Serological analysis of tumor antigens in cancer patients has been utilized in both diagnostic and predictive tests for cancer and metastasis. Mac-2 binding protein (Mac-2BP), a highly glycosylated, secreted 90-kDa protein has been reported to be detected in abundance in the sera of patients with various tumors, including cancers of the breast, lung, colon, ovary, pancreatic carcinoma and lymphoma.1, 2, 3, 4, 5, 6, 7, 8 Mac-2BP serum levels are also frequently found to be elevated in patients with viral infections, including human immunodeficiency virus and hepatitis C virus infections.9, 10 Mac-2BP has been detected in milk, urine and other biological fluids, and its expression levels do not appear to be influenced by age, gender or smoking habits.1 Interestingly, the Mac-2BP serum level has been found to be positively correlated with tumor progression. For example, the majority of breast tumor patients with a Mac-2BP serum level in excess of 11 μg/ml tend to exhibit metastatic spread to the liver, and consequently, poor survival rates.3 Thus, Mac-2BP may prove a useful prognostic marker for the detection of malignant progression, most notably tumor metastasis.
Mac-2BP binds to galectin-1, -3 and -7.11 Like the Mac-2BP, the galectin family proteins have also been shown to be abundantly expressed in malignant cell transformation and are associated with the progression of cancer. It has been suggested that the biological function of Mac-2BP bound to galectin family proteins involves cell adhesion via self-assembly,12, 13 as well as multicellular aggregation via the cross-linking of galectin proteins in adjacent tumor cells. The enhanced adhesive interactions between tumor cells and the extracellular matrix evidenced by Mac-2BP and galectins may contribute to the biosynthesis of new tumor colonies. Moreover, increased adhesion to the extracellular matrix may allow tumor cells to evade apoptosis. When Mac-2BP was used as an immobilized substrate, chemotherapy-induced apoptosis was found to be significantly reduced in Jurkat T lymphoma cells.8 These results support the notion that high levels of circulating Mac-2BP effectively eliminate the activity of chemotherapeutic drugs administered to lymphoma patients. Mac-2BP has also been identified as a potent immune stimulator and modulator, which exerts positive effects on the generation of natural killer/lymphocyte-activated killer (NK/LAK) cells from peripheral blood monocytic cells (PBMC) and suppresses asthma-associated inflammation via the downregulation of Th2-type cytokine levels.14, 15 Therefore, this protein is believed to play a crucial role in host cellular immune responses, as well as in tumor progression.
Most gastric carcinomas, regardless of histological type or tumor stage, also manifest potent telomerase activity.16 Some intestinal metaplasias and gastric adenomas have also evidenced slightly enhanced telomerase activity.17, 18 Recent reports suggest that the biological activity inherent in the maintenance of telomeres is sometimes dependent on the levels of negative regulators of telomere length, including TRF1, TRF2 and TIN2 mRNA, the levels of which are significantly reduced in gastric cancers when compared with what has been observed in noncancerous mucosal tissue samples.19 Therefore, the modulation of TERT and negative telomere length regulators both play crucial roles in the maintenance of telomeres during early gastric carcinogenesis. Thus, TERT expression levels might be employed as markers for the detection of “true precancerous lesions.”18
In this study, we examined the regulatory relationship between human telomerase reverse transcriptase (hTERT) and Mac-2BP and the clinical significance of Mac-2BP up-regulation in gastric cancer. We show that Mac-2BP expression is up-regulated by hTERT, and that its expression is significantly elevated in gastric cancer cell lines and gastric tumor tissues. We also provide evidence that increased Mac-2BP in gastric cancer is associated with clinical parameters indicative of poor prognosis.
Material and methods
Preparation of hTERT plasmid and construction of SW13/hTERT cell line
Human TERT (pcDNA 3-Neomycin) expression plasmid was kindly provided by Dr. Han Woong Lee (Sung Kyun Kwan University, School of Medicine, Suweon, Korea). The day before transfection, SW13 cells were plated in 60-mm culture dishes at 8 × 105 cells. The next day, 2 μg of purified TERT cDNA plasmid was introduced with Lipofectamine Plus™ Reagent (Invitrogen, Carlsbad, CA) and were further incubated for 24 hr. Transfected cells were selected by culturing in medium containing 1 mg/ml of G418 (Sigma, St. Louis, MO) for 2 weeks, and resistant cells were obtained by limiting the dilution for single cell cloning.
