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Carcinogenesis Advance Access originally published online on March 26, 2007
Carcinogenesis 2007 28(8):1622-1628; doi:10.1093/carcin/bgm064
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Mechanisms of growth arrest by zinc ribbon domain-containing 1 in gastric cancer cells

Liu Hong1,{dagger}, Yunping Zhao1,2,{dagger}, Ying Han1, Wei Guo1,2, Haifeng Jin1, Taidong Qiao1, Zheng Che1 and Daiming Fan1,*

1 State Key Laboratory of Cancer Biology and Institute of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi'an, 710032 Shaanxi Province, China
2 Department of Thoracic Surgery, Institute of Surgery Research, Daping Hospital. Third Military Medical University, Chongqing, China

* To whom correspondence should be addressed. Tel: +86 29 84775221; Fax: +86 29 82539041; Email: hlhyhj{at}126.com


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Previous studies by our laboratory indicated that zinc ribbon domain-containing 1 (ZNRD1) suppressed the growth of gastric cancer cells with a G1 cell cycle arrest. However, the precise molecular mechanism underlying the growth-inhibitory effect of ZNRD1 remained fragmentary. In the present study, we have demonstrated that ZNRD1 could significantly inhibit the in vitro and in vivo growth of gastric cell line MKN28. Human cDNA microarray, reverse transcription–polymerase chain reaction and western blot analyses were used to identify differentially expressed cell cycle-related genes in MKN28 cells over-expressing ZNRD1. ZNRD1-induced growth suppression was found at least partially to regulate various proteins and signaling pathways controlling G1 to S progression, including inhibition of cyclin D1 and CDK4, up-regulation of p21CIP1/WAF1 and p27Kip1 and acceleration of pRb dephosphorylation. Furthermore, ZNRD1 significantly inhibited the transcriptional activity of cyclin D1. p27Kip1 might play a pivotal role in ZNRD1-induced cell cycle arrest because the p27Kip1 anti-sense could block the cytostatic effects of ZNRD1. Moreover, ZNRD1 suppressed Skp2 expression via an increase in the protein instability, and induced significant decrease in cyclin E–CDK2 kinase activity. In addition, ZNRD1 could reduce tumor microvessel densities through inhibition of VEGF. Taken together, these results suggested that ZNRD1 might inhibit cell growth by targeting cell cycle-related genes and reducing tumor angiogenesis.

Abbreviations: FCM, flow cytometry; RT–PCR, reverse transcription–polymerase chain reaction; ZNRD1, zinc ribbon domain-containing 1


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
It was currently believed that the loss of normal cell cycle control played an important role in the genesis of most cancers. Cell cycle progression was governed by the actions of regulators, including positive regulators (cyclins and CDKs) and negative regulators (CKIs) (1). In normal cells, the proliferation was under strict regulation and these regulators worked in concert to ensure a regulated transition from one phase of the cell cycle to the next. In tumor cells, this exquisite balance between the positive and negative regulators was not maintained, thus contributing to the malignant phenotype. Therefore, the cell cycle regulatory proteins have been under intense investigation as the potential molecular targets of tumor therapy.

The connection between cancer and the cell cycle has been established in part due to the alteration in the expression and function of G1 cyclins in cancer cells and tissues. Cyclins D1–D3 and E and their respective kinase partners, CDK4/6 and CDK2, were responsible for regulating the transition from G1 to S phase. For example, cyclin D1, whose major function was the phosphorylation of the retinoblastoma gene product pRb (2), was a proto-oncogenic regulator of the G1–S phase checkpoint.

Zinc ribbon domain-containing 1 (ZNRD1) gene was cloned from human HLA, encoding a transcription-associated protein consisting of two zinc ribbon domains (3). Our laboratory has first found that ZNRD1 might be a regulator of gastric carcinogenesis. First, ZNRD1 was found to be down-regulated in human gastric cancer (4,5). Second, up-regulation of ZNRD1 might inhibit the growth of gastric cancer cell line AGS in vitro and possess anti-carcinogenic activity in vivo (6). Thirdly, down-regulation of ZNRD1 in normal gastric epithelium cell line GES could enhance cell growth significantly and promote cells from G1 to S phase. Lastly, up-regulation of ZNRD1 might induce the arrest of AGS cells in G1 phase as a consequence of inhibiting cyclin D1 expression (5,6). However, the precise molecular mechanism underlying the growth-inhibitory effect of ZNRD1 remained largely unknown. In this study, we provided the first evidence that ZNRD1 might inhibit cell growth by targeting cell cycle-related genes and reducing tumor angiogenesis. These results suggested that ZNRD1 had the eligible profile to be tested as a therapeutic agent for the treatment of gastric cancer.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 References
 
