Specific differences in gene expression profile revealed by cDNA microarray analysis of glutathione S-transferase placental form (GST-P) immunohistochemically positive rat liver foci and surrounding tissue

Shugo Suzuki1,3, Makoto Asamoto1, Kazunari Tsujimura1,2 and Tomoyuki Shirai1

1 Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan and 2 Chemicals Evaluation and Research Institute, Chemicals Assessment Center, Saitama, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Glutathione S-transferase placental form (GST-P), one of the glutathione S-transferases family of detoxification enzymes, is a very useful marker of rat liver pre-neoplastic lesions. We here investigated the gene expression profile in GST-P positive foci as compared with surrounding GST-P negative areas in the same liver of rats treated with diethylnitrosamine and then 2-acetylaminofluorene combined with partical hepatectomy. GST-P positive foci were harvested by laser microdissection and total RNAs were extracted to allow gene expression profiles to be assessed by cDNA microarray assays. Transaldolase, rat aflatoxin B1 aldehyde reductase and gamma-glutamylcysteine synthetase were found as up-regulated genes and regucalcin as a down-regulated gene, in line with findings for hepatocellular carcinomas. The results indicate that the approach adopted is useful for understanding mechanisms of hepatocarcinogenesis and identification of new markers for rat liver pre-neoplastic foci.

Abbreviations: 2-AAF, 2-acetylaminofluorene; Cy3, Cyanine 3; Cy5, Cyanine 5; DEN, diethylnitrosamine; GCS, glutamylcysteine synthetase; GST-P, glutathione-S transferase placental form; HCA, heterocyclic amine


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Glutathione S-transferase placental form (GST-P), one of the glutathione S-transferase family of important detoxification enzymes, has been identified as a reliable marker protein for pre-neoplasia in rat chemical hepatocarcinogenesis (1,2), superior to gamma-glutamyl transpeptidase (3,4). Using GST-P positive foci as end-point pre-neoplastic lesions in the rat liver, we have established a medium-term liver bioassay to detect hepatocarcinogens (57), with which we have already investigated over 300 chemicals (8). The development of cDNA microarrays, which are able to monitor thousands of genes simultaneously, now provides us with a powerful tool for high-throughput genetic analysis of carcinogenesis (9). Furthermore, laser capture microdissection (LCM) facilitates accurate sampling of specific types of cell or lesions (10,11). Therefore, a combination of LCM and microarray techniques was selected for analysis of global gene expression profile in immunohistochemically distinct cell populations in the present study. To rapidly generate large numbers of GST-P positive foci we applied our medium-term liver bioassay system (8) using 2-acetylaminofluorene (2-AAF) in the post-initiation stage with hepatectomy (12,13), comparing the lesions with surrounding GST-P negative areas in the same rat livers.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
2-AAF and diethylnitrosamine (DEN) were purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan), with purities of >98 and >99%, respectively.

Animals
Five-week-old male F344 rats were obtained from Charles River Japan (Atusgi, Japan). They were housed in plastic cages with hardwood chip bedding in an air-conditioned room at 23 ± 2°C and 55 ± 5% humidity with a 12 h light/dark cycle and maintained on a basal diet (Oriental MF, Oriental Yeast Co., Tokyo, Japan) and tap water ad libitum.

Experimental procedure
We used a modified medium-term liver bioassay (8). Animals were randomly divided into three groups of five rats each. All groups received an i.p. injection of DEN at a dose of 200 mg/kg body wt as an initiation procedure or saline as the control. Starting 2 weeks thereafter, they were administered a basal diet containing 100 p.p.m. 2-AAF or basal diet for 4 weeks. The rats treated with DEN were subjected to two-thirds partial hepatectomy at the end of experimental week 3. The original protocol of the medium-term liver bioassay is 8 weeks duration, but to obtain approximately equal areas of GST-P positive foci and negative lesions, the duration of 2-AAF administration was here reduced to 4 weeks. Body weights and food consumption were recorded every week and all surviving animals were killed under ether anesthesia at week 6. The livers were immediately excised, weighed and cut into 2- to 3-mm-thick slices, one from the caudate lobe and two from the right lateral lobe. The slices were fixed in ice-cold acetone for immunohistochemical examination of GST-P expression. The remaining livers were immediately frozen in liquid nitrogen and stored at -80°C until processed.

Immunohistochemistry for measurement of GST-P foci
The liver tissues fixed in ice-cold acetone were processed to paraffin embedded sections as described previously (3,14). Liver sections of 3 µM thickness were treated with rabbit anti-rat GST-P antibody (MBL, Nagoya, Japan) and then sequentially with secondary antibody and avidin–biotin complex reaction (Vectastain ABC elite kit, Vector Laboratories, CA). The sites of peroxidase binding were visualized with diaminobenzidine. Sections were then counterstained with hematoxylin for microscopic examination. Areas of GST-P positive foci >0.2 mm in diameter in the liver were quantitatively measured with an Image Processor for Analytical Pathology (IPAP-WIN, Sumika Technos Co., Osaka, Japan).

