Vol. 46 No. 6

SCIENCE IN CHINA (Series C)

December 2003

Primary functional identification of gene TMSG-1 MA Chunshu (৴ҝ೮)1, NING Junyu (ુࢊဳ)1, YOU Jiangfeng (ဎߞ‫)ע‬1, LIU Lin (ড ॿ)2, WANG Jieliang (ฆࠑ॥)1, CUI Xianglin (Ӂຳॾ)1, WU Bingquan (๑υᨧ)1 & ZHENG Jie (ᄂ ࠍ)1 1. Department of Pathology, Health Science Center, Peking University, Beijing 100083, China; 2. Analysis Center of Medicine and Hygiene, Health Science Center, Peking University, Beijing 100083, China Correspondence should be addressed to Zheng Jie (email: [email protected]) Received March 13, 2003

Abstract TMSG-1 was a tumor metastasis-related gene identified using mRNA differential display, whose expression level was lower in cancer cell lines with higher metastatic potential and in tumor tissue with metastasis. TMSG-1 was transfected to prostate cancer cell line (PC-3M-1E8) with high metastatic potential to observe the effects of increased expression of TMSG-1 on V-ATPase activity, intracellular pH and cell apoptosis. Subcellular localization of the encoded protein of TMSG-1 was determined by using GFP. Results showed that there were no differences of V-ATPase activity among parental PC-3M-1E8 cell line, pcDNA3 transfectant and anti-TMSG-1 transfectant, whereas the V-ATPase activity was significantly higher in TMSG-1 transfectant than that in parental PC-3M-1E8 cell line, pcDNA3 transfectant and Anti-TMSG-1 transfectant (p<0.001). Intracellular pH (pHi) was detected by using the pH-dependent fluorescence probe BECEF. Results showed the pHi was significantly increased in TMSG-1 transfectant. Cell apoptosis assay demonstrated cell apoptosis was significantly higher in -1 transfectant (p<0.01) and BCL2 expression was down regulated. Subcellular localization of TMSG-1 protein showed TMSG-1 was a transmembrane protein, which predicted TMSG-1 protein was located in cytoplasm system, such as endoplasmic reticulum and mitochondrial. These results indicated TMSG-1 up regulation in prostate cancer cell line could promote V-ATPase activity, increase pHi and cell apoptosis, and inhibit the expression of BCL2. Keywords: prostate cancer, gene transfection, pH, apoptosis. DOI: 10.1360/02yc0159

TMSG-1 (Tumor metastasis suppressor gene-1) is a cancer metastasis-related gene cloned by means of mRNA differential display from human prostate cancer cell lines with different metastatic potential[1], which has higher expression in non-metastatic cell line, whereas lower expression in highly metastatic cell line. In samples of primary gastric carcinoma, the TMSG-1 expression markedly decreased in gastric carcinoma with lymph node metastases. It was found that protein encoded by TMSG-1 could interact with the C subunit of V-ATPase[2], but the role it played was not clear. V-ATPase (Vacuolar-Type (H+)-ATPases) are ubiquitous proton-translocating pumps of eukaryotic cells[3]. The pumps are located in membranes of vacuoles, lysosomes, and other components of the endomembrane system, as well as in certain specialized plasma membranes. Protons are pumped out of the cytoplasm into the organelle or the

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extracellular space in order to regulate the intracellular acidity. C unit of V-ATPase plays an important role in structure and formation of the protons channel. Cytoplasmic pH of mammalian cells is mainly regulated by Na+/H+ exchange and HCO 3 -based transport mechanisms[4]. In most eukaryotic cells, these systems collaborate to maintain intracellular pH (pHi) within a proper values that are permissive for growth and function[5]. V-ATPase-mediated regulation of pHi has also been implicated in cancer drug resistance and the regulation of apoptosis[6,7]. BCECF is a new kind of pH-dependent fluorescence probe, which is the most widely used fluorescent indicator for pHi because of its high sensitivity and no effect on cell viability[8]. In this study, we attempted to analyze the function of TMSG-1 by analysis of subcellular localization of the encoded protein, V-ATPases activity, pHi, and cell apoptosis of the TMSG-1 transfectants. 1

Materials and methods

1.1

Cell culture Subclone PC-3M-1E8 was isolated from human prostate cancer cell line PC-3M by limiting dilution and was chosen for its highest metastatic potentials[5]. Cells were grown in RPMI 1640 medium supplemented with penicillin, streptomycin and 10% fetal calf serum. 1.2