Materials
The following materials were purchased from commercial sources: Dulbecco's modified Eagle's medium, trypsin/EDTA solution, cellfectin and G418 (Life Technologies, Grand Island, NY); LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA); Trizol reagent (Invitrogen); nitrocellulose membrane (Immobilon-P, Millipore, Bedford, MA); enhanced chemiluminescence agent (Amersham Life Science, Piscataway, NJ); anti-cyclins D1, D2, D3, anti-cyclin E, anti-CDK2, anti-CDK4, anti-CDK6, anti-p27Kip1, anti-p21CIP1/WAF1, anti-p57Kip2 antibodies, anti-Skp2, anti-CD31, anti-VEGF and goat anti-mouse immunoglobulin–horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Corp., Santa Cruz, CA); anti-p15INK4b, anti-p16INK4a and anti-p18INK4C (Upstate Biotechnology, Lake Placid, NY); anti-underphosphorylated pRB, p27Kip1 anti-sense oligonucleotides (TriLink BioTechnologies, San Diego, CA); p21CIP1/WAF1 anti-sense oligonucleotides (Eurobio, Les Ullis, France) and anti-ß-actin and cycloheximide (Sigma, St Louis, MO). Monoclonal anti-ZNRD1 was established by our laboratory (6).

Cell culture and transfection
Human gastric cancer cell line MKN28 was cultivated in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U/ml) and streptomycin (100 µg/ml) in a CO2 incubator (Forma Scientific).

MKN28 cells were grown on 6-well plates to 80% confluence and then transfected with the eukaryotic expression vector of ZNRD1 using the LipofectamineTM 2000 reagent as described previously (6). The cells transfected with pcDNA3.1 vector alone were served as negative control. Forty-eight hours later, cells were placed in growth medium containing G418 for clone selection.

Human Skp2 cDNA was generated by reverse transcription–polymerase chain reaction (RT–PCR) and subcloned into pcDNA3.1 expression vector (Invitrogen) as described previously (7) and then were transfected transiently into cells using the LipofectamineTM 2000 reagent. A mock vector-transfected clone was used as a control.

Cell growth assay
Cells were seeded on a 96-well plate at 3 x 104 cells per well. Each sample had four replicates. The medium was replaced at 2 day intervals. Viable cells were counted by the 3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyltetrazolium bromide (MTT) assay after 2, 4, 6 and 8 days.

Soft agar assay
Briefly, a volume of 2 ml of 0.5% agar was added to each well of 12-well plate and allowed to set. Cells were harvested, washed and mixed with the top-agarose suspension at a final concentration of 0.3%, which was then layered onto the bottom agar. Cells were incubated for 2 weeks at 37°C in 5% CO2 before counting colonies. Each assay was performed in triplicate.

Tumor growth in nude mice
Female athymic nu/nu mice, 5–6 weeks of age, were obtained from FMMU Experimental Animal Co. (Shaanxi, China) and housed in a pathogen-free facility for all of the experiments. The logarithmically growing cells were trypsinized and re-suspended in D'Hanks solution, and 5 x 106 cells in 0.2 ml were injected subcutaneously into the left flank of mice. Experimental and control groups had at least six mice each. Tumors were measured twice weekly with microcalipers, and the tumor volume was calculated according to the formula: volume = length x (width2)/2.

Cell cycle analysis
The cell cycle was analyzed by flow cytometry (FCM). Briefly, 1 x 106 cells were harvested and washed in phosphate-buffered saline, then fixed in 75% alcohol and kept at 4°C for 30 min. The suspension was filtered through 50 um nylon mesh, and the DNA content of stained nuclei was analyzed by a FACS Calibur flow cytometer (Becton Dickinson) equipped with a 15 mW, 488 nm air-cooled argon-ion laser. Data acquisition and analysis were performed using CellQuestPro software.

