Molecular profiling of genes up-regulated during promotion by phenobarbital treatment in a medium-term rat liver bioassay

Makoto Shibutani,1, Noriyuki Takahashi, Tsuneo Kobayashi, Chikako Uneyama, Naoya Masutomi, Akiyoshi Nishikawa and Masao Hirose

Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In search of genes that are steadily up-regulated during the promotion stage in carcinogenesis, suppression PCR subtractive hybridization and following northern blot screening were performed using a phenobarbital (PB)-promotion model based on a medium-term liver bioassay. Two weeks after a single injection of diethylnitrosamine (DEN; 200 mg/kg body wt, i.p.), rats were given 600 p.p.m. PB in the drinking water for up to 64 weeks. For comparison, animals fed 1 p.p.m. ethinylestradiol (EE) or 3000 p.p.m. butylated hydroxytoluene (BHT) in the diet at promotion stage were also included. Rats were subjected to partial hepatectomy (PH) at week 3. In addition, dose-dependence of PB at week 8 of promotion and responsiveness to representative non-genotoxic carcinogens without DEN initiation were examined. Fragments of a total of 67 different genes were isolated from the up-regulated gene population in the liver at day 10 of PB treatment by subtracting from basal expression of DEN + PH alone. Using northern blot screening for signal-detectable 48 genes, 16 genes showed up-regulation in the livers at week 8 of promotion, common to the PB and EE treatments with the levels being three times or more than the basal expression of unpromoted liver. The majority of these genes were also up-regulated at week 8 by BHT treatment, and were also constitutively expressed in the DEN(–), PH(–) untreated rat livers. Among the up-regulated genes common to the PB and EE promotion, and not responding to the non-genotoxic carcinogens in uninitiated liver, the following six genes showed overexpression in PB-promoted hepatocellular carcinomas at week 64, with the levels three times or more than untreated rat liver: ubiquitously expressed mammalian ABC half transporter, apolipoprotein A4, nuclear receptor binding factor-2, CD81, hypothetical protein (HSPC014) and one unidentified gene. These genes might be candidates for biomarkers in screening of non-genotoxic hepatocarcinogens by analysis in two-stage carcinogenesis models.

Abbreviations: APO, apolipoprotein; umat, ubiquitously expressed mammalian ABC half-transporter; BHT, butylated hydroxytoluene; CYP, cytochrome P450; DEN, diethylnitrosamine; DEHP, di-(2-ethylhexyl)phthalate; DL-E, DL-ethionine; EE, ethinylestradiol; EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST-P, glutathione-S-transferase placental form; gpld1, glycosylphosphatidylinositol-specific phospholipase D1; gstm2, glutathione-S-transferase mu2; HCC, hepatocellular carcinoma; LR1, laminin receptor 1; NRBF-2, nuclear receptor binding factor-2; PB, phenobarbital; PH, partial hepatectomy; DOC-1, deleted in oral cancer-1.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Advances in industrial technology have led to a recent upsurge in the number of new chemicals developed for a variety of purposes. Although the cancer risk to humans presented by such synthetic chemicals is regarded as low, toxicity and carcinogenic potential require evaluation in each individual case. Short-term in vitro/in vivo genotoxicity assays are usually employed for the prediction of carcinogenicity and have proved useful in the initial identification of potential genotoxic agents. However, their value is limited by the observation that ~60% of chemicals identified as carcinogens by in vivo long-term carcinogenicity studies produce mainly negative findings in genotoxicity tests (1,2). There are currently no reliable rapid means of evaluating the carcinogenic potential of new chemicals that fall into this latter group of compounds, termed non-genotoxic carcinogens.

Although the molecular events in the initial stage of chemical action in a target organ may vary with the carcinogen, there may be some common mechanism by which autonomous cellular proliferation is triggered. One approach to addressing this issue is molecular profiling to identify common factors/mechanisms that can serve as early biomarkers of carcinogenic potential for new chemicals.

