FHIT alterations in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine in rats
Toshifumi Tsujiuchi,1 ,
Yasutaka Sasaki,
Nao Murata,
Masahiro Tsutsumi,
Yoichi Konishi and
Dai Nakae
Department of Oncological Pathology, Cancer Center, Nara Medical University, Kashihara, Nara 634-8521, Japan
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Abstract
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Alteration of the FHIT gene was investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl) amine (BHP) in male Wistar rats. Animals at 6 weeks of age were given 2000 p.p.m. of BHP in drinking water for 12 weeks, then maintained without further treatment until killed at the end of week 25. A total of 25 lung adenocarcinomas were obtained and total RNAs were extracted from each for assessment of aberrant transcription of the FHIT gene by reverse transcription (RT)polymerase chain reaction (PCR) analysis. Aberrant transcripts were detected in 15 adenocarcinomas (60%) as absence in the regions of nucleotides (nt) -9 to 279, -98 to 279, -98 to 348 or -98 to 447. Genomic DNAs were also extracted from all 25 adenocarcinomas and exons 59 were examined for mutations, using PCRsingle strand conformation polymorphism (SSCP) analysis and sequencing. A mutation was detected in only one adenocarcinoma (4%), an ACC to ATC (Thr to IIe) transition at codon 76. Southern blot analysis of eight tumors did not show any evidence of gross rearrangement or deletion of the FHIT gene. Western blot analysis revealed reduced expression of Fhit protein in six out of 10 adenocarcinomas (60%). These results suggest that alteration of the FHIT gene may be involved in the development of lung adenocarcinomas induced by BHP in rats.
Abbreviations: BHP, N-nitrosobis(2-hydroxypropyl)amine; NSCLC, non-small cell lung cancer; PCR, polymerase chain reaction; RT, reverse transcription; SSCP, single-strand conformation polymorphism; nt, nucleotides; SCLC, small cell lung cancer.
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Introduction
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Lung cancer is one of the most common human malignancies, but the rate-limiting molecular events for its development are still largely obscure. Deletion of the short arm of chromosome 3 is frequently detected, suggesting the presence of tumor suppressor genes in this region (15). The FHIT gene has been identified as a tumor suppressor gene at chromosome 3p14.2, spanning the FRA3B common fragile site (68). It is >1 Mb in size and encodes a 1.1 kb cDNA with 10 small exons (7). On the basis of enzymatic properties, the human Fhit protein has been classified as a dinucleoside 5', 5'''-P1, P3-triphosphate hydrolase, which produces ADP and AMP from the diadenosine substrate in vitro (9,10). The FHIT gene and its product have also been suggested to somehow be involved in cell proliferative and apoptotic processes (11). The variety of alterations detected in human lung cancers as well as tumors in the head and neck, stomach, breast, kidney, pancreas and liver, include aberrant mRNA transcripts and reduction or even absence of Fhit protein expression, often in association with deletion of the FHIT gene (68,1215). It was recently suggested that environmental factors, particularly smoking in the lung cancer case, might increase the risks of such alterations occurring in the FHIT gene (1618). Because tobacco smoke contains chemicals obviously capable of causing lung cancers, such as nitrosamines (19), it can be hypothesized that they induce alterations of the FHIT gene, which may constitute rate-limiting molecular events. The present study was conducted to assess this hypothesis by examining alterations of the FHIT gene in lung adenocarcinomas induced by BHP in rats.
The experimental model employed for rat lung carcinogenesis features the induction of non-small cell lung cancers (NSCLCs) by BHP, with especially high yields of adenocarcinomas, mirroring the human situations (20,21). Because the step-by-step development of lung malignancies is accessible with this model, molecular mechanisms involved at each step can readily be investigated (2227). So far, we have detected a high frequency of mutations of Ki-ras, adenomatous polyposis coli (APC) and ß-catenin, but not Ha-ras and p53 genes in NSCLCs as well as their pre-neoplastic lesions (22,23). The genes participating in the transforming growth factor-ß (TGF-ß) signaling pathway, such as Smad2, Smad4 and TGF-ß type II receptor, may also be altered in adenocarcinomas (24,25). Furthermore, the genes for vascular endothelial growth factor as well as its receptors and midkine are overexpressed during the progression stage of BHP-induced lung carcinogenesis (26,27).