Construction of vector, gene transfections and clones selection TMSG-1 cDNA was isolated in our lab and cloned into pGEM-T-Easy vector. TMSG-1 cDNA fragment was digested with EcoR I, and inserted into the EcoR I sites of pcDNA3 (Invitrogen) by ligation with T4 DNA ligase. Introduce the ligated products into competent DH5D and select for

transformants by PCR and EcoR I restriction mapping. Direction of the inserted gene fragment was identified by BamH I restriction. Positive clones were further identified by sequence analysis. Recombinant plasmids were extracted by using Wizard Plus SV Minipreps Kit (Promega) and transfected into PC-3M-1E8 cell lines using LipofectAmine (GIBICOBRL). After 48ü72 h, positive clones were isolated by maintaining the cells in 800 Pg/mL G418 (GIBICOBRL). 1.3

Neor gene expression analysis by RT-PCR Total RNA of transfectants were isolated using TRIzol Kit (GIBICOBRL). Total RNA

1 Pg was reversely transcripted into cDNA with Oligo(dT) primer and 200 units of MMLV reverse transcriptase (Promega). The reaction mixture was incubated at 42ć for 60 min and terminated by heating at 75ć for 5 min. PCR with 2 PL reverse transcription reaction mixture was performed with primer sets (5Ą -AGA GGC TAT TCT GCT ATG AC-3Ą , 5Ą -GCT TCA GTG ACA ACG TCG AG-3Ą ) in the presence of Taq polymerase (Promega). The parameters for PCR were 94ć, 30 s; 94ć, 30 s; 58ć, 20 s; 72ć, 2 min (30 cycles).

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Northern blot analysis Total RNA was prepared from transfectant cells using TRIzol (GIBCO BRL). Electrophore-

sis of 15 Pg total RNA was performed by using a 2% denatured formaldehyde-agarose gel. RNA was blotted onto a nitrocellulose membrane and fixed to the membrane by baking the membrane for 90 min at 80ć. pcDNA3-S-TMSG-1 plasmid was digested with Xho I and Hind III. TMSG-1 cDNA fragment was purified with NcleoTrap Kit (Clontech) as template of transcription in vitro. Sense and antisense TMSG-1 cDNA probes were radiolabeled using the Riboprobe in vitro Transcription Systems (Promega). Reaction conditions were 5hBuffer 4 PL, 100 mmol/L DTT 2 PL, RNasin inhibitor 0.5 PL, ATP, GTP, CTP (2.5 mmol/L) 4 PL, 100 Pmol/L UTP 2.4 PL, linear template 1 Pg, T7 or SP6 RNA Polymerase 1 PL, [D-32P]UTP 5 PL (10 PCi/PL, Yahui), 37ć, 60 min. DNase 1 PL was added at 37ć for 15 min. Filter was prehybridized at 42ć for 1 h and then hybridized by addition of denatured probe for 16 h. The filter was washed in 2hSSC/0.1%SDS and 0.2hSSC/0.1%SDS at 42ć separately, each twice for 15 min, subsequently exposed to films for 1—3 d. To confirm that equal amounts were loaded in each sample, the filter was later hybridized with a E-actin probe, which was labeled according to the recommended protocol of the Prime-a-gene Labeling System (Promega). 1.5 V-ATPase activity analysis 1.5.1 Preparation of microsomes. Microsomes were prepared as described by Wang[9]. Cultured cells were collected and homogenized with a homogenizer. Homogenizing medium contained 10 mmol/L Hepes (pH 7.4), 0.25 mol/L sucrose, 1 mmol/L DTT, 1 mmol/L PMSF, 1 mmol/L EDTA, 10 mmol/L KCl. After homogenization, the mixture was centrifuged at 700 g for 2 min, and then 10000 g for 15 min. Supernatant was collected. The pellet was resuspended in the medium and homogenized again, centrifuged at 700 g for 2 min, and then 10000 g for 15 min. Supernatant was collected, and then centrifuged at 80000 g for 60 min to pellet the microsomal fraction. The pellet was resuspended in the medium (10 mmol/L Hepes (pH 7.4), 0.25 mol/L sucrose, 1 mmol/L DTT, 1 mmol/L PMSF), and stored at 80ć. All these manipulations were performed at 4ć. 1.5.2

V-ATPase activity analysis.

