U94 alters FN1 and ANGPTL4 gene expression and inhibits tumorigenesis of prostate cancer cell line PC3

Background Insensitivity of advanced-stage prostate cancer to androgen ablation therapy is a serious problem in clinical practice because it is associated with aggressive progression and poor prognosis. Targeted therapeutic drug discovery efforts are thwarted by lack of adequate knowledge of gene(s) associated with prostate tumorigenesis. Therefore there is the need for studies to provide leads to targeted intervention measures. Here we propose that stable expression of U94, a tumor suppressor gene encoded by human herpesvirus 6A (HHV-6A), could alter gene expression and thereby inhibit the tumorigenicity of PC3 cell line. Microarray gene expression profiling on U94 recombinant PC3 cell line could reveal genes that would elucidate prostate cancer biology, and hopefully identify potential therapeutic targets. Results We have shown that stable expression of U94 gene in PC3 cell line inhibited its focus formation in culture, and tumorigenesis in nude mice. Moreover gene expression profiling revealed dramatic upregulation of FN 1 (fibronectin, 91 ± 16-fold), and profound downregulation of ANGPTL 4 (angiopoietin-like-4, 20 ± 4-fold) in U94 recombinant PC3 cell line. Quantitative real-time polymerase chain reaction (QRT-PCR) analysis showed that the pattern of expression of FN 1 and ANGPTL 4 mRNA were consistent with the microarray data. Based on previous reports, the findings in this study implicate upregulation of FN 1 and downregulation of ANGPTL 4 in the anti tumor activity of U94. Genes with cancer inhibitory activities that were also upregulated include SERPINE 2 (serine/cysteine protease inhibitor 2, 7 ± 1-fold increase) and ADAMTS 1 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, 7 ± 2-fold increase). Additionally, SPUVE 23 (serine protease 23) that is pro-tumorigenic was significantly downregulated (10 ± 1-fold). Conclusion The dramatic upregulation of FN 1 and downregulation of ANGPTL 4 genes in PC3 cell line stably expressing U94 implicate up-regulation of FN 1 and downregulation of ANGPTL 4 in anti tumor activity of U94. Further studies are necessary to determine functional roles of differentially expressed genes in U94 recombinant PC3 cell line, and hopefully provide leads to potential therapeutic targets in prostate cancer.


Background
Prostate cancer is the most common form of malignancy in US males. An estimated 29,900 fatalities out of 230,110 new cases are expected in the year 2004 [1]. Androgen ablation is currently the mainstay in prostate cancer therapy, but its efficiency is marred by the relapse of some advanced-stage prostate cancer cells into an androgen refractory state [2,3]. Advanced-stage prostate cancer progression is usually aggressive and correlates with poor prognosis [2,4,5]. Hence, insensitivity to androgen ablation by advanced-stage prostate cancer invariably constitutes a major problem in clinical therapy. Therefore there is an urgent need for the development of targeted therapeutic strategies in advanced-stage prostate cancer.
Knowledge of the genes that are associated with prostate cancer is important for designing an effective therapeutic strategy. However, present knowledge of the molecular biology of prostate cancer is inadequate to define etiologic genes [5][6][7]. Consequently, current therapeutic strategies in prostate cancer are inefficient [8][9][10][11][12][13][14][15][16][17][18][19][20], and an effective targeted therapy remains elusive. This situation prompted our laboratory to embark on studies to provide alternative leads for the development of efficacious and targeted anti prostate cancer agent(s). In our approach, we investigated the anti tumor activity of U94 protein (U94) in prostate cancer cell line, PC3.
Previous studies demonstrated that U94 suppressed transformation by oncogenes [22,29]. Data from these studies showed that an NIH 3T3 cell line stably expressing U94 gene suppressed transformation by the oncogene H-ras, when compared to the parental NIH 3T3 cell line treated under similar conditions [29]. We were motivated by the findings in previous studies to determine the anti tumor potential of U94 in the human prostate tumor cell line PC3.
In this paper we report that the expression of U94 protein in PC3 cells inhibited foci formation ( Figure 2; Table 1), and the tumorigenicity of recombinant PC3 cell line in athymic nude mice ( Figure 3). Moreover, gene expression analyses (Figures 4 and 5), and QRT-PCR (Table 2) revealed dramatic upregulation of FN 1 (~91-fold) and profound downregulation of ANGPTL 4 (~20-fold) in 2 separate recombinant PC3 cell lines stably expressing U94. Our study also demonstrated the differential expression pattern of several other genes in the presence of U94. This is the first study to report the inhibitory potential of U94 on the tumorigenicity of advanced-stage prostate cancer cell line PC3.