Cell cycle synchronization
Cells were synchronized by double thymidine block. Briefly, cells were treated with 2 mM thymidine for 16 h. Then cells were washed two times with Hanks buffered saline solution and incubated for an additional 8 h in the absence of thymidine. Cells were incubated a second time with 2 mM thymidine for 16 h to arrest cells at the G1/S boundary of the cell cycle. Lastly, cells were harvested at 4 h intervals for 16 h and were analyzed by FCM.

cDNA microarray analysis
The gene expression was compared between MKN28-Z3 and MKN28-vector cells for three times. RNA was extracted from 80 to 90% confluent cells using Trizol and purified with RNeasy spin columns (Qiagen, Valencia, CA) according to the manufacturer's instructions. Quality of the RNA was ensured before labeling by analyzing 20–50 ng of each sample using the RNA 6000 NanoAssay and a Bioanalyzer 2100 (Agilent, Palo Alto, CA). Samples with a peak ratio of 1.8–2.0 were considered suitable for labeling. Cy3- or Cy5-labeled cDNA was generated and the Cy3/Cy5 single-stranded cDNA/cot1 DNA pellet was re-suspended in hybridization buffer and then the hybridization mix was applied to Agilent Human 1A oligonucleotide arrays. Detailed labeling and hybridization protocols were available for download (8). Arrays were read on an Agilent Microarray Scanner (Agilent Technologies), and data were collected using Genepix Pro 4.0 software (Axon Instruments, Foster City, CA).

The ratios of gene expression were considered to be significant if they were 4 or 0.25 in at least two independent experiments. Genes were assigned to functional families based on information from LocusLink and PubMed.

RT–PCR and western blot
All of the PCR products were separated on ethidium bromide-stained agarose and visualized with UV. A measure of 60 ug of lysates were electrophoresed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted on a nitrocellulose membrane. The primers for RT–PCR were showed as supplement data.

Reporter gene assay
The pGL3-cyclin D1 vector (promoter of cyclin D1: –127 to –99) and the control vector were the generous gift from Dr Chenguang Wang (Georgetown, MA) (9). Briefly, MKN28 cells were co-transfected with indicated amounts of pcDNA3.1/ZNRD1 plasmids (0.3, 0.5 and 0.8 µg), pGL3-cyclin D1 and pRL-TK vector using the Fugene transfection reagent (Roche, Indianapolis, IN). After 48 h, luciferase reporter assays were performed following the protocol of Promega Corp. Cells were then processed for ß-galactosidase staining with the PanVera (Madison, WI) ß-galactosidase Staining Kit according to manufacturer's protocols. A total of 300 cells per well were counted, and the percentage of blue cells was determined.

p21CIP1/WAF1 and p27Kip1 anti-sense oligonucleotides
The sequences of the p27Kip1 anti-sense and the mismatch control oligonucleotides were 5'-UGGCUCUCCUGCGCC-3' (targets 306–320 bp of murine Kip1) and 5'-UCCCUUUGGCGCGCC-3', respectively (10). The sequences of anti-p21CIP1/WAF1 oligonucleotides (5'-TCCCCAGCCGGTTCTGACAT-3') and the control oligonucleotides (5'-ATGTCAGAACCGGCTGGGGA-3') have been shown to be effective at inhibiting p21CIP1/WAF1 expression (11). Briefly, 20 µl of Cellfectin and oligonucleotides (2 µM final concentration) were incubated in 1 ml of serum-free RPMI 1640 for 30 min and subsequently added to the monolayer with 1 ml of RPMI 1640 supplemented with 10% fetal calf serum. The oligonucleotide/Cellfectin solution was then decanted and the monolayer washed once with RPMI 1640 (5% fetal calf serum). Forty-eight hours after treatment with oligonucleotides, the cells were either subjected to cell cycle analysis by FCM or lysed and subjected to immunoblot procedures.

Protein stabilization
For p27Kip1 and Skp2 protein stabilization, we measured their half-life by treatment with cycloheximide for 0, 1, 2 and 4 h. After treatment, cells were collected and the protein was examined by western blot analysis as described above.

Kinase assay
CDK2 and cyclin E-associated histone H1 kinase activity was determined as described previously (12). Briefly, 200 µg of the protein lysate was precleared with protein A/G-plus agarose beads and then CDK2 and cyclin E proteins were immunoprecipitated using anti-CDK2 and anti-cyclin E antibodies, respectively. The kinase reactions were initiated by re-suspending the beads in 25 µl of kinase buffer containing 2 µg of histone H1. The reaction was stopped by adding 2x sodium dodecyl sulfate sample buffer. After boiling for 5 min, the reaction products were electrophoretically separated on a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and were visualized by autoradiography.