As well as genotoxic carcinogens, most non-genotoxic carcinogens are known to show tumor-promoting activity in two-stage carcinogenesis models (3). Therefore, it is reasonable to perform molecular profiling during the promotion stage using representative non-genotoxic carcinogens to identify molecules up- or down-regulated with the development of neoplastic lesions by non-genotoxic mechanisms. For this purpose, analysis using microarray technique or subtractive cDNA hybridization could be considered. Although the numbers of genes identified in rat species are small as compared with those in humans and mice at present, global gene expression analysis by high-density microarray, when it covers genes of whole genome, would be a powerful tool to identify gene cluster(s) involved in a biological response (4). Another technique, subtractive cDNA hybridization has been a powerful approach to identify and isolate cDNAs of differentially expressed genes in any species (57). Among the methods developed for this purpose, a PCR-based suppression subtractive hybridization (SSH) is now widely used to obtain both rare and highly abundant transcripts by selective amplification of target cDNA fragments (differentially expressed) and simultaneous suppression of non-target DNA amplification (810). It is primarily based on a technique called suppression PCR, and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. As a result only one round of subtractive hybridization is needed and the subtracted library is normalized in terms of abundance of different cDNAs.

In the present investigation, we focused on up-regulated genes in the early stage of liver promotion by phenobarbital (PB), a well studied non-genotoxic hepatocarcinogen (11,12), using SSH, and selection of steadily up-regulated genes at later stages of promotion utilizing a medium-term liver bioassay system to identify biomarkers for the rapid screening of carcinogens involving non-genotoxic mechanisms. In addition, the effects of ethinylestradiol (EE) and butylated hydroxytoluene (BHT), regarded as positive and negative controls for tumor-promoting activity in the liver (13,14), were examined by northern blotting for gene screening.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals and treatment
Male 4-week-old F344/DuCrj rats were purchased from Charles River Japan (Kanagawa, Japan), and maintained with pelleted basal diet (CRF-1; Oriental Yeast Co., Tokyo, Japan) and tap water until 5 weeks of age when the study was started. The rats were housed in plastic cages with hardwood-chip bedding in an air-conditioned room at 24 ± 1°C, 55 ± 5% humidity with a 12 h light/dark cycle. For the gene isolation and subsequent gene screening, a two-stage liver carcinogenesis model utilizing a medium-term liver bioassay protocol was employed in this study (Figures 1 and 2GoGo; refs 3,15). In brief, animals were given a single i.p. injection of diethylnitrosamine (DEN: Nacalai Tesque, Kyoto, Japan; 200 mg/kg) dissolved in 0.9% NaCl solution to initiate hepatocarcinogenesis and after a 2 week recovery period, received sodium salt of PB (Wako Pure Chemicals Industries, Osaka, Japan) at a concentration of 600 p.p.m. in the drinking water. This dose showed clear tumor-promoting activity in a previous two-stage hepatocarcinogenesis study (4). The rats were subjected to two-thirds partial hepatectomy (PH) at week 3, and maintained for up to 64 weeks to induce hepatic tumors.



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Fig. 1. Experimental design for the suppression PCR subtractive hybridization to isolate genes up-regulated in the liver of rats at day 10 of PB treatment in our medium-term liver bioassay.

 


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Fig. 2. Experimental designs for northern blot screening of genes up-regulated in the livers of rats in our medium-term liver bioassay. Two-step screening was employed, i.e. comparison of liver expression levels between animals treated with PB, EE, BHT or no chemical at week 8 in the medium-term bioassay protocol during the promotion stage (first screening); comparison of expression levels in the HCC produced by PB promotion at week 64 and those in the livers of untreated control and DEN + PH + PB treated rats at week 8.