Expression and intracellular localization of U94 protein
First, we wanted to determine whether U94 could be expressed in PC3 cell line. We transfected PC3 cell line with plasmid pBKU94, which contained U94 DNA insert and a selectable geneticin (G418)-resistant vector cassette. pBKCMV vector transfected PC3 cells served as control. Immunoblot analyses, using the U94 polyclonal antibody AB679 as depicted in Figure 1, showed that U94 (56 kDa) protein was expressed in the nuclear fraction (lane 3) and not the cytoplasmic fraction (lane 2) of stably transfected PC3 cell line. No immunoreactivity was detected in the nuclear fraction of vector transfected PC3 cell line (lane 1). Figure 1 lane 4 shows the high molecular weight Rainbow marker.

Inhibition of focus formation by U94 protein expression
In order to monitor the effect of U94 on tumor formation, we investigated focus formation by PC3 cell line as an index of a neoplastic phenotype. Focus formation was observed as dense foci of intensive cell growth in culture, consisting of refractive cells that rounded up and piled on top of each other [49]. Three PC3 cell lines were used in this study: U94 transfected, vector cassette transfected, and parental PC3 cell line. For each cell line, 1 × 10 6 cells/ 60 mm culture dish was seeded and grown to confluence.
Focus formation was examined 10 days post confluence. The result of this study (Table 1) showed a drastic reduction in focus formation by PC3 cells expressing U94: the number of foci were reduced ~35-fold and 40-fold in comparison with the control vector transfected and parental PC3 cell lines, respectively. Figure 2 shows large and widespread foci in the culture of control vector transfected and parental PC3 cell lines. The culture of recombinant PC3 cell line expressing U94 protein showed only few foci, grossly reduced in size. Our findings suggest that U94 may exhibit anti tumor activity in vitro.

Expression of U94 inhibits tumorigenicity of PC3 cell line in athymic nude mice
In order to determine whether U94 inhibits the tumorigenicity of PC3 cell line in vivo, we inoculated 5 × 10 6 PC3 cells (U94 transfected cells as test, or vector cassette transfected cells as control) subcutaneously behind the neck, into athymic nude mice. Animals were examined for tumor formation on days 7, 14, 17, 21, and 28 after inoculation. Our result showed that tumor formation was inhibited in mice that were inoculated with PC3 cell line stably expressing U94 protein ( Figure 3). The control animals that were inoculated with PC3 cell line transfected with vector cassette developed tumors, and tumor size increased progressively with time as shown in Figure 3. Statistical analysis, using a repeated measures analysis of variance (ANOVA), demonstrated that tumor volume in test and control animals were significantly different (P < 0.05). A comparison of tumor volume between test and control mice, using Paired Student's t-test to supplement ANOVA, further showed that the average tumor volume of test and control animals were significantly different (P < 0.05) on each day tumor volumes were determined. These findings demonstrate that U94 significantly (P < 0.05) inhibited the tumorigenicity of PC3 cell line in athymic nude mice, and corroborate our data ( Figure 2) from focus formation assay.

Microarray gene expression profiling in PC3 cell line stably expressing U94
We performed microarray gene expression profiling on recombinant PC3 cell line stably expressing U94 to determine whether U94 affected expression of genes involved in tumorigenesis. We used two clones of U94 recombinant PC3 cell lines as test samples, and PC3 cell line transfected with plasmid vector as our reference. The effect of U94 on gene expression was analyzed using two-color comparative fluorescence assays on glass slide microarrays containing ~6,000 cancer related genes. Our data demonstrated the differential expression of 78 genes: 31 genes were upregulated ( Figure 4) while 47 genes were downregulated ( Figure 5). These results show the mean values for two clones of U94 recombinant PC3 cell lines. Notably, the microarray results revealed dramatic upregulation of FN 1 (91 ± 16-fold), and profound downregulation of ANGPTL 4 (20 ± 4-fold) in PC3 cell lines stably expressing U94 protein. Although a majority of the differentially expressed genes showed a 2-3 fold change in expression level in the presence of U94, we decided to consider for further studies only genes with ≥ 6-fold change. The microarray data is deposited in the Gene Expression Omnibus of the NCBI, and is available at the NCBI web site http://www.ncbi.nlm.nih.gov/entrez/ query.fcgi?db=ncbisearch. There was no immunoreactivity in all cytoplasmic extracts, and nuclear extracts of control cells. U94 protein was detected using the polyclonal anti-U94 antibody, AB679 (Amersham, England). The positions of 46 kDa and 66 kDa markers, and the U94 protein are indicated on the right.