Quantification of tumor microvessel density
Tumor microvessel densities were quantified by anti-CD31 immunohistochemistry as described previously (13). Briefly, tumor sections from nude mice were cut using a LEICA cryostat and the paraffin sections (4–6 µm thick) were mounted on positively charged Superfrost slides and dried overnight. The immunostaining was done according to standardized protocols. CD31 localization was revealed by using a peroxidase reaction with 3,3'-diaminobenzidine as the chromogen. After completion of the immunostaining cycle, slides were counterstained in hematoxylin, rinsed, dehydrated and coverslipped for microscopic evaluation.

Regions of highest vessel density (‘hot spot’ regions) were scanned at low magnification (40x to 100x) and counted at higher magnification (200x). Three such hot spot fields were counted in each tumor section, and the mean microvessel density value was recorded. Any endothelial cell or endothelial cell cluster that was clearly separated from adjacent microvessels was considered as a single, countable microvessel.

Detection of VEGF in cell culture and tumor homogenates
Cells were cultured for 48 h, and then supernatants were taken from the cells. Secreted VEGF was determined by using the ELISA development kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Each data point was presented as the mean of triplicate wells. Tumor extracts were prepared by homogenizing and sonicating non-necrotic areas of pancreatic tumors in anti-protease lysis buffer (Roche Applied Science). To quantitate VEGF levels in tumor homogenates, lysates obtained from the homogenized tumor tissues were subjected to the ELISA assay.

Statistics
The data were expressed as the means ± standard deviations. Comparisons between groups were made using the Student–Newman–Keuls test or the Kruskal–Wallis test. All data were analyzed using the SPSS software package (SPSS, Chicago, IL). A value of P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 References
 
Up-regulation of ZNRD1 inhibited the growth and tumorigenecity of MKN28 cells
As Figure 1A showed, MKN28 cells were transfected with the recombinant plasmids containing the full ORF of wild-type ZNRD1, and three resistant clones (MKN28-Z1, MKN28-Z2 and MKN28-Z3) with significantly increased ZNRD1 expression and one vector-transfected control clone (MKN28-vector) were selected. Cell growth was assayed over 9 days using a biomass assay (Figure 1B). Up-regulation of ZNRD1 significantly decreased the proliferation of MKN28 cells (P < 0.05).


Figure 1
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  		Fig. 1. ZNRD1 suppressed growth of gastric cancer cells in vitro and in vivo. (A) Western blot analysis of the parental cells MKN28 (P), the vector transfectants MKN28-V (V) and three ZNRD1-over-expressing transfectants MKN28-Z1, -Z3 and -Z3 (Z1, Z2 and Z3). Total cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted with an antibody to ZNRD1. ß-Actin was used as loading control. (B) The growth rate of the cells was detected using MTT assay as described in Materials and methods. The value shown was the mean of three determinations. (C) Colony numbers of the cells were detected in soft agar. The data represented the mean ± standard deviation of three independent experiments. (D) Tumorigenicity of the cells in BALB/c nu/nu mice was detected as described in Materials and methods. Each group had at least 6 mice. The volumes of tumors were monitored at the indicated time.

 
MKN28 cells and their transfectants were seeded in soft agar and colon formation was assessed after 2 weeks. As shown in Figure 1C, the parental cells and the clones carrying the empty vector formed numerous colonies compared with ZNRD1-over-expressing cells, which showed restricted growth. Tumorigenesis was found profoundly decreased in ZNRD1-over-expressing MKN28 cells (Figure 1D), suggesting that ZNRD1 might be directly involved in inhibiting tumor growth. Taken together, these data implied that ZNRD1 might play a suppressor role to reverse the malignant growth potential of MKN28 cells in vitro and in vivo.

ZNRD1 suppressed growth through a G1 cell cycle arrest
The percentage of cells in G1, S and G2 phases was examined by FCM (Table I). Up-regulation of ZNRD1 induced an increase in the number of cells in G1 phase with a concomitant decrease in the proportion of cells in S phase.


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  		Table I. The percentage of gastric cancer cells in G1 and S phases (%)

 
As shown in Table I, we further analyzed cell cycle distribution of transfectants by blocking the cell progression of G1 to S phase using thymidine. After blocked by thymidine, the cells were mostly kept in G1 phase (0 h). After 4 h releasing, the cells were entering into the S phase, and the releasing rate of cells transmitted from G1 to S phase was much lower in ZNRD1-over-expressing cells than that of control cells. After 8 h releasing, most cells went into G2 phase, and no significant difference was found between ZNRD1-over-expressing cells and control cells. Twelve hours later, cells came back from G2 to G1. ZNRD1-over-expressing cells seemed a little faster than control cells. By 16 h later, no significant difference was found between ZNRD1-over-expressing cells and control cells. Taken together, these data strongly implied that ZNRD1 might induce G1 arrest in gastric cancer cells.