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Materials and methods
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Animals and treatment
A total of 28 male Wistar rats, were purchased at 5 weeks of age from Japan SLC Inc. (Shizuoka, Japan) and housed 35 to a plastic cage with white flake bedding in an air-conditioned room, with a constant temperature of 25°C, and a 12-h lightdark cycle. Food and water were given ad libitum throughout the study. After a 1-week acclimation period on basal diet in pellet form (CF-2 Diet; Clea Japan, Tokyo, Japan), 25 animals received drinking water containing BHP (Nakalai Tesque Co. Ltd, Kyoto, Japan) at a concentration of 2000 p.p.m. for 12 weeks and then drinking water without BHP. In order to obtain normal lung tissue, the remaining three animals were maintained free from the carcinogen exposure throughout the experimental period. Normal lung tissue was used as a control to eliminate contamination with cancerous tissue macroscopically undetected. All rats were killed by exsanguination from the abdominal aorta under light ether anesthesia 25 weeks after the beginning of the experiment.
Tissue preparation
Upon sacrifice, the lungs were immediately excised, and grossly apparent tumors were dissected from their surrounding tissue. Samples were frozen in liquid nitrogen and stored at -80°C until analysis. Portions of the tumors were fixed in 10% neutrally buffered formalin at 4°C and embedded in paraffin. Two serial thin sections were made, one with 3 µm thickness to be stained with hematoxylin and eosin for histological examination, and the other with 5 µm thickness for DNA extraction.
5' and 3' RACE for the rat FHIT gene cDNA
The 5' and 3' RACE analyses were performed, because the length of the previously cloned sequence for the rat FHIT gene cDNA is not sufficient to examine for aberrant transcription. The primers used for 5' and 3' RACE were designated from the rat FHIT gene cDNA (GenBank accession number AF170064).
5' RACE was performed with the 5' RACE System (Gibco BRL, Gaithersburg, MD, USA) according to the manufacturer's instructions. Total RNA was extracted from frozen normal lung tissue using an ISOGEN kit (Nippon Gene Inc., Toyama, Japan), and 1 µg of total RNA was reverse-transcribed (RT) using a downstream gene-specific primer, 5'-CACGAGGACATGGCCAGGTACAAC-3'. A homopolymetric tail was then added to the 3'-end of the resultant cDNA using terminal deoxynucleotidyl transferase and dCTP and PCR was performed with a nested gene-specific primer, 5'-CACCAGGGCAAAGGACAGTTCGGT-3' and an abridged anchor primer and UAP (from the RACE kit).
The 3' RACE System (Gibco BRL) was used for 3' RACE. First-strand cDNA was generated from 1 µg of rat normal lung tissue total RNA using a 3' RACE adaptor primer with a polydeoxythymidylate tail. Subsequent rounds of PCR were performed using nested gene-specific primers, 5'-CATGTCCACATTCTTCCCAG-3' and 5'-GGAGACTTCCGCAGGAATGA-3' and a universal adaptor primer (from the RACE kit).
All RACE products were subcloned with a TOPO TA cloning kit (Invitrogen Corporation, CA, USA) and sequenced with Sequencing Pro (TOYOBO Co. Ltd, Tokyo, Japan).
RTPCR amplification
Total RNA was extracted from frozen tissue of 25 adenocarcinomas, and first-strand cDNAs were synthesized from 5 µg aliquots with a Ready-To-Go Your-Prime First-Strand Beads (Pharmacia Co. Ltd, Tokyo, Japan). To eliminate possible false positives caused by residual genomic DNA, all samples were treated with DNase.