5 Pg microsome protein was added to 200 PL reaction

medium (containing 10 mmol/L Hepes-Tris pH 7.0, 0.2 mol/L sucrose, 50 mmol/L KCl, 1 mmol/L CDTA, 3 mmol/L ATP, 0.1 mmol/L ammonium molybdate, 5 Pmol/L valinomycin, 5 Pmol/L Nigericin, 5 Pg/mL Oligomycin, 1 mmol/L vanadate). The reaction was started with 5 mmol/L MgSO4 at 37ć for 60 min. To evaluate V-ATPase activity, the release of phosphate was measured in a 96-well microplate reader by colorimetric malachite green assay according to Chan et al.[10]. Absorbency (590 nm) of samples in reaction medium of malachite green was measured to determine the V-ATPase activity by a calibration curve obtained by using potassium dihydrogen phosphate solutions of known concentrations.

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1.5.3 Determination of protein concentration. According to the Bradford assay, bovine serum albumin (BSA) was used to obtain the calibration curve. 1.6 Intracellular pH determination 1.6.1 Preparation of pHi calibration. High potassium Hepes buffer (25 mmol/L Hepes, 5 mmol/L NaCl, 140 mmol/L KCl, 1.8 mmol/L CaCl2, 1 mmol/L MgCl2, 5.5 mmol/L Glucose, 5 Pmol/L Nigericin) at pH 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 was prepared. Cultured cells were washed with the high potassium Hepes buffer of different pH, each twice for 5 min. Cells were incubated in the same solution containing 1 Pmol/L BCECF/AM for 30 min at 37ć in 5% CO2. When the incubating solution was discarded, the cells were digested with trypsin and resuspended in PBS, centrifuged at 100 g at 4ć for 10 min. Then the supernatant was discarded. Cells were suspended in PBS, centrifuged at 100 g at 4ć for 10 min, and the supernatant was discarded . 0.5 mL PBS was added to the suspended cells and constantly stirred, and then filtered through a nylon net. 1.6.2 Flow cytometric analysis. Intracellular pH was estimated by fluorescence intensity ratio (FIR) obtained at 520 nm/640 nm fluorescence emission ratios (excited at 488 nm). Calibration curve was obtained by using high potassium Hepes buffer of different pH to get a linear relationship between pH values and 520 nm/640 nm ratio. 1.7

Apoptosis analysis Cells were digested with trypsin and centrifuged at 100 g for 10 min at 4ć, and the super-

natant was discarded. Cells were suspended in iced PBS, and centrifuged, with the supernatant discarded. 1 mL PBS was added to the suspended cells and constantly stirred, filtered through a nylon net, and centrifuged, with the supernatant discarded. Apoptotic cells were labeled according to the Apoptosis Detect Kit (Biosea). Cells were resuspended in 200 PL binding buffer. 10 PL Annexin-V-FITC and 5 PL PI were added, mixed gently, and incubated at 4ć for 30 min. 300 PL binding buffer was added to the solution to start the analysis. 1.8

Western blot analysis Whole -cell lysates were made by lysing cells in the buffer containing 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.02% NaN2, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40. 50 Pg protein was separated by SDS-PAGE gels and transferred to membranes. The membranes were probed with specific antibody and secondary antibody (Santa Cruz), and protein was visualized by using ECL (Santa Cruz). 1.9 Subcellular localization and transmembrane analysis 1.9.1 Bioinformatic analysis. Subcellular localization and transmembrane analysis can be found at wide web site http: //www.ch.embnet.org and http://psort.nibb.ac.jp. 1.9.2 Subcellular localization of TMSG-1 by GFP. TMSG-1 cDNA fragment was oriented into the Apa I and Kpn I sites of pEGFP-C1 to construct pEGFP-C1-TMSG-1, which can express

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the GFP-TMSG-1 fusion protein. Positive clones were identified by restriction mapping and sequence analysis. Recombinant plasmids and pEGFP-C1 were transfected into PC-3M-1E8 cell lines using LipofectAmine (GIBICOBRL), and the distribution of GFP was observed under a laser confocal microscope. 1.10

Statistical analysis Results were reported as x fSD, followed by Student’s t test.

2

Result

2.1

Identification of recombinants Recombinants were identified by agarose gel electrophoresis, and the plasmid size of about 6.9 kb was primarily regarded as positive clones. Recombinant DNA were restricted with EcoR I and two bands were visual in agarose gel electrophoresis. The fragments of about 5.4 kb and 1.5 kb were positive clones. After BamH I digestion, fragments of sense plasmid were 0.44 kb and 6.46 kb and fragments of antisense plasmid were 1.0 kb and 5.9 kb. pcDNA3 fragments digested by Sma I and Hind III were 1.2 kb and 4.2 kb (fig. 1). Positive recombinants were identified by sequence analysis.

