was no significant variation in the ability of vector control and S100A2-expressing cells to attach substratum or in the apoptotic index induced by serum withdrawal (data not shown). Therefore, G1- and S-phase stalling by S100A2 as shown in the cell cycle analysis is likely responsible for the slower growth rate of S100A2-expressing cells in growth curve analysis and in vivo tumor formation. The involvement of S100A2 in cell cycle regulation was consistent with previous findings of cell cycle–regulated pattern of S100A2 mRNA in cycling cells and a biphasic induction of S100A2 mRNA during cell cycle progression triggered by growth factor induction (11). Although the interaction of S100A2 with p53 resulting in subsequent activation of p53 transcriptional activity may contribute to slower cell growth, little or no p53 could be detected in the KB cells (24, 25). The antitumor effect of S100A2 we reported is likely through a p53-independent pathway. More studies are needed to define the role of S100A2 in the mechanism of cell cycle progression.

Among 217 deregulated genes, the expression of COX-2 mRNA and protein was consistently repressed by S100A2. COX-2 is a key enzyme that catalyzes the rate-limiting step in prostaglandin biosynthesis and is inducible in response to inflammatory cytokines, growth factors, and tumor promoters (26). Expression of COX-2 is increased at inflammatory sites and in various types of human cancer, particularly colorectal, breast, prostate, and oral cancers (26, 27). Transgenic mice expressing human COX-2 in mammary glands develop focal mammary gland hyperplasia, dysplasia, and metastatic tumors (28). Conversely, knocking out expression of COX-2 notably reduces the development of intestinal tumors and skin papillomas in mice (29, 30). COX-2 thus plays an important role in inflammation and carcinogenesis (31).

More than 15% of all malignancies are initiated by inflammation. Long-term use of nonsteroidal anti-inflammatory drugs decreases cancer risk (40-50% reduction in colon cancer; ref. 32). A link between inflammation and cancer has long been suspected, but its molecular nature remained ill-defined until a recent finding that recruitment of host inflammatory cells by interleukin-8, a transcriptional target of oncogene Ras, plays a critical role tumor angiogenesis and growth (33). The hypothesis that overexpression of S100A2 exerts its tumor suppression function through COX-2 down-regulation is further strengthened by the finding that partial restoration of COX-2 expression attenuated the antitumor effect of S100A2. Incomplete restoration was probably due to lower COX-2 expression levels in S100A2-expressing cells transfected with COX-2 than in KB vector control cells (Fig. 7A). Alternatively, other downstream effectors may be also critical for the antitumor effects mediated by S100A2. Consistent with the latter notion, both the induced expression of TM and the reduced expression of cyclin D1 could not be attenuated by ectopically expressed COX-2 in S100A2-expressing cells (data not shown). The exact role of other S100A2-deregulated genes, including TM and cyclin D1 in the antitumor effect of S100A2, remains to be characterized.

Expression of COX-2 protein can be regulated by Ras oncogene and tumor suppressor p53 in positive and negative manners, respectively (34, 35). The mechanisms involved in deregulation of COX-2 expression include an alteration of mRNA stability and transcriptional promotion via direct binding to COX-2 promoter. We were not able to detect any direct modulation of COX-2 promoter activity by S100A2 using dual-luciferase reporter assays (data not shown). Although several upstream regulatory elements for transcription factors have been characterized in the promoter region of COX-2 gene (36), none of these transcription factors was in the list of differentially regulated genes triggered by overexpression of S100A2. Suppression of COX-2 by overexpression of S100A2 is probably through an indirect mediation. More studies are needed to define the role of reduced COX-2 expression in the S100A2-expressing SCC cells.