Nested RTPCR analysis was performed to analyze for aberrant transcription of the FHIT gene as described previously (7,8,14), using the primers 1F, 1R, 2F and 2R (Table I
) designated according to the resultant sequences obtained from the above 5' and 3' RACE analyses. The first round of PCR amplification was performed in 10 µl of reaction mixture consisting of 1 µM of primers 1F and 1R, 200 µM of each dNTP, 1x PCR buffer (Perkin Elmer, Applied Biosystems Division, Foster City, CA, USA), 2.5 U of Ampli Taq (Perkin Elmer) and 0.5 µl of synthesized cDNA mixture under the following reaction conditions; a primary denaturation step for 2 min at 95°C, 30 cycles of 30 s denaturation at 95°C, 30 s annealing at 58°C and 1 min extension at 72°C and a final extension for 10 min at 72°C. The amplified product was diluted 20-fold in TE buffer, and 1 µl of the dilutant was subjected to the second round of PCR amplification with the above conditions except the annealing temperature was 63°C, using primers 2F and 2R (Table I
). PCR products were then separated on 2% NuSieve agarose gels (BMA, Rockland, ME, USA) containing 0.05 µg/ml ethidium bromide. Each nested RTPCR assay was repeated at least twice for confirmation using the originally extracted RNA.
PCRSSCP analysis
DNA was extracted from paraffin embedded sections of 25 adenocarcinomas and three frozen normal lung tissues as described previously (22,23). PCRSSCP analysis was then conducted to seek for mutations in the FHIT gene. Exons 58, -F and -R primers (Table I
), were designed for mouse FHIT gene exons 58 (GenBank accession numbers AF047700, AF047701 and AF047702, respectively) (28) and rat FHIT cDNA sequences. Because no information is available for exon 9 in terms of mouse FHIT gene, exon 9-F and -R primers (Table I
) were designed for rat cDNA sequence, considering the 3'-end of mouse FHIT gene exon 8 sequence (AF047702 as above). Briefly, PCR for SSCP was performed in 10 µl of reaction mixture containing 1 µl of each primer, 1x PCR buffer (Perkin Elmer), 200 µM of each dNTP, 68 nM of [
-32P]dCTP, 2.5 U of Ampli Taq (Perkin Elmer) and an appropriate amount of template DNA. Each mixture was subjected to 32 cycles of amplification, 30 s at 95°C for denaturation, 30 s at 5566°C for annealing and 1 min at 72°C for extension (see Table I
). To rule out PCR artifacts, PCR amplification was repeated from individual original DNAs at least once. PCR products were diluted with 90 µl of loading solution containing 90% formide, 20 mM EDTA and 0.05% xylene cyanol and bromophenol blue, denatured at 90°C for 2 min and applied to 5, 6, 8 and 10% polyacrylamide gels containing 0.5x Trisborate EDTA buffer with or without 5% glycerol. Electrophoresis was performed at 40 W for ~2.5 h at 30°C. Gels were then dried on filter paper and exposed X-ray films at 80°C.
Cloning and DNA nucleotide sequencing
Following the RTPCR and the PCRSSCP analyses, DNA fragments from aberrant transcription bands and mobility-shifted bands in the gel were extracted and reamplified. The obtained PCR products were cloned with a TOPO TA cloning kit (Invitrogen) and recombinant plasmid DNA clones were sequenced using Sequencing Pro (TOYOBO). In each experiment, 510 clones from different bacterial colonies were investigated.
Preparation of a cDNA probe and Southern blot analysis
A 0.88 kb cDNA fragment for the rat FHIT gene was obtained by RTPCR, using primers 1F and 1R (Table I
). Total RNA was extracted from frozen normal lung tissue and subjected to 32 cycles of 1 min at 95°C for denaturation, 1 min at 58°C for annealing and 2 min at 72°C for extension. The amplified product was separated on 1% agarose gels containing 0.05 µg/ml ethidium bromide and the DNA fragment extracted from the gels was cloned and confirmed by sequencing, then used as the hybridization probe for FHIT.