 
In the same manner, animals for northern blot screening were fed a diet containing either 1 p.p.m. EE (Schering AG, Berlin, Germany) or 3000 p.p.m. BHT (Wako Pure Chemicals) in the promotion stage and maintained for 8 weeks (Figure 2Go). A promoting effect in the liver including generation of hepatocellular carcinomas (HCCs) was observed with an EE of 0.5 p.p.m. in the diet (14); while no promotion of glutathione-S-transferase placental form (GST-P)-positive foci was noted with 2500 or 5000 p.p.m. dietary concentrations of BHT in our medium-term liver bioassay (13). Based on the data, the doses of EE and BHT were determined.

To analyze the dose-dependence of gene expression with PB treatment, additional groups of animals were administered PB in the drinking water at concentrations of 9.5, 37.5, 150 or 600 p.p.m., and killed at week 8 of the experiment.

To analyze the expression changes of selected genes in the livers of rats that received representative non-genotoxic carcinogens without initiation, 6-week-old male SD:IGS/DuCrj rats (Charles River Japan) were fed a diet containing 600 p.p.m. PB, 2.5 p.p.m. EE, 20 000 p.p.m. di-(2-ethylhexyl)phthalate (DEHP; Sigma-Aldrich, St Louis, MO), 2500 p.p.m. DL-ethionine (DL-E; Wako Pure Chemicals) or 600 p.p.m. thioacetamide (TAA; Wako Pure Chemicals) for 4 weeks (28 days). The doses of PB, EE, TAA and DL-E were determined to be equivalent levels or excess over the levels to show tumor-promoting activity in the liver of SD rats (14,1618). High-doses of DEHP in the carcinogenicity studies are shown to be hepatocarcinogenic in mice and rats, and 20 000 p.p.m. in diet is excess over the carcinogenic dose in rats (1923). Groups of animals that received PH at the first day of experiment, or hepatotoxic dose of non-carcinogens, such as 10 000 p.p.m. acetaminophen (APAP; Sigma-Aldrich) or 1000 p.p.m. {alpha}-naphthyl isothiocyanate (ANIT; Wako Pure Chemicals) were also included (24,25).

In every experiment, two animals were killed at each time point under deep anesthesia with ether by exsanguination from the posterior vena cava and abdominal aorta for sampling of liver tissues/tumors.

The animal protocols were reviewed and approved by the Animal Care and Use Committee of National Institute of Health Sciences, Japan.

Suppression PCR subtractive hybridization
Fragments of liver genes up-regulated at day 10 of PB treatment were obtained by subtracting cDNAs from the corresponding DEN+PH animal (Figure 1Go). At this time point, the modifying effects of PB on gene expression associated with PH-derived cellular regeneration could be predicted. Subtraction was carried out with poly(A+) RNAs derived from single animals. Approximately 200 mg of frozen liver tissue was disintegrated under liquid nitrogen using a Mikro-Dismembrator S (B. Braun Biotech International GmBH, Melsungen, Germany). Poly(A+) RNAs were extracted from the ground liver using the FastTrackTM 2.0 Kit (Invitrogen Co., Carlsbad, CA) and cDNA subtraction was carried out using a PCR-SelectTM cDNA Subtraction Kit (Clontech, Palo Alto, CA) according to the manufacturers' instructions. Subtracted cDNAs were ligated into the pT7 blue-T vector (Novagen, Madison, WI), and isolated clones were subjected to nucleotide sequencing using a T7 promoter sequence as a primer.

Northern blot screening
Northern blot analysis was performed for screening of the genes isolated. As a first screening, liver mRNA levels in animals treated with PB, EE and BHT at 8 weeks in the medium-term bioassay protocol were compared with that in animals treated with DEN + PH without chemical administration during the promotion stage (Figure 2Go). Isolated poly(A+) RNA (1 µg) was analyzed on 0.8% formaldehyde–agarose gels, transferred to Hybond-N nylon membranes (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and probed with 32P random-primed cDNA fragments of isolated up-regulated genes, followed by phosphorimaging analysis using BAS1500 (Fuji Film Co., Tokyo, Japan).