Primary small cell carcinoma, SW13, was purchased from ATCC (American Type Culture Collection) and was maintained in L-15 medium Leibovitz (Sigma, St. Louis, MO) containing 10% FBS and antibiotics, in a humidified incubator at 37°C. SW13/pcDNA3, SW13/hTERT clones and HEK293 were maintained in DMEM medium (GIBCO, Grand Island, NY) supplemented with 10% FBS and antibiotics in a humidified incubator with an atmosphere of 5% CO2 and a temperature of 37°C. The human gastric cancer cell lines, SNU-1, -16, -216, -484, -620 and -638, were obtained from the Korean Cell Line Bank at the Cancer Research Center, Seoul National University (Seoul, Korea) and were cultured in RPMI-1640 medium (Hyclone, Road Logan, Utah) containing 10% FBS and antibiotics. K562, MCF-7 tumor cells were also maintained in RPMI-1640 medium.
RT-PCR and northern blot analysis
The total RNA of various tumor cells containing SW13/pcDNA3 and SW13/hTERT was isolated by the guanidinium method. In brief, first-strand cDNAs were synthesized using a ProSTAR™ First-Strand RT-PCR kit (Stratagene, La Jolla, CA). hTERT and Mac-2BP cDNA were generated via PCR amplification. The PCR conditions were as follows: 94°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec for 28 cycles, followed by 5 min of incubation at 72°C. The primer pairs utilized were as follows: hTERT sense; 5′-ATG AAG TTC CTG CAC TGG CTG AT-3′, hTERT antisense; 5′-AGT TGA GCA CGC TGA ACA GT-3′, Mac-2BP sense; 5′-ACA CGG TCA TCC TGA CTG C-3′, Mac-2BP antisense; 5′-ACA GGG ACA GGT TGA ACT GC-3′. For Northern blot analysis, the extracted total RNA was run on 1.2% agarose–formaldehyde gel, transferred to nylon membranes, and hybridized with [32P]-labeled probes. Hybridization was detected via autoradiography.
Western blot analysis
For Western blot analysis, the proteins were separated via gel electrophoresis with 8% SDS-PAGE and the separated proteins were transferred to PVDF membranes (Hybond-P, Amersham Biosciences, Buckinghampshire, UK). The membranes were then probed with anti-serum against hTERT (Calbiochem, CA), and the membrane was reprobed with β-actin antibody in order to normalize the quantity of the loaded samples.
The BD Atlas™ cDNA Expression Array System (BD Biosciences Clontech, Palo Alto, CA) was used in accordance with the instructions of the manufacturer. In brief, radioisotope-labeled target cDNA was generated from total RNA from SW13/pcDNA3 and SW13/hTERT. To prepare the total RNA and cDNA probes, the BD Atlas Pure Total RNA Labeling System (No. K1038-1) was employed. Membranes harboring 1,176 different cDNA PCR products were hybridized overnight with the [32P]-labeled first-strand cDNA probes at 68°C. The hybridized blots were washed with 80–100 ml of prewarmed wash solution 1 (2× SSC, 1% SDS) for 20 min at 68°C, and washed for an additional 30 min with prewarmed wash solution 2 (0.1× SSC, 0.5% SDS) at 68°C. The array blots were then exposed to X-ray film for 1 day at −70°C. The positive signals from the internal control genes were used to normalize and compare the signals from all genes.
Harvesting of culture supernatants from SNU cell lines
To evaluate the secreted Mac-2BP, the SNU cells were fully grown in 15-cm tissue culture dishes or T75 flasks. The cells were washed and then incubated for 24 hr in serum-free medium. The supernatants were then harvested and centrifuged in order to eliminate any remaining intact cells. The samples were dialyzed with PBS (MW cutoff 50,000 Da; Spectrum Laboratories, Rancho Dominguez, CA) for 24 hr and concentrated with Amicon Centriplus® (Millipore, Bedford, MA01730). The protein concentrations of the samples were determined using a BioRad protein assay kit (BioRad Laboratories, Richmond, CA).