In conclusion, S100A2 possesses a profound antitumor effect in vitro and in vivo using SCC as a cancer model. Using oligonucleotide microarray analysis, we found that COX-2 was significantly suppressed in S100A2-expressing stable clones compared with vector control cells. Ectopic expression of S100A2 also significantly suppressed the expression of COX-2 in transiently transfected SCC cells. Consistent with a role of COX-2 in tumor progression, partial restoration of COX-2 expression in S100A2-expressing cells attenuated the S100A2-mediated antitumor effect in vitro and in vivo. To our knowledge, we are the first to suggest that the antitumor effect of S100A2 is in part mediated by down-regulation of COX-2.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Materials
Human Caucasian DOK were purchased from and maintained as described by the European Collection of Cell Culture. NOK, five Caucasian oral cancer lines (KB, CAL-27, SCC-9, SCC-15, and SCC-25), three Taiwanese oral cell lines (OC-2, OC-3, and OEC-M1), and a human nasal septum SCC line (RPMI-2650) were maintained as described previously (17, 37, 38). HSC-3 cells derived from human tongue carcinoma with lymph note metastasis were from the Japanese Collection of Research Bioresources Cell Bank of Japan and maintained as described previously (39). All other culture medium, fetal bovine serum, and antibiotics were purchased from Life Technologies (Grand Island, NY). LipofectAMINE 2000 transfection reagent, TRIzol reagents, and reagents for reverse transcription-PCR were from Invitrogen (Carlsbad, CA). Oligonucleotide primers were from MDbio, Inc. (Taipei, Taiwan). Antibodies to S100A2 and COX-2 were from BD Transduction Laboratories (San Jose, CA). The antibodies to cyclin D1 and actin were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-{alpha}-tubulin antibodies were from NeoMarker (Fremont, CA). Chemiluminescence Reagent Plus was from NEN Life Science Products (Boston, MA). RNase A, propidium iodine, and other chemicals were purchased from Sigma (St. Louis, MO). Matrigel was from BD Biosciences (Bedford, MA). COX-2 cDNA expression vector was a generous gift of Dr. Kan Wai-Ming (Department of Pharmacology, National Cheng Kung University, Tainan, Taiwan).

Semiquantitative Reverse Transcription-PCR
DNA-free total RNA was isolated from cultured cells using TRIzol reagent, and 1 µg was reverse transcribed with oligo(dT12-18) primers. The cDNA mixture was then used as template for gene-specific PCR. The primer sequences were 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3' for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 5'-TCAAGCTGAGTAAGGGGG-3' and 5'-ATCCATGGCAGGAAGTCAAG-3' for S100A2. The PCR conditions were 94°C (15 seconds), 50°C (30 seconds), and 72°C (30 seconds). The cycle number for linear amplification of GAPDH and S100A2 was 21 and 32, respectively. The experiment was independently repeated thrice.

Construction of a Mammalian Expression Vector
A mammalian expression vector for myc-tagged S100A2 was generated as follows: the coding region of human S100A2 was PCR-amplified and cloned in-frame into the EcoRI and BamHI sites of pcDNA3.1-myc/His-A(+) (Invitrogen). The myc-tagged S100A2 cDNA was then subcloned into a pOPI3CAT-derived mammalian expression vector driven by a Rous sarcoma virus promoter. Automatic DNA sequencing analysis verified the direction and coding sequence of S100A2.

Transfection and Establishment of Stable Clones
KB or the indicated cells were seeded overnight into 35-mm culture dishes in appropriate growth medium supplemented with 10% fetal bovine serum before transfection with 1 µg S100A2-expressing vector or empty vector using LipofectAMINE 2000. For transient transfection, cells were harvested for isolation of total RNA or protein 24 to 48 hours after transfection. For stable transfection, stable clones were selected 24 hours after transfection over a 2-week period in growth medium containing 800 µg/mL G418. S100A2-expressing clones were then expanded into individual cell clones. Stable clones expressing COX-2 were established accordingly.

Growth Rate Assay
Parental KB and stable clones (1.6 x 104 cells per well) were seeded in duplicate onto 24-well culture dishes. Cells were harvested for direct counting at the indicated days after seeding. This experiment was independently repeated thrice.

Cell Cycle Analysis and Double Thymidine Block
Vector and S100A2-2 clones (1 x 106 cells seeded per 60-mm dish in growth medium and incubated 16 hours) were processed as follows: medium was changed to starvation medium containing 2 mmol/L thymidine, incubation for 10 hours, cells were washed twice, cells were fed with regular growth medium for 14 hours, cells were fed with starvation medium with 2 mmol/L thymidine for another 10 hours, and both adherent and floating cells harvested for flow cytometry at 4-hour intervals for 24 hours. Approximately 1 x 106 cells were washed with PBS, fixed with 70% cold ethanol, treated with 100 µg/mL RNase A, and stained with 40 µg/mL propidium iodine. The cell cycle distribution was analyzed by FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). The percentage of cells present in each phase of the cell cycle was determined using ModFit software (Verity Software House, Topsham, ME).