Effect of ZNRD1 on molecules regulating the G1 to S transition
The gene expression was compared between MKN28-Z3 and MKN28-vector cells (data were showed as supplement data). After up-regulation of ZNRD1, MKN28-Z3 cells showed alteration of various types of genes, including those involved in transcription, signal transduction, development, protein kinases, cell structure, cell adhesion, angiogenesis, drug resistance, DNA repair, cell cycle, apoptosis and inflammatory response. Totally five cell cycle-related genes, including cyclin D1, CDK4, p21CIP1/WAF1, p27Kip1 and RB1, were found altered. As all of them played key roles in regulating the transition from G1 to S phase, we focused our attention on the effect exerted by ZNRD1 on molecules involved in the G1 to S transition. As the results of RT–PCR showed (Figure 2A), a strong reduction in the expression of cyclin D1 was observed in MKN28-Z3 cells, whereas no differentiation was observed on cyclin D2, cyclin D3 and cyclin E expression. ZNRD1 also selectively decreased the level of CDK4 and increased the level of the CDK inhibitors p21CIP1/WAF1 and p27Kip1. The results were consistent with that of microarray analysis, and were further confirmed by western blot (Figure 2B), suggesting transcriptional and translational regulation of these genes by ZNRD1.


Figure 2
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  		Fig. 2. G1 growth arrest induced by ZNRD1 was associated with modulation of cell cycle regulatory proteins. Cyclins D1, D2, D3, E; CDK 2, 4, 6; CDK inhibitors p15INK4b, p16INK4a, p18INK4C, p21CIP1/WAF1, p27Kip1, p57Kip2 and underphosphorylated pRB were detected in gastric cancer cells by RT–PCR (A) or western blot (B). (C) Luciferase reporter assay was detected by co-transfection of the reporter gene of cyclin D1 (0.2 µg per well) with increasing amounts of ZNRD1 expression vector (0.3, 0.5 and 0.8 µg) in MKN28 cells. Luciferase activities were normalized against the activities of the control vector pRL-TK. Results were presented as means ± standard deviations (n = 3).

 
Effect of ZNRD1 on RB protein family members
pRb played a key role in the G1–S transition of the cell cycle and released the transcription factors bound by RB, resulting in their subsequent binding to the promoter regions of various genes. In replicating cells, Rb was successively phosphorylated. Dephosphorylated pRb could bind to and inactivate E2F, thereby repressing transcription of multiple genes involved in S phase progression (14). As shown in Figure 2B, the minor increase in the amount of underphosphorylated pRb might contribute to the growth arrest of ZNRD1-over-expressing transfectants.

Effect of ZNRD1 on cyclin D1 promoter activity
To elucidate the regulatory effects of ZNRD1 on the promoter activity of cyclin D1, luciferase reporter assays were performed. As shown in Figure 2C, co-transfection of the cyclin D1 reporter gene with increasing amounts of ZNRD1 expression vector resulted in an essentially linear decrease in cyclin D1 promoter activity, indicating that ZNRD1 might be involved in regulation of cyclin D1 transcription.

p27Kip1 played a pivotal role in ZNRD1-induced growth inhibition
p21CIP1/WAF1 and p27Kip1 had a dual function in the cell cycle: inhibition of CDK–cyclin formation, particularly cyclin E–CDK2, as well as facilitating the assembly of cyclin D–CDK4/6 complexes (15). To clarify the hypothesis that increased p27Kip1 and p21CIP1/WAF1 were responsible for the ZNRD1-induced growth inhibition, ZNRD1-over-expressing transfectants were treated with p21CIP1/WAF1 or p27Kip1 anti-sense oligonucleotides (or the appropriate sense control). As shown in Figure 3A, compared with cells without anti-senses transfection, the expression of p27Kip1 and p21CIP1/WAF1 in anti-sense-treated cells was significantly decreased. FCM analysis was used as a measure of the cell cycle. As shown in Figure 3B, when p21CIP1/WAF1 was down-regulated, the percentage of G1 cells was slightly but insignificantly lower (P > 0.05). However, as shown in Figure 3C, anti-sense oligonucleotides of p27Kip1 significantly blocked the increase in the G1 cell fraction induced by up-regulation of ZNRD1 (P < 0.05), whereas mismatch controls did not. Taken together, p27Kip1 might play a partial role in mediating ZNRD1-induced growth arrest, whereas p21CIP1/WAF1 might play a relatively minor role.