ELISA for Mac-2BP
The cells were suspended in a lysis buffer (0.01% Nonidet P-40, 10 mM Tris, pH 7.6, 50 mM KCl, 5 mM MgCl2, 2 mM dithiothreitol, 20% glycerol plus protease inhibitor cocktail) (Sigma, St. Louis, MO) and sonicated for two pulses at 50 J/Watts. The lysates were centrifuged for 20 min at 12,000 rpm, and the quantities of Mac-2BP in the supernatants and in the cytosol of the cells were measured via ELISA, using an s90k/Mac-2BP ELISA kit (Bender Med Systems GmbH, Vienna, Austria) in accordance with the instructions of the manufacturer.
Intracellular staining for Mac-2BP and hTERT
Intracellular Mac-2BP and hTERT in the SW13/hTERT and SNU cell lines were detected by using flow cytometry. In brief, the cells were washed with an ice-cold FACS staining buffer (0.05% BSA, 0.02% sodium azaid in PBS) and fixed for 15 min in 2% paraformaldehyde in PBS on ice. After fixation, the cells were washed once with an ice-cold FACS staining buffer and were resuspended in a permeabilization buffer (0.1% saponin and 0.05% sodium azaid in PBS) for 15 min on ice, followed by incubation with anti-hTERT polyserum (Calbiochem, CA) and anti-Mac-2BP monoclonal antibody (Alexis, San Diego, CA) for 30 min on ice. After 3 washes, the cells were incubated in FITC-conjugated IgG in a permeabilization buffer for 30 min on ice, followed by 3 more washes. Cell pellets were then resuspended in an FACS staining buffer and were analyzed for intracellular Mac-2BP and hTERT levels using FACS Caliber and CellQuest software (Becton Dickinson, Mountain View, CA).
Introduction of siRNA for specific hTERT into SW13/hTERT
Unmodified 21-nucleotide synthetic RNAs were chemically synthesized (Samchully Pharm, Seoul, Korea). The siRNA sequences were as follows: hTERT-siRNA (position 1983∼2001 bp) sense, 5′-GAA CGU UCC GCA GAG AAA ATT-3′; antisense, 5′-UUU UCU CUG CGG AAC GUU CTT-3′; siRNA-control (GL2) sense, 5′-CGU ACG CGG AAU ACU UCG ATT-3′; antisense, 5′-UCG AAG UAU UCC GCG UAC GTT-3′. The transient transfection of synthetic siRNA was accomplished through the use of Lipofectamine Plus™ Reagent (Invitrogen, Carlsbad, CA).
Immunohistochemistry was carried out for evaluation of the modulated proteins present in the tumor tissues. Four micrometer sections were taken from routinely processed paraffin-embedded human gastric carcinoma tissues. Mouse anti-Mac-2BP monoclonal antibody was employed as the primary antibody. In brief, paraffinized sections of the gastric tissues were deparaffinized with xylene and rehydrated in a graded alcohol series. Antigenic enhancement was then conducted via submersion in a 0.1 M citrate buffer (pH 6.0), followed by microwaving. The sections were treated with 3% hydrogen peroxide in methanol in order to quench endogenous peroxidase activity and were then incubated with 1% BSA blocking solution. The primary antibody was incubated for 90 min at room temperature. After washing, the tissue section was allowed to react with biotinylated secondary antibody, followed by incubation with the streptavidin–horseradish–peroxidase complex. The tissue section was immersed in 3-amino-9-ethyl carbazole as a substrate. In the negative controls, the primary antibody was replaced with either nonimmune mouse IgG of the same isotype or a diluted antibody solution. Moderate to strong cytoplasmic staining was considered to indicate a positive reaction. The distribution of Mac-2BP was then scored on a semiquantitative scale, as follows: negative (<10% of tumor positive), focally positive (10–50% of tumor positive) and diffusely positive (>50% of tumor positive).