Fig. 1. Restriction mapping of recombinants. 1, Lambda DNA/EcoR I+Hind III marker; 2, pcDNA3/Sma I + Hind III; 3, pcDNA3-S-TMSG-1/BamH I; 4, pcDNA3-

Fig. 2.

Neor gene expression of RT-PCR in transfectants. 1,

100 bp marker; 2, 1E8; 3, 1E8 (pcDNA3); 4 and 5, (pcDNA3-AS-TMSG-1); 6 and 7, (pcDNA3-S-TMSG-1).

AS-TMSG-1/BamH I.

2.2

RT-PCR Expression of Neor gene in pcDAN3 was determined using RT-PCR. Amplification of 210 bp DNA fragment could be detected in positive recombinant, whereas no DNA amplification was found in negative control (fig. 2), which indicated the integration and expression of pcDAN3 vector in these transfectants.

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2.3

Northern blot Expression of sense and antisense TMSG-1 was observed by using Northern blot. Sense and antisense transfectants were determined by using antisense and sense probe. Results showed expression levels of sense and antisense TMSG-1 were higher in 1E8 (s-TMSG-1) and 1E8 (as-TMSG-1), but lower in 1E8 and 1E8 (pcDNA3)(fig. 3). The expression levels of E-actin were similar in all cell lines.

Fig. 4.

V-ATPase activity assays. The V-ATPase activ-

ity in 1E8 (s-TMSG-1) was 4.98f0.04 Pmol pi /(Pg proteingmin), which was significantly increased comFig. 3.

Northern blot. The antisense and sense expres-

sion of TMSG-1 were higher in 1E8 (as-TMSG-1) and

pared with the aforementioned cell line groups (P < 0.001).

1E8 (s-TMSG-1) transfectants respectively. E-actin was blotted as an internal control.

2.4

V-ATPase activity assay V-ATPase activity in 1E8, 1E8 (pcDNA3) and 1E8 (as-TMSG-1) was 2.40f0.04 Pmol pi /Pg

proteingmin, 2.32f0.05 Pmol pi /Pg proteingmin and 2.23f0.22 Pmol pi/Pg proteingmin respectively, whereas that in 1E8 (s-TMSG-1) was 4.98f0.04 Pmol pi /Pg proteingmin, which was significantly higher than aforementioned cell line groups (P<0.001). 2.5

Intracellular pH determination Calibration curve obtained by FIR and corresponding pHi of standard 1E8 cells indicated a linear correlation between pHi (6.5—9.0) and FIR. The regression equation was Y = 1.1166X– 4.1468. According to the FIR, the corresponding pHi were obtained (table 1). Results showed FIR in 1E8 (s-TMSG-1) significantly increased compared with 1E8 (pcDNA3) (P<0.01), and there were no differences among 1E8, 1E8 (pcDNA3) and 1E8 (as-TMSG-1). Compared with 1E8 (pcDNA3), pHi in 1E8 (as-TMSG-1) decreased about 0.333f0.042 and obviously increased about 0.735f0.112 in 1E8 (s-TMSG-1).

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Table 1 FIR and pHi of transfectants Cell line

FIR

pHi

1E8

4.815f0.205

7.936f0.259

1E8 (pcDNA3)

4.755f0.163

7.972f0.205

1E8 (Antisense)

4.383f0.106

7.639f0.175

1E8 (Sense)

5.575f0.092*

8.707f0.126

Results showed FIR in 1E8(s-TMSG-1) was significantly increased compared with 1E8 (pcDNA3) (P < 0.01), and no differences were found among 1E8, 1E8 (pcDNA3) and 1E8 (as-TMSG-1). Compared with 1E8 (pcDNA3), pHi in 1E8 (as-TMSG-1) were decreased about 0.333f0.042 and obviously increased about 0.735f0.112 in 1E8 (s-TMSG-1).

2.6

Apoptosis assays Under the normal culture conditions, the apoptotic population is 0.57f0.12%, 0.41f0.15%,

0.22f0.10% in 1E8, 1E8 (pcDNA3) and 1E8 (as-TMSG-1) respectively, and there is no significant difference among them. Apoptotic population was 5.66f1.18% in 1E8 (s-TMSG-1), increased significantly compared with other groups (P < 0.01) (fig. 5).

Fig. 5.

Apoptosis assays of transfectants. Apoptotic population was 5.66f1.18% in 1E8 (s-TMSG-1), increased significantly

compared with other groups (P < 0.01).