Figure 3
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  		Fig. 3. p27Kip1 played important roles in ZNRD1-mediated cell growth inhibition. The MKN28-Z1 (Z1), MKN28-Z2 (Z2) and MKN28-Z3 (Z3) cells (Z) were treated with p21CIP1/WAF1 anti-sense oligonucleotide, p27Kip1 anti-sense oligonucleotide (AS) or the appropriate sense control (C). (A) Total protein levels of p21CIP1/WAF1 and p27Kip1 after 48 h of anti-sense treatment. Total cell lysates were prepared, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted for p21CIP1/WAF1 and p27Kip1. The nitrocellulose was subsequently blotted with an antibody to ß-actin as a loading control. (B) The p21CIP1/WAF1 anti-sense played a minor role in mediating ZNRD1-mediated G1 phase arrest. Forty-eight hours after transduction, the DNA content of cells was determined by propidium iodide staining and FACS analysis. The percentage of cells in G1 stage of the cell cycle was showed. (C) p27Kip1 played a pivotal role in mediating ZNRD1-mediated growth arrest. The p27Kip1 anti-sense significantly blocked the increase in the G1 phase induced by up-regulation of ZNRD1 (P < 0.05), whereas mismatch controls did not. The data represented the mean ± standard deviation of three independent experiments. *, P < 0.05 versus Z cells and C cells.

 
Effect of ZNRD1 on the stability of p27Kip1 and Skp2
As shown in Figure 4A, the stability of p27Kip1 seemed increased in the ZNRD1-over-expressing transfectants (Z3). However, since Z3 cells have higher p27Kip1 level at 0 h than control cells, whether ZNRD1 affected the stability of p27Kip1 was still questionable from this data. Recent studies have shown that the stability of p27Kip1 was regulated by the F-box protein Skp2 of the SCFSkp2 complex (16). Moreover, Skp2 expression correlated inversely with p27Kip1 in human tumors (17). Consistent with the results of microarray, the results of RT–PCR and immunoblot analyses revealed that the ZNRD1-over-expressing transfectants expressed a lower level of Skp2 accompanied by a higher level of p27Kip1 (Figure 4B). The data further confirmed that ZNRD1 enhanced the protein instability of Skp2 (Figure 4A). We next tested the importance of Skp2 suppression in ZNRD1-induced growth inhibition. As shown in Figure 4C, Skp2 protein levels were significantly higher in Skp2-transfected cells compared with cells transfected with an empty plasmid. The results of FCM (Figure 4D) showed that up-regulation of Skp2 significantly inhibited ZNRD1-induced G1 phase arrest.


Figure 4
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  		Fig. 4. Effect of ZNRD1 on p27Kip1 stability, Skp2 stability and cyclin E–CDK2 kinase activity. (A) Half-life of p27Kip1 and Skp2 in MKN28 cells (P), MKN28-V cells (V) and MKN28-Z3 cells (Z3). Cells were treated with 150 µM cycloheximide for 0, 0.5, 1 and 2 h, and then were processed for immunoblotting with the indicated antibodies. (B) The expression of Skp2 was detected in gastric cancer cells by RT–PCR (up) and western blot (down). (C) Western blot analysis of lysates from Z3 cells transfected with Skp2 expression vector (S) or empty control vector (C) for 2 days. (D) Up-regulation of Skp2 significantly reversed ZNRD1-induced G1 phase arrest (P < 0.05). Forty-eight hours after transfection, the cell cycle distribution of cells was determined. The data represented the mean ± standard deviation of three independent experiments. *, P < 0.05 versus Z3 cells and C cells. (E) Cyclin E was immunoprecipitated and subjected to an in vitro kinase assay using histone H1 as substrate to determine the activity of cyclin E–CDK2.

 
Effect of ZNRD1 on cyclin E–CDK2 kinase activity
As shown in Figure 4E, compared with the parental cells and the empty vector cells, up-regulation of ZNRD1 led to significant inhibition in cyclin E–CDK2 kinase activity.