Blood samples were obtained from the Dankook University College of Medicine with informed consent having been provided by all subjects. In this study, sera from 36 patients with gastric carcinoma and from 9 healthy donors were used. The samples were allowed to clot and were then stored at −20°C until assaying. The gastric patient's group consisted of 24 (67%) men and 12 (33%) women, with a mean age of 61.5 years (range: 41–79 years). According to the TNM classifications and histopathologic grading system of the International Union Against Cancer, there were 11 Stage I, 7 Stage II, 11 Stage III and 7 Stage IV tumors. Tumor grading classified 6 well differentiated tumors, 22 moderately differentiated tumors and 8 poorly differentiated tumors. Six of the men and three of the women had normal gastric sera samples. The mean age of the donors was 44 years (range: 23–60 years). We acquired the approval of the University Ethics Committee and informed consent from each patient and volunteer before performing this experiment.
Differences between gastric patients and normal donor group were tested using Student's t-tests. The p-value indicated a significant difference (p = 0.0012). Correlations between Mac-2BP levels and other clinicopathological features were evaluated using the Mann–Whitney U test.
Mac-2BP was induced by hTERT
To investigate hTERT in terms of its function other than as the reverse transcriptase in gastric carcinogenesis, we searched for genes for which expression is regulated by hTERT. We constructed an hTERT-overexpressing cell line, SW13/hTERT, in the telomerase-negative small cell carcinoma cell line,20 SW13, by stable transfection with an expression vector for full-length hTERT cDNA. RT-PCR analysis proved that SW13 expressed no hTERT mRNA, which was then increased by the introduction of full-length hTERT cDNA (Fig. 1a). Western blot analyses also showed that TERT protein was increased by 2- to 3-fold in the hTERT-overexpressing cells (Fig. 1b).
We then employed BD Atlas™ cDNA expression arrays in order to identify the genes modulated by hTERT overexpression. The Mac-2BP mRNA was screened out as one of the clones that were abundantly induced because of hTERT overexpression (Fig. 2a). RT-PCR and Northern blotting verified that the Mac-2BP transcript had been induced by hTERT expression (Fig. 2b). As evaluated by ELISA, the Mac-2BP protein level was also elevated by 4-fold in the cytosol of cells in which hTERT had been introduced (Fig. 2c). Intracellular staining of Mac-2BP also revealed a 2-fold increase in intensity compared with that of the control (Fig. 2d). These results suggest that hTERT expression exerts profound effects on the induction of Mac-2BP transcripts and protein.
To evaluate whether hTERT expression would up-regulate Mac-2BP directly or indirectly, we introduced small interfering RNA in order to neutralize the exogenously provided hTERT in SW13/hTERT (Fig. 3a) and found that the siRNA-mediated knock-down of hTERT resulted in the reduction of the Mac-2BP protein level to about one-third (Fig. 3b). These results suggest that Mac-2BP expression is regulated by the modulation of hTERT.
Correlation of the Mac-2BP expression with that of hTERT in gastric carcinoma cell lines
We investigated whether Mac-2BP expression is up-regulated in gastric cancer cells and whether its up-regulation is correlated with hTERT level. Mac-2BP expression was evaluated in human gastric cancer cell lines established from Korean cancer patients.21, 22 As shown in Figure 4a, the 2.2-kb Mac-2BP transcript was found in a number of SNU cell lines, including SNU-1, -16, -216, -620 and -638. It was not detected in SNU-484 and was very weakly expressed in the untransformed gastric cell line, HS677.St. At the transcription level, endogenous hTERT transcript showed a similar expression pattern to that of Mac-2BP in SNU cell lines. The hTERT expression pattern in SNU cell lines has been reported previously.23 The expression of Legumain, a molecule which is known to be frequently overexpressed in human solid tumors,24 was evaluated as the control.