Migration Assay
Culture dishes (35 mm), coated overnight with type I collagen (5 µg/mL) from rat tails, were seeded with 20,000 cells in growth medium. After 16 hours, cells were refed with MEM containing 1% fetal bovine serum and 20 mmol/L HEPES (pH 7.0), placed on a heated (37°C) microscope stage, and filmed for 6 hours using a time-lapse video camera. The movement of randomly chosen cells over 15 minutes was evaluated by exporting the video images to the graphics program, The University of Texas Health Science Center at San Antonio ImageTool software. Only isolated cells and the nondividing cells that did not leave the frames during recording were evaluated. For each clone, the migration paths of 10 to 15 cells were measured, summed, averaged, and represented as pixels.

Invasion Assay
Invasion assays were done in 24-well Transwell units with 8-µm-pore polycarbonate membranes (Corning Costar, Cambridge, MA). Cells (1 x 105 per well) were added to upper chambers (filter coated with 1 mg/mL Matrigel) in 100 µL of the growth medium. Lower chambers were filled with 500 µL growth medium. After 24-hour incubation, cells that remained in the Matrigel or attached to the upper side of the filter were removed with cotton swabs. Cells that had migrated through the membrane to the lower surface were stained with Giemsa solution and counted in five different fields under a light microscope at x400 magnification. Each experiment was done in duplicate wells and repeated twice, and results were expressed as mean ± SD.

Soft Agar Assay
Briefly, 5 x 103 cells trypsinized to form a single-cell suspension were seeded in duplicate onto 60-mm plastic culture dishes in MEM containing 0.35% agarose overlying a solidified 0.7% agarose underlayer. After the cell-containing layer solidified, 0.7% agarose was overlaid. Fresh growth medium was added every 4 days. Plates were incubated at 37°C in 5% CO2 for 21 days. Following staining with Giemsa solution, blue colonies (≥0.5 and <0.5 mm) were enumerated for each stable clone.

In vivo Tumorigenesis Assay
The tumorigenic activity of cells was examined in 8-week-old nude BALB/c nu/nu mice (5-7 per group). Tumor cells (3.3 x 106) were suspended in 0.2 mL PBS and subcutaneously injected into the flank using a 27-gauge needle. Tumors were generally palpable at 5 to 7 days after inoculation and tumor volumes were measured (using a caliper and calculated as length x width2 x 0.5) twice weekly until day 28. The mice were euthanized 28 days after inoculation, and the tumors were excised, weighed, and used for isolation of total proteins. This experiment was independently repeated twice to thrice.

Western Blot Analysis
Cells were lysed in boiled lysis buffer containing 1% SDS and 10 mmol/L Tris-HCl (pH 7.4). Following centrifugation at top speed for 10 minutes, the protein concentration in each lysate was measured (Bradford assay). Equal amounts of total protein from each sample were fractionated by SDS-PAGE, blotted onto a polyvinylidene difluoride membrane, and probed with the indicated antibody and then a secondary antibody. The hybridized immunocomplex was detected by Renaissance Chemiluminescence Reagent Plus.

Microarray Hybridization and Data Analysis
The mRNA expression profiles of vector and S100A2-2 clones were analyzed in triplicate independent experiments. Raw data were normalized and analyzed using GeneSpring software version 7.2 (Silicon Genetics, Redwood City, CA). After data transformation (to convert any negative value to 0.01), two types of normalization were done: a per-chip on 50th percentile method and a per-gene on median normalization method. To identify genes with statistically significant differences between vector and S100A2-2 clones in three biological repeats, one-way ANOVA nonmultiple testing corrections with the similarity >90% were done.

Statistical Analysis
Values are mean ± SD of experiments done in duplicate or triplicate. Statistical significance was tested by Student's t test for either paired or unpaired data as appropriate. Statistical significance was defined as P < 0.05, P < 0.01, or P < 0.001.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Dr. David D. Donner for helpful suggestions and comments about the article and Dr. Tung-Yiu Wong for help in isolation of NOK.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: National Science Council in Taiwan grant NSC-93-2314-B006-041 and Department of Health grant DOH-TD-B-111-004 (S.-T. Tsai); NSC94-3112-B-006-004-Y (Y.-T. Jin); NSC94-2320-B-006-012 (L.-W. Wu).

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

Received 12/30/05; revised 5/ 1/06; accepted 5/24/06.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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R. D. Kirschner, K. Sanger, G. A. Muller, and K. Engeland
Transcriptional activation of the tumor suppressor and differentiation gene S100A2 by a novel p63-binding site
Nucleic Acids Res., May 1, 2008; 36(9): 2969 - 2980.
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