Effect of ZNRD1 on tumor vasculature
The formation of a microvascular endothelium might play a critical role in the growth of established tumors. To further determine whether ZNRD1 affected tumor angiogenesis, we analyzed the vascularization of nude mice tumors by immunostaining with CD31, which was a marker on vascular endothelial cells. A significant reduction in the number of endothelial cells was observed within the tumors formed by ZNRD1-over-expressing transfectants (Figure 5A).


Figure 5
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  		Fig. 5. Effects of ZNRD1 on tumor angiogenesis and VEGF production. (A) Tumor microvessel densities (means) in sections from tumors formed by MKN28 cells (P), MKN28-V cells (V) and MKN28-Z1, -Z2, -Z3 cells (Z1, Z2 and Z3). Tumor samples were immunostained with antibodies against CD31. The areas of tumor capillary vessels/high-power field were calculated, and the microvessel density was expressed as the mean percentage of vessel areas/field from three highly vascularized areas. The quantitative microvessel density count in ZNRD1-over-expressing tumor was significantly less than those of P group or V group. Mean ± standard deviation, n = 3. *, P < 0.05 versus P or V group. (B) The expression of VEGF165 in one random section from each tumor group was detected by RT–PCR (up) and western blot (down). The results revealed strong reductions of VEGF in ZNRD1-over-expressing tumors (Z1, Z2 and Z3). (C) Effect of ZNRD1 on production of VEGF by cancer cells. Forty-eight hours after cell culture, conditioned medium was taken from each culture well and subjected to ELISA assay for human VEGF concentration. Results presented were representative of three independent experiments. (D) VEGF levels in tumor homogenates of one random section from each tumor group. Supernatants obtained from the homogenized tumor samples were subjected to ELISA assay for VEGF. Each column was presented as the mean of triplicate ELISA wells.

 
Effect of ZNRD1 on the production of VEGF
The expression of VEGF165 was found significantly decreased in the tumors formed by ZNRD1-over-expressing transfectants (Figure 5B). As shown in Figure 5C, up-regulation of ZNRD1 significantly inhibited VEGF production by MKN28 cells. These results suggested that ZNRD1 might contribute to the angiogenic phenotype of MKN28 cells. To further determine whether such anti-angiogenic effect also occurred in vivo, we examined the levels of VEGF in the tumor homogenates. As shown in Figure 5D, the levels of VEGF in tumors formed by ZNRD1-over-expressing transfectants were significantly reduced. Because VEGF was a survival factor for endothelial cells (18), the observed reductions in VEGF secretion probably contributed to reduced microvessel densities observed in vivo.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 References
 
The zinc ribbon domain was a ubiquitous motif in archaeal and eucaryal transcription, and was proved to be a functional domain required for biological activities in TFIIS and TFIIB (19–21). The zinc ribbon domain at the C-terminal of ZNRD1, similar to the zinc ribbon motif of human yeast TFIIS (22), was well conserved throughout evolution, including archaea, yeast, drosophila, nematodes, amphibians and mammals. And the analogous Cys4 structural motifs might play important roles in promoting cleavage of the nascent transcript and read-through past the block to elongation (23). Recently, we have found that ZNRD1 expression was related to multidrug resistance and carcinogenesis of gastric cancer (4–6,24–27). There might be a balance. ZNRD1 was down-regulated in gastric cancer samples without chemotherapy, where it was under the balance, so up-regulation of ZNRD1 could reverse the malignant phenotype of gastric cancer cells. ZNRD1 was up-regulated in vincristine-resistant gastric cancer cells compared with drug-sensitive ones, where it was above the balance, so down-regulation of ZNRD1 could reverse the multidrug resistance phenotype of gastric cancer cells. Taken together, ZNRD1 might be associated with transcription regulation and might play potential roles in mediating some physiological and pathological functions.

The present study was aimed at investigating the effect of ZNRD1 on gastric cancer cells and, more importantly, examining the mechanisms governing these effects. The results confirmed with our previous reports that ZNRD1 could reverse the malignant phenotype of gastric cancer cells by causing cell cycle arrest in G1 phase (5,6). Passage through the G1 checkpoint, called the restriction point, allowed cells to progress through the cell cycle in an autonomous and mitogen-independent manner. Cyclins belonging to the D and E families and their respective kinase partners, CDK4/6 and CDK2, were involved in G1 restriction point control. Cyclin D1–CDK4 complexes governed G1 progression, whereas cyclin E–CDK2 complexes controlled entry into S phase. Cyclins were regulated not only at the transcriptional and translational levels but also by their rate of degradation via the ubiquitin pathway (28). An additional level of negative control was produced by the expression of CDK inhibitors, including KIPs (p21CIP1/WAF1, p27Kip1 and p57Kip2) and INKs (p15INK4b, p16INK4a, p18INK4C and p19ARF) (29). Here we clearly showed for the first time that ZNRD1 might restrain cell proliferation by altering the intracellular levels of cyclin D1, CDK4, underphosphorylated pRb, p27Kip1 and p21CIP1/WAF1.