Quantitative real-time PCR
We performed QRT-PCR to confirm the microarray data. In one clone of U94 recombinant PC3 cell line, the QRT-PCR data ( Table 2) showed that the changes in expression levels of FN 1 and ANGPTL 4 mRNA were 183 ± 27-fold increase and 76 ± 13-fold decrease, respectively. In the second clone of U94 recombinant PC3 cell line, the corresponding changes in expression levels were 467 ± 32-fold increase and 67 ± 5-fold decrease, respectively. The observed differences in the fold changes between the microarray and QRT-PCR data may be due, at least in part, to differences in detection sensitivity of the two techniques, as well as the subtle differences in experimental conditions and physiological conditions in the microenvironment of the cells in culture. Nevertheless, the trend observed from QRT-PCR data was consistent with the trend from microarray data. Additionally, the results from both techniques showed elevation of SERPINE 2 and ADAMTS 1 expressions (Table 2 and Figure 4), and downregulation of SPUVE 23 (Table 2 and Figure 5). TGM-2 (transglutaminase 2) showed 8 ± 2-fold decrease by microarray, but we did not perform QRT-PCR.

Discussion
In the present study, we have demonstrated for the first time that U94 protein inhibited focus formation and tumorigenicity of the prostate cancer cell line PC3. This study is particularly interesting because PC3 cell line is a derivative of advanced-stage prostate cancer metastasis to bone and is insensitive to androgen ablation therapy. Insensi-tivity to androgen ablation therapy is associated with aggressive progression of the cancer, and ultimately fatal in less than 24 months [2,6]. Therefore the anti tumor activity of U94 in PC3 cell line is novel and interesting, and may have a translational application.
The impetus for our study on anti tumor activity of U94 in PC3 cell line was given by previous findings [22,29] that U94 suppressed transformation by oncogenes. Apparently, U94 shares this functional activity with its homologue Rep 78/68 of AAV-2 [24]. However, the mechanism(s) of transformation suppressor activity is not understood. A previous report showed that U94 lost its activity when translation termination linkers were inserted at codons 25, 125 and 245 of its nucleotide sequence [29]. This finding implicates U94 protein expression in anti tumor activity in recombinant PC3 cell line. Therefore we performed immunoblot analysis and demonstrated that U94 protein (56 kDa) was expressed, and localized to the nucleus ( Figure 1, lane 3) in PC3 cell line. Nuclear localization of U94 protein suggests that U94 might exert activity, probably on gene expression, in the nucleus of PC3 cell line. This view is in consonance with previous findings [22,29] that U94 inhibited gene expression. A previous study [29] showed that U94 suppressed the P97 promoter, which controls the expression of the E6 and E7 transforming genes of human papillomavirus 16 (HPV-6). Therefore we suspect that the tumor suppressor activity of U94 in our study was exerted by inhibition of gene expression. It is interesting to note that foci A B C the expression of U94 protein does not affect the growth pattern of NIH 3T3 cell line [29]. This observation is supported by another report [25] that lymphoid cells stably expressing U94 had the same morphology and growth characteristics as parental cell line. Thus previous findings suggest that U94 is not toxic to cells, and we speculate that the same would be true for the PC3 cell line.
In the current investigation, we examined the effect of stable expression of U94 protein on focus formation, a malignant phenotype, by recombinant PC3 cell line.
Focus formation by PC3 cell line stably expressing U94 was inhibited drastically (~30-to 40-fold) in comparison to that of control parental and vector transfected cell lines ( Table 1). As shown in Figure 2, widespread and large foci were formed by control PC3 cell lines in contrast to background foci formed by U94 recombinants. It is possible that the few foci observed in the background originated from spontaneous transformation and/ or leakage during clonal selection. Furthermore, our studies demonstrated that the anti cancer activity of U94 was sustainable in vivo as tumor development was significantly (P < 0.05) inhibited in mice that were treated with U94 recombinant PC3 cell line (Figure 3). Previous studies linked prostate malignancy to the activities of oncogenes [39][40][41][42][43][44][45][46][47][48]. Therefore the inhibition of focus formation and tumorigenicity in our study supports the hypothesis that U94 probably inhibited oncogenic activities in prostate cancer cell line PC3, and thereby exhibited anti cancer activity. Our microarray data identified FN 1 that was dramatically elevated (~91fold) ( Figure 4) and ANGPTL 4 that was profoundly reduced (~20-fold) ( Figure 5) as genes of interest in this study. Up-regulation of FN 1 in the current study was actually unexpected because previous reports [22,29] suggested that U94 inhibited gene expression. In contrast, our findings suggested that U94 actually altered gene expression in PC3 cell line positively or negatively.
Although it is not known how U94 mediated gene expression, our data is interesting because ANGPTL 4 is pro-angiogenic [50], and reduced expression could negatively impact tumorigenesis. Additionally, previous reports [51][52][53][54] implicated elevated FN 1 and/ or its derivatives in paradigms of tumor inhibition.
Fibronectin (FN) is a major component of extracellular matrix (ECM), where it is assembled as insoluble polymers, and is present in the blood as a soluble dimer [55]. Fibronectin 1 (FN 1) is a homologue of FN, and contains a self-assembly domain, which induces FN 1-FN 1 polymerization [56][57][58]. Therefore the terms FN 1 and FN are used interchangeably in regard to polymerization in this report.
FN 1-FN 1 interaction is reported [59,60] to induce conformational changes that increase the binding ability of FN 1 to receptor(s). We speculate that the tremendous upregulation of FN 1 transcription in the presence of U94 led to elevated translation and secretion of protein product in PC3 cells. The increased level of FN 1 protein in turn accelerated FN 1-FN 1 polymerization [56][57][58]. We suspect that polymeric FN 1 binding to PC3 cell surface mitigated malignant signaling [61]. The potential antimalignancy activity of FN 1 is evident from a recent report [62] that exogenous FN 1 could reverse transformed phenotype. Hence, FN 1 interaction with PC3 cell surface might have contributed, at least in part, to inhibition of focus formation in vitro ( Figure 2) and tumorigenesis in vivo ( Figure 3).
Strong support for in vivo anti tumor activity of polymeric FN 1 is given by a previous report [53] demonstrating that systemic administration of polymeric FN 1 exhibited anti tumor activity in mice bearing various types of tumors. Further support is provided by a recent report [59] showing that anastellin, a component of FN