We also quantitated the intracellular level of Mac-2BP and hTERT using a flow cytometer and found that SNU-620 and -638 cells abundantly expressed Mac-2BP when compared with SNU-484. However, hTERT protein levels showed a slightly different pattern compared to mRNA; we suggest that hTERT may hold potential for post-transcriptional modifications, as well as transcriptional regulations25, 26, 27 (Fig. 4b). Quantitative ELISA also showed that more than 0.4 μg/mg of Mac-2BP protein was present in the cytosol of SNU-620 and -638 cells (Fig. 4c). These results indicated that the Mac-2BP transcript and its protein products were differentially expressed in the SNU cell lines. It is worthwhile to note that the SNU-620 and -638 cells, derived from metastatic tumors, expressed Mac-2BP at higher levels than other tumor cell lines. Using ELISA, we then determined the level of secreted Mac-2BP in the culture supernatant of the SNU cell lines. As is shown in Figure 4d, the levels of secreted Mac-2BP appeared to be proportional to that of the cytosolic form, and the quantity was approximately 10-fold higher than that in the cytosolic fraction. A significant amount of secreted Mac-2BP was also detected in hTERT-overexpressing cells.
Mac-2BP expression in human gastric tumor tissues
Mac-2BP expression in human gastric tumor tissues was analyzed by immunohistochemical staining with anti-Mac-2BP monoclonal antibody. As shown in Figure 5, Mac-2BP expression was not detected in normal gastric mucosa; however, it was abundantly expressed in gastric tumor tissues, especially in the cytosolic regions of mucosa cells. In this study, Mac-2BP expression was measured in 22 samples of gastric cancer tissues containing normal mucosal tissue; of these, 70–75% of the cancerous tissues showed increased Mac-2BP expression compared with the normal mucosal tissues. These data support the suggestion that Mac-2BP expression may be associated with the progression of gastric cancer.
Serum Mac-2BP protein levels in gastric cancer patients
To investigate the potential of Mac-2BP as a serological marker for gastric cancers, we analyzed sera obtained from 36 gastric cancer patients and 9 healthy donors using ELISA (Fig. 6). The average Mac-2BP level in the sera of the gastric cancer patients was 11 μg/ml, with a range of 2.4–22 μg/ml. This was a significantly higher value than what was measured from healthy blood donors (p = 0.0012). The clinical profiles of patients with a serum Mac-2BP level above the cut-off level (11 μg/ml) are shown in Table I. Statistical analysis of the relationship between the serum Mac-2BP level and clinicopathological parameters revealed that the serum Mac-2BP level was not significantly affected by age or gender. However, patients with distant metastasis tended to have higher serum Mac-2BP concentrations (p = 0.05), and stage III/IV patients also expressed higher Mac-2BP levels than the early stage patients (p = 0.04). Thus, the elevation of serum Mac-2BP levels appears to be closely associated with malignant progression of gastric cancers.
Table I. Correlation Between Clinicopathological Features and Serum Mac-2BP Levels in Patients With Gastric Cancer
Number (percentage) of gastric cancer patients with Mac-2BP levels above the cutoff value of 11.2 μg/ml.
p values were determined by Mann–Whitney U test.
I + II
III + IV
Gastric cancer is the second most common cause of cancer-related mortality in the world. Gastric cancer is most frequently encountered in Japan, China and South America, while it occurs at relatively low rates in Western Europe and the United States.28 In the search for new tools for detecting and eliminating gastric cancers, a number of tumor antigens have been identified, and many of these have been employed as immunotherapeutic agents.29, 30, 31 However, in the clinical context, the therapeutic effects of the currently employed agents remain unsatisfactory.