For a second screening, mRNA levels in HCCs produced by PB promotion at week 64 were compared with those in the livers of untreated controls and DEN + PH + PB-treated rats at week 8 (Figure 2Go). Identification of tumors was made by histopathological assessment of parts of nodules isolated at autopsy. Total cytoplasmic RNAs were isolated by RNA STAT-60 (Tel-Test `B', Friendswood, TX), and 15 µg/lane were analyzed on formaldehyde–agarose gels. After transfer to nylon membranes, visualization of 18S and 28S ribosomal RNAs was accomplished by methylene blue staining. Similarly, total RNAs were used for expression analysis in the dose–response study of PB and feeding study of non-genotoxic carcinogens without initiation treatment.

Each membrane was re-probed four times with other gene fragments for screening after stripping with a boiled 0.1% SDS solution, and re-probed finally with a 0.68 kb gene fragment of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously (26). In case of signal-detectable genes, another identical northern blot analysis was performed using RNA samples of different liver tissues/HCCs. Band intensities obtained by northern blots were analyzed using the ONE-D/ZERO-Dscan software program (Scanalytics, Fairfax, VA).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Identification of isolated genes
To obtain up-regulated genes at an early time point in promotion by PB, 80 clones, sized 84–879 bp were isolated from the up-regulated gene fragment pool of rat liver after PB treatment for 10 days (Figure 1Go). On sequence analysis of each clone, a total of 67 different genes were found to be isolated. The other 13 clones contained either only poly(A+) tail short sequences (three clones), or sequences overlapping with identical genes such as those encoded the fibrinogen B ß chain, cytochrome P450 (CYP) 2B2, f-spondin, transferrin, guanydinoacetate methyltransferase and fetuin-like protein. Among 67 genes, 48 genes showed high homology with sequences of mammalian genes already registered (Table IGo), and the other 19 clones did not show any sequence homology with mammalian genes registered in the EMBL/GeneBank. According to the classification scheme based on the biological function of encoded protein (27), the listed 48 genes were classified into the following categories: metabolism (16 genes); cell signaling/communication (five genes); cell structure/motility (three genes); protein expression (two genes); cell/organism defense (two genes); gene expression (one gene); unclassified [19 genes including five expressed sequence tag (EST) clones].


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Table I. Identification of genes found to be up-regulated in the livers of rats treated with phenobarbital for 10 days in the medium-term liver bioassay
 
PB is known to induce a variety of genes including those biotransformation enzymes. Among the identified genes isolated from the up-regulated gene pool after 10 days of PB treatment in the present study protocol, the following have already been reported to be up-regulated by short-term PB treatment: UDP-glucuronosyl transferases (4,2830), aldehyde dehydrogenases (4,29), CYP2B2 (4,29), CYP3A (4,29,31), CYP4B1 (32) and apolipoproteins (APOs) (4,30,33,34). In addition, modification of microfilaments and microtubules in hepatocytes occurs with short-term PB treatment (35,36), presumably involving altered expression of genes for elements such as tubulin or actin.

First screening of genes
To identify genes still up-regulated at the time point when GST-P-positive foci, putative pre-neoplastic liver lesions, appeared with promoter treatment, northern blot screening was performed for all genes obtained at the 8 week time point by PB, EE or BHT treatment (Figure 2Go). Among the 48 signal-detectable genes, 21 genes showed increased expression in the PB- or EE-treated rat livers, as compared with the level of DEN + PH alone (Table IIGo, Figure 3Go). Many of the up-regulated genes also showed increased expression with BHT. Among these genes, the following 16 genes showed up-regulation common to the PB and EE treatments with the relative expression levels being three times or more than those of DEN + PH alone: CYP2C39, CYP3A, ß-alanine synthase, glycosylphosphatidylinositol-specific phospholipase D1 (gpld1), glutathione-S-transferase mu2 (gstm2), APOA4, ubiquitously expressed mammalian ABC half transporter (umat), hemolytic complement, nuclear receptor binding factor (NRBF)-2, CD81, hypothetical protein, mlrq-like protein and four unidentified clones. In addition, {gamma}-actin and deleted in oral cancer-1 (DOC-1R) also retained increased, but less pronounced expression by PB and EE treatments. No specific signals could be detected for 19 genes even at two different hybridization temperatures of 55 and 68°C, and the expression with the other 27 genes remained unchanged or was reduced by promoter treatment for 8 weeks.