The results of FCM analysis showed that p27Kip1 might play a pivotal role in ZNRD1-induced growth inhibition. It was well known that reduced expression of p27Kip1 was frequently found in various cancers, and the lack of p27Kip1 was suggested to be due to an enhancement of its degradation (30). The increase in the cellular abundance of p27Kip1 was primarily due to a decrease in the rate of its degradation (31). The F-box protein Skp2 played a central role in p27Kip1 ubiquitination, which was thought to be mainly responsible for p27Kip1 degradation. Once p27Kip1 was phosphorylated on T187, it would be recruited to Skp2, then multiple ubiquitin molecules would be attached to the lysine residues of p27Kip1 and finally the ubiquitinated p27Kip1 proteins would be degraded by the 26 S proteasome (32). The data clearly showed that up-regulation of ZNRD1 inhibited the expression of Skp2 by increasing its instability. This reduction of Skp2 correlated with an increase of p27Kip1 and a decrease of cellular proliferation. Up-regulation of Skp2 recovered cell cycle progression in ZNRD1-over-expressing cells, providing evidence that Skp2 was central to ZNRD1-induced proliferation defect.

Cyclin D1 was known to play at least two important roles in facilitating the transition from G1 phase into S. First, it served as the regulatory subunit of CDK4 and contributed to its stability, allowing the initial phosphorylation of pRb in mid-G1. Second, it acted in complex with its partner kinase to sequester the CIP/KIP kinase inhibitors, relieving their inhibitory effect on cyclin E–CDK2 (33). The decrease in the steady-state levels of cyclin D1 in ZNRD1-over-expressing cells and the ability of ZNRD1 to reduce the cyclin D1 promoter activity argued that ZNRD1 might be a transcriptional regulator of cyclin D1. The reduction of cyclin D1 could be reasonably linked to the mechanisms of the observed cell cycle arrest both through the resulting inability of unbound CDK4 to phosphorylate pRb and through the potential decrease in the association of cyclin D–CDK4 complexes with p21CIP1/WAF1 and/or p27Kip1. With limited sequestration of the CIP/KIP proteins, increased levels of p27Kip1 and/or p21CIP1/WAF1 would be free to decrease the ability of cyclin E–CDK2 complexes. Cyclin E–CDK2 activity was required for phosphorylation of pRb, which was minimally phosphorylated in early G1. The increased hypo-phosphorylated form of pRb might exert a growth-suppressive effect by indirectly influencing transcription of cell cycle-related genes through sequestration of critical transcription factors, such as E2F.

The results of immunohistochemical analysis revealed a reduction of vascular density within the tumors formed by ZNRD1-over-expressing transfectants. Such an enhanced anti-angiogenic effect might contribute to the inhibition of the tumorigenesis in nude mice. VEGF expression was associated with an increase in tumor vasculature and a decrease in gastric cancer patient survival (34). The results of in vitro and in vivo studies revealed that up-regulation of ZNRD1 might inhibit the VEGF production by tumor cells. Therefore, the effects of ZNRD1 on VEGF-mediated angiogenesis could prove to be beneficial in patients with gastric cancer.

It was becoming increasingly clear that the signals that govern cellular processes, such as entry and exit from the cell cycle, multidrug resistance and angiogenesis, functioned in complex regulatory networks rather than simple linear pathways and that these networks might be wired differently in different cells or tumor types. The precise mechanism by which ZNRD1 brought about these changes, and which of these changes were primary or secondary ones, remained to be elucidated. Mapping these pathways would aid in the identification of target molecules that could modulate gastric carcinogenesis.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
References
 
Supplementary data can be found at http://carcin.oxfordjournals.org/.


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
This study was supported in part by grants from the National Scientific Foundation of China (30400203).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received January 10, 2007; revised February 24, 2007; accepted March 14, 2007.


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