Telomerase activity and hTERT mRNA expression have been found to be closely correlated with the progression of gastric cancer.23, 32 In the present study, we have shown for the first time that hTERT could up-regulate the expression of Mac-2BP and have identified it as a serological marker associated with malignant progression of gastric cancers. The tumor-associated antigen, Mac-2BP, was originally isolated from the culture media of breast carcinoma cells33 and was identified as a member of the macrophage scavenger receptor cysteine-rich domain (SRCR) family of proteins. The majority of the proteins in this family, including CD5, CD6, M130, complement factor 1, WC1 and several other proteins, play biological roles in both immune defense and immune regulation.34 Additionally, Mac-2BP appears to function as a ligand for galectin proteins and mediates cell-to-cell aggregation, as well as cell-to-matrix aggregation via interactions with β-integrin. Mac-2BP has also been shown to inhibit chemotherapeutic drug-induced apoptosis.8, 35Via these interactions, Mac-2BP may be intimately involved in the metastatic activity and chemoresistance of cancer cells. The elevated Mac-2BP levels in cancerous tissues may play roles in the higher incidence of metastasis and poor survival rates seen in patients with several different types of cancer.3, 5 Meanwhile, Mac-2BP also appears to perform a function relevant to the activation of host cellular immune responses, via the generation of IL-1, IL-6 and other cytokines in peripheral blood mononuclear cells (PBMC), the stimulation of activity of NK and LAK,2, 36 and the up-regulation of major histocompatibility complex (MHC) class I molecules.37
Here, we have shown that Mac-2BP expression was controlled, to some degree, by intracellular hTERT in SW13 cells (Fig. 3). The Mac-2BP expression was also similarly induced by hTERT in other cells such as human breast cancer (MCF-7) and human embryonic kidney cell lines (HEK293), which suggests that hTERT-induced Mac-2BP expression was not specific to SW13 cells (data not shown). The up-regulation of Mac-2BP by hTERT might also be associated with the progression of gastric cancer, as it was expressed in abundance in human gastric carcinoma cell lines, particularly in those with metastatic potential where hTERT mRNA expression levels were similarly high. We also showed that secreted Mac-2BP could be detected in the culture supernatant of tumor cell lines by using ELISA techniques. The level of secreted Mac-2BP protein was up to 10-fold higher than that of cytosolic Mac-2BP. In addition, an immunohistochemical approach in gastric tumor tissues revealed that Mac-2BP may prove to be a valuable clinical biomarker, as it was expressed at substantial levels in gastric tumor tissues, but was barely detectable in normal tissues. The serum reactivity of Mac-2BP of gastric cancer patients was also significantly higher than that of healthy donors. These results suggest that Mac-2BP is specifically overexpressed in gastric tumor tissues, compared with normal tissues. Our population studies showed that the serum Mac-2BP level was significantly associated with distant metastasis (p = 0.05) and later tumor stages (p = 0.04) in gastric cancer. Therefore, Mac-2BP does, indeed, appear to be a valuable prognostic marker for gastric cancers.
Our study left some of the flow cytometric results of hTERT in Figure 4b questionable, as the hTERT protein level was shown to be slightly different from the mRNA level. Therefore, we tried to understand the relationships between hTERT and Mac-2BP from various points of view. It might be considered that the discrepancy between hTERT mRNA and protein levels and the Mac-2BP-inducing activity in some of the stomach cancer cell lines was due to alternative splicing of hTERT mRNA, although we did not solve this matter thoroughly.25, 26, 27 As TERT activation is been known to be a limiting step for the induction of telomerase activity in most cells, the transcriptional regulation of TERT has recently become the focus of a great deal of attention. However, the up-regulation of TERT levels does not always coincide with higher enzymatic activity.38, 39 Additionally, the specific pattern of hTERT mRNA variants in human development provides evidence that alternative splicing is nonrandom and participates in the regulation of telomerase activity.40 Hara et al. reported cases of renal cell carcinoma in which the telomerase activities in protein levels were not always in parallel with the hTERT mRNA levels.41 The sample, which expressed only the spliced transcripts, rarely showed a high level of telomerase activities. It is also known that the abundance of full-length transcripts, when compared with spliced variants, affects the telomerase activities.42 It is possible that alternatively-spliced variants of hTERT would work on the regulation of the full-length hTERT transcripts. It was recently demonstrated that ectopically-expressed hTERT alpha variants inhibited the endogenous telomerase.43, 44 Therefore, it may eventually be found that hTERT expression can be regulated at multiple levels, including the transcription and alternative splicing of hTERT transcripts. In addition to these noted effects of hTERT mRNA metabolism on the ultimate telomerase activity, the inactivity of the Mac-2BP promoter can be another possible reason for the discrepancy between hTERT and Mac-2BP protein levels in cells such as SNU-484.
In conclusion, the data obtained in this study indicate that Mac-2BP was overexpressed in gastric tumors, particularly in those with metastatic potential, and that both ELISA and immunohistochemical staining with anti-Mac-2BP can be employed as effective diagnostic tools for gastric cancers.