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Table II. Results of first screening of the isolated genes by northern blotting
 


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Fig. 3. Representative northern blots obtained from the first screening of genes isolated by suppression PCR subtractive hybridization.

 
Second screening of genes
To select for genes still up-regulated in the tumor tissue, northern blotting analysis was performed using total RNAs from HCCs produced by PB treatment at 64 weeks in the medium-term bioassay (Figure 2Go). The expression level was compared with those of livers of PB-treated rats in the medium-term bioassay and untreated normal animals. Among the genes selected at the first screening, many genes were constitutively expressed in untreated rat livers at levels similar to those with PB after DEN + PH treatment (Table IIIGo, Figure 4Go). Among the 21 genes selected by the first screening as listed in Table IIGo, the following eight genes retained high expression levels in PB induced HCCs (three times or more than the levels in untreated rat liver): APOA4, umat, laminin receptor (LR)-1, {gamma}-actin, NRBF-2, CD81, hypothetical protein and unidentified clone #2 (cloned sequence, Figure 5Go). Increased expression of DOC-1R was also observed in HCC (2.7-fold increase). Those showing striking up-regulation by promoter treatment and in HCCs were APOA4, umat, NRBF-2, CD81, hypothetical protein and clone #2.


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Table III. Results of second screening of the isolated genes by northern blotting
 


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Fig. 4. Representative northern blots obtained from the second screening of genes selected on the first screening.

 


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Fig. 5. Sequence data for a cDNA fragment of the unidentified clone #2. The size of the isolated fragment is 666 bp.

 
Dose-dependence with PB treatment
On the basis to the expression analysis by first screening, six genes (gstm2, APOA4, DOC-1R, CD81, hypothetical protein and clone #2) were selected for examination of dose-dependence with PB treatment at week 8 in the medium-term liver bioassay (Figure 6Go). With an increase of the PB dosage, dose-related incremental expression was observed with gstm2, CD81, hypothetical protein and clone #2. In the case of APOA4 and DOC-1R, an apparent up-regulation was observed at 600 p.p.m.



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Fig. 6. Analysis of dose-dependency with PB treatment on the expression of six genes selected at first screening. northern blot analysis was performed with the livers of rats at week 8 of PB promotion in the medium-term liver bioassay. Values were calculated as ratios of promotion (–) control after normalization with the intensity of 18S ribosomal RNA and represent the means of duplicate samples.

 
Responsiveness to non-genotoxic carcinogens without initiation
To examine whether selected genes in the present two-stage hepatocarcinogenesis model are available for the rapid screening of non-genotoxic carcinogens, expression analysis was performed in the livers of rats simply administered chemicals for 4 weeks without DEN initiation. In addition to the above six genes selected for the dose-response study, NRBF-2 was also examined. Different from the expression pattern in the two-stage carcinogenesis model, selected genes did not show up-regulation by treatment with PB or EE without initiation except for the increased expression of gstm2 in PB-treated animals (Figure 7Go). Every gene examined showed constitutive expression in untreated rat livers, and did not show specific expression pattern to non-genotoxic carcinogens.



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Fig. 7. Expression changes of selected seven genes by exposure to representative non-genotoxic carcinogens without initiation. Northern blot analysis was performed with the livers of rats at week 4 of treatment with PB, EE, DEHP, DL-E or TAA. Expression changes by hepatotoxicants (APAP or ANIT) or PH alone were also examined. Values were calculated as ratios of untreated control after normalization with the intensity of GAPDH signal and represent the means of duplicate samples.