to PC3 cells surface induces the inhibition of focus formation in culture and
Upregulated genes in PC3 cell line stably expressing U94 Figure 4 Upregulated genes in PC3 cell line stably expressing U94. Two clones of G418 resistant PC3 cell lines transfected with plasmid pBk-U94 (tests 1 and 2) and a clone transfected with plasmid pBK-CMV (reference) were used for cDNA microarray studies. Each experiment was performed in triplicate and the results are mean ± SD. Notice the dramatic upregulation of FN 1 (91 ± 16-fold). A subset of other genes was also upregulated, but genes of interest in this study (> 6-fold change) include: SER-PINE 2 (7 ± 1-fold); and ADAMTS 1 (7 ± 2-fold).
inhibition of tumorigenicity in mice. However, in addition to FN 1, our results also implicated ANGPTL 4 in the anti tumor activity of U94 in PC3 cell line.
ANGPTL 4 was shown in a chicken chorioallantoic membrane assay to induce a strong pro-angiogenic response, independent of VEGF gene [50]. Since angiogenesis is implicated in vascular development, and neovascularization is the hallmark of tumor progression [63,64], an inhibitor of angiogenesis could greatly impact tumor therapy. In the current study we have demonstrated that ANGPTL 4 was profoundly inhibited (downregulated about 20-fold) in U94 recombinant PC3 cell line. It is therefore expected that downregulation of ANGPTL 4 would exert a negative effect on vascular development, and thereby inhibit PC3 cell line tumorigenicity in vivo.
Although it is not clear how U94 mediates the expression of ANGPTL 4, recent reports [65][66][67] show that angiogenesis is regulated by ECM signals. Interestingly, other reports [53,59] suggest that the anti-angiogenic property of polymeric FN 1 is mediated by induction of ECM signals. Therefore, it appears that there may be a casual or causal relationship between anti tumor activity of polymeric FN 1 and the inhibition of ANGPTL 4 in U94 recombinant PC3 cell line. Since ANGPTL 4 supports vascular development [50], we speculate that ANGPTL 4 did not mediate the inhibition of focus formation by PC3 cell line in this study.
In addition to FN 1 and ANGPTL 4, we also chose for further studies a subset of other genes that expressed differentially > 6-fold. Genes in this category included SERPINE 2 (elevated ~7-fold), ADAMTS 1 (upregulated ~7-fold) and SPUVE 23 (downregulated ~10-fold). SERPINE 2 encodes a serine proteinase inhibitor, and was recently implicated in anti cancer activity [68]. ADAMTS 1 is an active metalloproteinase associated with ECM [69]. It is essential for normal growth [70], but also displays antiangiogenic activity [71]. In consonance with previous reports [68,71], data from the current study suggest that SERPINE 2 and ADAMST 1 probably exerted anti tumor activity. Previous studies [72,73] showed that the expression of SPUVE 23, a serine protease, is associated with increased malignant potential. Therefore we propose that downregulation of SPUVE 23 in U94 recombinant PC3 cell line is tantamount to anti tumor activity.
In conclusion, the findings in this study have suggested that U94 exhibits anti tumor potential in PC3 cell line. The dramatic elevation of FN 1 expression and reduction of ANGPTL 4 expression in U94 recombinant PC3 cell line can be interpreted as evidence of the mechanism of U94 anti tumor activity. Therefore data from our study seem to support the anti tumor hypothesis of FN 1 previ-  ously reported by other workers [51,52,54,55,74]. Moreover, this report identifies ANGPTL 4 and SPUVE 23 as potential therapeutic targets in prostate tumorigenesis.
Hopefully, further studies on the microarray data reported herein might elucidate the complex genetic alterations that underlie advanced-stage prostate tumorigenesis, and thereby provide leads for defining targeted therapeutic strategies for advanced-stage prostate cancer.