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2.7

Bax and BCL2 protein detection As illustrated in fig. 6, expression levels of Bax protein had no difference among all cell groups, expression levels of BCL2 protein also had no difference among 1E8, 1E8 (pcDNA3) and 1E8 (as-TMSG-1), but obviously decreased in 1E8 (s-TMSG-1). Densitometric analysis of Western blot showed BCL2/Bax ratio was 0.49f0.01, 0.39f0.03, 0.42f0.02, 0.08f0.01 in 1E8, 1E8 (pcDNA3), 1E8 (as-TMSG-1) and 1E8 (s-TMSG-1) groups, respectively. BCL2/Bax ratio was lower in 1E8 (s-TMSG-1) than that in other groups.

Fig. 6. Western blot analysis of expression of BCL2 and Bax. (a) Expression levels of Bax protein had no difference among all cell groups. Expression levels of BCL2 protein in 1E8 (as-TMSG-1) obviously decreased in 1E8 (s-TMSG-1). (b) BCL2/Bax ratio was lower in 1E8 (s-TMSG-1) than that in other groups.

2.8

TMSG-1 protein analysis and localization TMpred software suggested the protein was a transmembrane protein and identified four

transmembrane domains (aa33ü48, aa64ü79, aa114ü130, aa158ü174). PSORTII software analysis predicted TMSG-1 protein was located in cytoplasm (Reliability: 94.1%). The definite location was endoplasmic reticulum 66.7%, mitochondrial 1.1%, plasma membrane 11.1%, Golgi 11.1%. Microscope observation showed that TMSG-1-GFP fusion protein was expressed in cytoplasm and took on granular and annular distribution, while GFP protein was expressed in nucleolus and cytoplasm, and had permeating distribution (fig. 7). 3

Discussion

The full length of TMSG-1 cDNA sequence is 2 kb, encoding a protein of 230 amino acids. Primary analysis indicated that TMSG-1 protein was a transmembrane protein, located in cytoplasm system, such as endoplasmic reticulum and mitochondrial. It was found recently that encoded protein of TMSG-1 could interact with the C subunit of V-ATPase[2]. The C subunits were hydrophobic subunits of 16ü17 kd[3], involving the formation of a proton channel. 2/3 amino acid residues of C subunits formed 4 alpha helix, which were separated by hydrophilic annular zone outside the membrane. 6 C subunits were symmetrically arranged and formed a hexmater stalk

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Fig. 7. Subcellular localization of TMSG-1 by GFP. TMSG-1-GFP fusion protein was expressed in cytoplasm and took on granular and annular distribution (b), while GFP protein was expressed in nucleolus and cytoplasm, and had permeating distribution (a).

with a channel of 15ü20 nm in diameter in the center. A glutamic acid residue in the fourth transmembrane alpha helix of C subunit was the binding site of protons, which played a key role in the translocation of protons. V-ATPase activity increased obviously after transfection of TMSG-1 cDNA. It was indicated that up regulation of TMSG-1 in 1E8 cells could promote the activity of V-ATPase. Functions of V-ATPase are hydrolysis of adenosine triphosphate (ATP) generates an electrochemical potential across the membrane that drives the transport of ions and solutes for the regulation of pHi[5]. Results showed that up regulation of TMSG-1 could increase the pHi obviously and it might be correlated with the promotion of V-ATPase activity. V-ATPase is an H+ exchanger. Recently the effects of Na+/H+ exchanger on pHi, cell proliferation, apoptosis are drawing more and more attention. Apoptosis of cultured thymocytes in vitro was companied with the increasing of pHi caused by the activation of Na+/H+ exchanger, and the pHi was higher in apoptotic cells than that in nonapoptotic cells[11]. Spontaneous apoptosis happened under the condition of intracellular alkalinization and seldom in acidification. In the studies of HL-60, Br-A23187 and thapsigargin caused apoptosis by inducing alkalinization through activating Na+/H+ exchanger, while dimethyl amiloride prohibited apoptosis induced by Br-A23187 and thapsigargin through inhibiting alkalinization[12]. Up regulation of TMSG-1 promoted cell apoptosis under normal cultural condition and inhibited expression levels of BCL2, decreasing the BCL2/Bax ratio obviously. These results indicated TMSG-1 might induce apoptosis by suppressing the expression of BCL2. Although the exact roles of TMSG-1 gene in cancer metastasis are still unclear, our results suggested that regulating the pHi by increasing the V-ATPase activity and inhibiting expression of BCL2 protein to induce the apoptosis might be one of its functions.

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Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 30170363), the Major Science and Technology Program of Ministry of Education, the Major State Basic Research Development Program of People’s Republic of China, the National Program for Key Science and Technology Projects and 211 Key Project.

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