 

    Discussion
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 Abstract
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 Materials and methods
 Results
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 References
 
This study was performed to identify specific genes steadily expressed in the liver during the promotion stage of hepatocarcinogenesis mediated by PB treatment. Many of the up-regulated genes in the PB- and/or EE-treated rat livers at 8 weeks were also up-regulated with administration of BHT, intended for use as a negative control for tumor promotion (13,37). However, BHT is reported to cause DNA methylation in the rat liver (38) and inhibit gap junctional intercellular communication in cultured hepatocytes (39), indicating a link to carcinogenic processes. Considering chemopreventive effects due to the inhibition of bioactivation and binding of carcinogenic chemicals to DNA (4042), BHT might exert intricate actions involving both carcinogenic and anticarcinogenic mechanisms. Therefore, it is still possible that genes up-regulated by BHT-treatment in the present study could be playing roles in hepatocarcinogenic processes.

In the present study, the first comparison was made between PB treatment after DEN + PH and DEN + PH alone. However, many of the selected genes were found to be expressed constitutively in the livers of untreated rats, with no marked differences in the expression level. Furthermore, without DEN initiation, most of the genes selected did not increase the expression by treatment with PB or EE for 4 weeks. Among gene probes in Gene Chip® Rat Genome U34A Array (Affymetrix, Santa Clara, CA), several genes selected at screenings in the present study, such as APOA4, CYP3A, ß-alanine synthase and {gamma}-actin are included. By microarray analysis using U34A arrays, we also found unchanged or rather decreased expression of these genes in the liver of rats treated with PB for 4 weeks (T.Arimura and M.Shibutani, manuscript in preparation). These results suggest suppressive effects on gene expression triggered by DEN or DEN + PH treatment, reversed by PB treatment in the promotion stage. DEN is a mitoinhibitory hepatocarcinogen, which induces p53 in the rat liver (43). Selective down-regulation of p21waf1/cip1, a downstream effector of p53, in GST-P-positive foci as compared with surrounding hepatocytes (44) may suggest a selective involvement of signaling relevant to the G1–S check point. Induction of reversible or irreversible G1 cell-cycle arrest elicits differential gene expression patterns that could contribute to a quiescent or senescence-like phenotype (45). In addition, overexpressed p21 in senescent cells has been shown to directly interact with the E2F complex to cause negative regulation of E2F transcription factor activity to down-regulate cell proliferation-related genes (46). Eight weeks is set as endpoint of medium-term bioassay to detect tumor-promoting activity by analysis of generated GST-P-positive foci (3), and generation of GST-P-positive foci is very low at the 8 week time point without promoter treatment (12). Our observations may suggest differences in transcriptional activity between normal and DEN-induced mitoinhibitory cellular populations. Although the mechanisms of gene expression recovery with promoter treatment remain to be clarified, differences in the dose level of PB to induce genes examined in our dose-response study may underline the complex and subtle cellular response to PB, which indicates a regulatory cascade at many different levels of gene regulation (30).

With the second screening, a number of genes were found to be up-regulated in HCCs (3-fold or more): APOA4, umat, LR1, {gamma}-actin, NRBF-2, CD81, hypothetical protein and one unidentified. Among them, the gene for hypothetical protein has only recently been registered and its function remains unknown. In the case of APOA4, there have been no carcinogenesis-related reports to our knowledge. With actins, qualitative alteration has been investigated in detail in cancer cells in response to interactions with their environment (4751), but reports on subclass expression in association with carcinogenesis are limited (52,53).