Materials and methods
Cells and transfection PC3 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and plasmid U94 DNA was prepared as previously described [22,29]). All cells were cultured in HAM's F12 medium (Cell gro/Mediatech, VA, USA) supplemented with 2 mM glutamine, 100 U of penicillin-streptomycin per ml (Invitrogen, Gaithersburg, MD, USA), and 10% Fetal Bovine Serum (FBS, HyClone, Logan, UT, USA), at 37°C and 5% CO 2 . Plasmid U94 DNA was cloned into the HindIII site of pRc-RSV vector (Invitrogen, Gaithersburg, MD, USA) or HindIII/ BamHI site of pBK-CMV (Stratagene, Cedar Creek, TX, USA) vector. Both pRc-RSV and pBK-CMV vectors contain a geneticin (G418; Mediatech Inc, Herndon, VA, USA) selectable marker. U94 DNA sequence in the constructs was confirmed by DNA sequencing. The pBK-U94 construct was specifically used in experiments that necessitated strong expression of U94 protein e.g. immunoblotting, because previous findings showed that U94 mRNA and protein were expressed at very low levels [23,24,75]. All plasmid DNA were prepared by doublebanded cesium chloride gradient ultracentrifugation. U94 construct (pRc-U94 or pBK-U94), or plasmid vector cassette (pRc-RSV or pBK-CMV) was used for transfection of PC3 cells. For transfections, 5.5 × 10 5 PC3 cells were plated in 60 mm culture dish, and grown over-night (50%-70% confluence). Transfection was performed by the calcium phosphate-based ProFection Mammalian Transfection method (Promega, Madison, WI, USA) in accordance with manufacturer's protocol. Stably transfected PC3 cells were selected with G418 (600 µg/ ml), and expanded to establish U94 recombinant PC3 cell line. Clonal selection was performed on G418 resistant healthy colonies using a clonal cylinder. In order to minimise culture driven genetic changes [76], transfected cells were discarded after 8 passages.

Protein extraction, and immunoblot analysis
Nuclear fraction from cellular extract was prepared as described previously [77]. Confluent PC3 cell line (10 7  Protein determination in the extracts was performed using BCA protein assay kit (Pierce, Rockford, IL, USA). Following sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), resolved proteins were electroblotted onto polyvinylidene difluoride (PVDF) membrane. The membrane was blocked in 5% non-fat milk solution on a rocker for 30 min, and rinsed quickly