Immunohistochemistry for RNA extraction
Six-micron serial frozen sections cut on a standard cryostat with a clean blade, were mounted on slides with films (Leica Microsystems K.K., Tokyo, Japan) and fixed in ice-cold acetone for 15 min. The immunohistochemical staining was performed with the DAKO ENVISIONTM System (DAKO Co., Tokyo, Japan). The sections were treated with rabbit anti-rat GST-P antibody with 1:200 diluted RNasein (Promega, Tokyo, Japan) for 30 min at 4°C and briefly rinsed with 1x phosphate-buffered saline (PBS), and then sequentially exposed to secondary antibody with 1:200 diluted RNasein and 2.5 µM EDTA in PBS. The sites of peroxidase binding were demonstrated with diaminobenzidine at room temperature.

Laser capture microdissection
After immunostaining of frozen tissue, microdissection was performed using the AS LMD system (Leica Microsystems K.K). GST-P positive foci and negative areas were resected with laser beams separately and collected into tubes.

RNA extraction and amplification
Total RNAs were isolated using ISOGEN (Wako, Tokyo, Japan), according to the manufacturer's instruction. The RNA amplification was performed using a MessageAMPTM aRNA Kit (Ambion, Austin, TX). For comparison of gene expression profiles with cDNA microarrays between unamplified and amplified RNAs, we collected RNA (~10 µg of total RNA) from 50 slides of rat liver for the unamplified sample, 0.2 µg of total RNA being isolated from one slide section.

Transcript profiling
Quality of total and amplified RNAs was examined with a high-resolution electrophoresis system, the Agilent 2100 BioanalyzerTM (Agilent Technologies, Palo Alto, CA).

Reverse transcription
cDNA was synthesized with SuperScript II-reverse transcriptase used random primers (Promega), dUTP labeled with Cyanine 3 (Cy3) or Cyanine 5 (Cy5) (PerkinElmer Life Sciences Japan Co., Tokyo, Japan), and 10 µg of amplified RNA as the template. Labeled cDNAs were purified on QIAquick columns (QIAGEN K.K., Tokyo, Japan).

Hybridization of cDNA microarray chip
Using a Rat cDNA Microarray Kit (Agilent Technologies), gene expression analysis was performed. The slides were hybridized with Cy3- or Cy5-labeled cDNA for 16–18 h at 65°C, washed in 0.5x SSC 0.01% SDS buffer for 5 min at room temperature, then 0.06x SSC buffer for 2 min, and desiccated with a centrifuge. The slides were scanned with a DNA Microarray Scanner (Agilent Technologies) at two wavelengths to detect emission from both Cy3 and Cy5. We collected RNA from GST-P positive foci and surrounding GST-P negative areas separately from the same liver sections of individual rats. Differences in expression of RNA between the foci and surrounding as pairs from the same animal were analyzed independently for the four rats. The genes with significantly different expression levels were revealed by Significance Analysis of Microarray (delta = 4.58; http://www-stat.stanford.edu/~tibs/SAM/).

Real-time quantitative reverse transcription (RT)–PCR
cDNAs were synthesized with SuperScript II-reverse transcriptase primed with oligo(dT) primers from 0.5 µg of total RNA as the template. Amplification was performed using a Light Cycler DNA Master SYBR Green I mix (Roche Diagnostics, Mannheim, Germany) containing 3.5 mM MgCl2 with 50 cycles of 1 s at 95°C, 5 s at 58°C and 15 s at 72°C. Fluorescence detection was at 87°C after each cycle in a Light Cycler apparatus (Roche Diagnostics). After the final cycle, melting curve analysis of all samples was conducted within the range from 60 to 95°C. An external standard curve was generated by dilutions of the target PCR product (10-4–10-9 times, six points).


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 Materials and methods
 Results
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Animal experiment
During the experimental period, significant suppression of body weight gain was observed in the group of rats given DEN and 2-AAF. The final body weights treated with 2-AAF were significantly lower than with the basal diet. Relative liver weights of rats treated with DEN and 2-AAF were significantly higher than those given basal diet alone. There was a significant difference in food consumption between rats given 2-AAF and their counterparts receiving basal diet (Table I).


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Table I. Final body, liver weights and food consumption data

 
The numbers and areas of GST-P positive liver cell foci were clearly increased by treatment with DEN and 2-AAF (Table II). The foci were sufficiently large to be easily recognized macroscopically after GST-P staining. Single GST-P positive cells were observed in the 2-AAF alone group (Figure 1).


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Table II. Quantitative results for GST-P positive liver cell foci

 


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Fig. 1. Immunohistochemistry of GST-P in rat livers; (A) control; (B) 2-AAF; (C) DEN + 2-AAF.

 
Degradation of RNA during immunostaining procedures
Including the duration of fixation with acetone, immunostaining procedures were minimized to 1.5 h in order to avoid degradation of RNA. We also performed staining at 4°C and added RNase inhibitor to antibody solutions. However, RNA quality and the 28S/18S ratio nevertheless gradually decreased, the latter by half at the stage of secondary antibody treatment (Figure 2).