Umat is a recently identified gene encoding a protein belonging to the ATP binding cassette superfamily (54). With the sequence similarity to hmt1 and atm1 transporters, umat might be involved in metal ion homeostasis. Gene transcripts of P-glycoprotein-related protein (PRP), sized ~3 kb, have been reported to be increased during experimental hepatocarcinogenesis (55). Most of the sequence of this gene shows 98% homology with the 3'-half of umat, whose transcript size is estimated as 3.4 kb, suggesting that the overexpression of 3 kb transcripts of PRP in neoplastic lesions described by Furuya et al. might be identical to the umat transcript in the present study.

NRBF-2 is a rat gene recently identified by yeast two-hybrid screening using the mouse peroxisome proliferator-activated receptor {alpha} as a bait, is considered to interact with this and several other nuclear hormone receptors to effect gene activation, when tethered to a heterologous DNA binding domain. NRBF-2 mRNA is widely expressed in a variety of organs, but most profoundly in the liver or tumor cell lines of liver cell origin, and transcriptional activation by this factor is considered to be cell type-specific on the basis of reporter gene assays (56). Strong induction of this gene during the promotion stage in the present study may suggest involvement of nuclear hormone receptor(s) in hepatocarcinogenesis.

CD81 is a member of the tetraspanin superfamily, widely expressed cell-surface proteins involved in a variety of cellular functions, including adhesion, activation, proliferation and differentiation (57). Rockett et al. (58) reported its up-regulation in the rat but down-regulation in the Guinea-pig liver following 3 day treatments with WY-14,643, a representative peroxisome proliferator, suggesting differential regulation of this gene in sensitive and resistant species, respectively.

Although the LR1 mRNA was not remarkably increased at week 8 by PB treatment, overexpression in HCCs was observed here. The encoded protein has been identified as a p40 ribosome-associated element, and is now regarded as the 37 kDa precursor of the 67 kDa laminin receptor, which is up-regulated in fetal liver and in DEN-induced rat liver tumors (59). Moreover, it is consistently up-regulated in aggressive carcinomas and the responsible gene is located on chromosome 3 in the locus 3p21.3 which, interestingly, is a hot spot for genetic alterations in several cancers and particularly in small cell lung carcinomas in humans (60).

Although the ratio of up-regulation was not strong by promoter treatment, DOC-1R showed increased expression in HCC (2.7-fold increase) in the present study. DOC-1R is a recently identified human gene that is located in 11q13, a cytogenetic region rich in cancer-related genes, and is homologous with the DOC-1 gene (61). DOC-1 was originally isolated by subtractive hybridization from a cDNA library enriched for gene expression lost during oral carcinogenesis with a hamster oral cancer model (62). Ectopically expressed p12DOC–1 protein in cultured cells has been shown to act as a growth suppressor by negative regulation of DNA polymerase {alpha}–primase complexes and cyclin-dependent kinase 2 (63,64). Although similar growth suppressive effects have been suggested for DOC-1R protein (61), its function and underlying molecular events are still unknown.

In summary, we identified a number of genes, which were consistently up-regulated during PB-promoted hepatocarcinogenesis in the present medium-term liver bioassay. Those showing striking up-regulation with promoter treatment and in HCCs, such as umat, APOA4, NRBF-2, CD81, hypothetical protein and one unidentified example may be linked to hepatocarcinogenetic processes. Considering the constitutive expression in untreated rat liver and no specificity for non-genotoxic carcinogens without initiation, expression changes of these genes might be specific to the promotion stage after initiation, and therefore these genes might be utilized in the initial screening of non-genotoxic hepatocarcinogens by analysis in the present two-stage model. Further work will be required on the availability of identified genes for short-term detection of other genotoxic or non-genotoxic carcinogens as well as the target organ specificity.


    Notes
 
1 To whom correspondence should be addressed Email: shibutan{at}nihs.go.jp Back


    Acknowledgments
 
This work was supported by a Grant-in-Aid for Scientific Research 11660319 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 11, 2001; revised March 5, 2002; accepted March 7, 2002.





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