Genes
Forward Primer Reverse primer Gene specific primers for QRT-PCR were designed using Primer Express Version 2.0 (Applied Biosystems, Foster City, CA) according to the sequence information provided for the cDNAs on the microarray.
in Tris/sodium chloride/EDTA/Tween 20 (TNET, 0.2 M Tris pH 7.5; 0.05 M EDTA; 1.0 M NaCl; 1% Tween 20) wash solution. Then the membrane was washed twice in TNET on a rocker for 10 minutes, before it was probed with U94 primary antibody AB679 (Rabbit antiserum, 1:1000 dilution in TNET; Amersham) on a rocker for 1 hour. This was followed by three 10-minutes washes in TNET before anti rabbit-HRP-tagged secondary antibody (1:10,000 dilution in TNET; Amersham) was added and incubated for 1 hour. Three 10-minutes washes in TNET were performed on a rocker, before the detection of immunoreactive proteins using ECL (Amersham, England) reagent.

Focus formation assay
Two clones of U94 transfected cell lines were used for studies. Vector transfected and parental PC3 cell lines were used as controls. Cells were plated at 1 × 10 6 cells/ 75 cm 2 culture flask in duplicate and grown to confluence. Focus formation was visually detected by observing dense foci of intensive cell growth, consisting of refractive cells that rounded up and piled on top of each other [49]. The number of foci in each flask was noted on the 10th day after the cells were confluent. Average counts of foci in duplicate flasks were determined for each cell type.

Tumorigenicity assay
The tumorigenicity of PC3 cell line stably expressing U94 was tested in athymic Ncr nu/nu mice. The control animals were treated with vector transfected PC3 cell line. In all cases 5 × 10 6 cells were inoculated subcutaneously behind the neck, into athymic nude mice as earlier described [78]. The mice were monitored every 2 or 3 days for the appearance of tumors, and tumor volume was measured on days 7, 14, 17, 21 and 28 post inoculation. Tumor sizes were evaluated by tumor volume (length × width × height, in cm). In all cases confluent cells were used. There were two animal experiments. In the first (n = 3 per group), data entries were made on days 0, 7, 17, 21, and 28, while in the second (n = 4 per group) entries were made on days 0, 7, 14, and 17 post inoculation. Data were pooled from the two experiments and reported. The Animal Welfare Committee, Georgetown University, approved the protocol for the animal studies.

Quantitative real-time polymerase chain reaction (QRT-PCR)
To verify the expression pattern of the differentially expressing genes identified from microarray experiments, QRT-PCR was performed as described previously (79). Equal amounts of total RNA from test and reference cell lines were treated with DNase 1 (Invitrogen, Gaithersburg, MD, USA), and reverse transcribed using random hexamers and SuperScript II (Invitrogen, Gaithersburg, MD, USA) to prepare the first strand cDNA samples for QRT-PCR analyses. The RT product was diluted 5-fold, and 1 µl is equivalent to 1x concentration. Gene specific primers (Table 3) were designed by Primer Express Version 2.0 (Applied Biosystems, Foster City, CA, USA) according to the sequence information provided for the cDNAs on the microarray. The primers were BLASTed against the non-redundant and EST mouse sets from NCBI to confirm specificity. QRT-PCR was performed in triplicate using SYBR ® Green I chemistry on 7900 HTS Sequence Detection System (Applied Biosystems, Foster City, CA, USA) according to manufacturer's instructions. The temperature cycle for QRT-PCR was set up as following: 50°C for 2 minutes; 95°C for 10 minutes; 95°C 15 seconds and 60°C for 1 minute for 40 cycles. A final dissociation cycle running at 95°C for 15 seconds, 60°C for 15 seconds and 95°C for 15 seconds was set up for monitoring the specificity of amplification. The relative standard curve method was used for quantifying gene expression level, in which the C T values of a series of fixed amounts of test sample (or reference sample) cDNAs (0.01x , 0.1x and 1x as described above) were plotted against these amounts of cDNAs. The C T value for a gene at 0.1x concentration in the reference sample (or test sample) was fitted onto the standard curve to obtain the respective expression level. A smaller C T value indicates a higher expression level, and vice versa. Genes showing C T values ≥ 40 were considered to be non-expressing. The final gene expression data were reported after normalising to that of 18S RNA.

Statistical analysis
A repeated measures analysis of variance (ANOVA), supplemented by Paired Student's t-test, was used to evaluate the differences in tumor volume between U94 treated and control vector treated mice. A value of p < 0.05 was considered statistically significant. SAS software (v8.2, SAS Institute, Cary, NC, USA) was used for ANOVA. Experimental data, where applicable, are represented as mean ± SD.