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Fig. 2. Quantity and quality of RNA determined with an Agilent 2100 BioanalyzerTM. (A) Relative extraction volume; (B) relative 28S/18S. **, ***, Significantly different from the acetone value (as 1.0) at P < 0.01 and P < 0.001, respectively.

 
cDNA microarray analysis
There was a good correlation in gene expression profiles with amplified RNA and unamplified RNA as templates (data not shown; correlation coefficient = 0.705), the value being in the same range as for rat individual differences (correlation coefficient = 0.61–0.87). We detected a number of alterations in gene expression patterns between the GST-P positive foci and surrounding GST-P negative area (Table III), and then confirmed the variation in expression of five particular genes by quantitative RT–PCR. These were rat transaldolase, rat aflatoxin B1 aldehyde reductase (AFAR), gamma-glutamylcysteine synthetase catalytic heavy subunit (GCSc), gamma-glutamylcysteine synthetase regulatory light subunit (GCSr), rat regucalcin and GST-P (Table IV).


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Table III. Significant difference between GST-P positive foci and GST-P negative areas; the ratio is GST-P foci vs GST-P negative areas

 

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Table IV. mRNA expression levels determined by quantitative RT–PCR analysis

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study demonstrated clearly that total RNA can be readily extracted from immunostained slides for RNA amplification and cDNA microarray analysis. Fend et al. reported previously methods for RNA extraction from immunostained frozen sections of several human organs (15) and showed three factors, the fixative used, staining time and addition of RNase inhibitor, to be most crucial for maintaining RNA quality. We here also employed an RNase inhibitor and minimized the duration of the immunostaining in order to maximize RNA contents. However, RNA degradation was still apparent after treatment with the antibodies, particularly the secondary antibody, suggesting RNase remained even after the treatment with RNase inhibitor and EDTA. By minimizing degradation we could obtain sufficient RNA for amplification and analysis by cDNA microarrays and quantitative RT–PCR.

As a result we found 37 genes to be up-regulated and 10 genes to be down-regulated in the GST-P positive foci. According to the gene ontology (16), almost all are associated with metabolism in the biological process category, and with catalytic activity in the molecular function category. The important roles of metabolism are confirmed by the inclusion of GSTs, GCS, aldo-keto reductases, glutathione synthetase and P450 s (1719).

Among the up-regulated genes, four were also reported to demonstrate increased expression in hepatocellular carcinomas in man and rats. The activity of one of them, transaldolase, which contributes to the supply of ribose 5-phosphate for DNA synthesis and cell proliferation, was earlier found to be increased in liver tumors compared with normal liver in the rat, but transketolase showed no relationship to tumor proliferation rate (20). AFAR, earlier identified as an up-regulated gene in aflatoxin B1-induced rat pre-neoplastic nodules and hepatomas (17,21), is included in the aldo-keto reductase superfamily, and may function in detoxification of chemical carcinogens. GCS, which catalyses the first step in the pathway for glutathione synthesis, is a heterodimer of catalytic heavy and regulatory light subunits (22,23). We found both of them to be up-regulated. The enzyme has also been shown to be increased substantially in tumor cell lines resulting in resistance to different chemotherapeutic drugs and radiation (2426). The mechanism of resistance is associated with the glutathione redox system, one of the most important antioxidant systems. Glutathione levels are regulated by GCS (27,28). The present work further demonstrated one protein, regucalcin, to be appreciably down-regulated in GST-P positive foci. Its expression was earlier found to be decreased in rats treated with 2-AAF alone, suggesting that regucalcin might play some role in the early stages of hepatocarcinogenesis. The protein has been shown to inhibit DNA and RNA synthesis due to inhibition of Ca2+ in rat liver (29,30). Its mRNA has been reported to be down-regulated in hepatoma cells (31). Quantitative RT–PCR also here confirmed that GST-P RNA expression was significantly higher in GST-P positive foci compared with GST-P negative areas, but such a clear difference was not detected in the cDNA array analysis, suggesting a limitation with the latter.

In conclusion, the present results indicate that the approach adopted may be useful for analysis of the mechanisms of hepatocarcinogenesis and identification of new markers for rat liver pre-neoplastic foci. We are now expanding our study to include GST-P positive foci induced by different carcinogens to target common mechanisms in early hepatocarcinogenesis. In future studies, relationships between alteration of major genes and development of GST-P positive foci should be investigated, to provide information relevant to dose– response issues in carcinogenesis.


    Notes
 
3 To whom correspondence should be addressed Email: shugo{at}med.nagoya-cu.ac.jp Back


    Acknowledgments
 
We would like to express our gratitude to Mrs Yasuko Yoshida of NGK INSULATORS for analysis of cDNA microarray signals. This work was supported in part by Development of a High Precision Summary Toxicity (Hazard) Assessment System for New Energy and Industrial Technology Development (NEDO), Japan, and the Ministry of Health, Labour and Welfare, a Grant-in-aid from the Ministry of Health, Labour and Welfare for the Second Term Comprehensive 10-Year Strategy for Cancer Control, Japan, and a grant from the Society for Promotion of Toxicological Pathology of Nagoya, Japan.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 8, 2003; revised November 7, 2003; accepted November 15, 2003.





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