Genomic DNAs were extracted from the frozen tissues of eight adenocarcinomas using a DNeasy tissue kit (Qiagen, Hilden, Germany). After digestion with the restriction enzyme BamHI (TaKaRa, Kyoto, Japan), which does not cut rat FHIT cDNA, 10 µg of DNA samples were fractioned by size in 1% agarose gels, blotted onto Hybond-XL membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK) and hybridized with the [
-32P]dCTP radiolabeled FHIT probe using a Rediprime II random prime labeling system (Amersham Pharmacia Biotech). Blots were washed and then placed in contact with Hyperfilm MP (Amersham Pharmacia Biotech) at 80°C.
Western blot analysis
Proteins were extracted from frozen tissues of 10 adenocarcinomas, and western blot analysis was carried out as described previously (29). Briefly, aliquots of 20 µg of total protein were electrophoresed on 12% SDSpolyacrylamide gels, transferred to Hybond ECL (Amersham Pharmacia Biotech) and probed with a rabbit polyclonal anti-Fhit antibody (IBL, Gunma, Japan), which was obtained by the immunization of synthesized peptides corresponding to amino acid residues 130147 of human Fhit protein. Mouse monoclonal anti-human-actin antibody (Santa Cruz Biothechnology Inc., CA, USA) was used as an internal control. The membranes were developed with a chemiluminescence system (ECL detection reagents; Amersham Pharmacia Biotech).
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Results
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All 25 rats treated with BHP developed adenocarcinomas, and a total of 25 adenocarcinomas >5 mm in diameter were obtained as one each from every rat for the RTPCR and PCRSSCP analyses. Among them, 10 were sized >8 mm in diameter and could thus be employed for additional Southern and western blotting. We detected 105 bp of 5' upstream region and 155 bp of 3' downstream region sequences of the rat FHIT gene cDNA by the 5' and 3' RACE analyses and thus designated primers for the nested RT-PCR (Figure 1
). Using 25 adenocarcinomas and three normal lung tissues, we reverse-transcribed mRNAs and amplified cDNA by nested PCR as described previously (7,8,13).

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Fig. 1. Nucleotide and deduced amino acid sequences of the rat FHIT gene cDNA. Those in bold face represent 105 bp of the 5' upstream region and 155 bp of the 3' downstream region detected by the 5' and 3' RACE analyses. Arrows indicate the regions used for the nested RTPCR primers.
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The nested RTPCR analysis revealed that, whereas only the normal product band was amplified at 823 bp in all three normal lung tissues, one or two abnormal-sized bands additionally appeared representing aberrant RTPCR products in 15 out of 25 adenocarcinomas (60% incidence). Representative RTPCR results for one normal lung tissue and 15 adenocarcinomas are shown in Figure 2A
. Such abnormal-sized transcripts were seen at four different positions of 535, 446, 377 and 278 bp. Sequencing analysis revealed that these aberrant fragments were due to absences in the regions of nt -9 to 279, -98 to 279, -98 to 447 and -98 to 348 (Figure 2B
). All normal sized-transcripts demonstrated normal sequences (data not shown).

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Fig. 2. Aberrant transcription of the FHIT gene. (A) Representative results of the nested RTPCR analysis. M, 100 bp DNA size marker; N, normal lung tissue. (B) Patterns of aberrant transcripts detected by the sequencing analysis. Arrows indicate the junctions between nts -10 and 280 (a), -99 and 280 (b), -99 and 349 (c) and -99 and 448 (d) in the aberrant transcripts.
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Using primers to amplify exons 59 (Table I
), the PCR products showed clear single bands in 1% agarose gels (data not shown). Representative results of PCRSSCP analysis and sequencing analysis for FHIT mutations are shown in Figure 3A and B
. One out of 25 adenocarcinomas (4% incidence) showed an abnormal band shift for exon 6 (Figure 3A
). Sequencing analysis revealed the mutation to be an ACC to ATC (Thr to IIe) transition at codon 76 (Figure 3B
). No mutations were found in exons 5, 79 and no homozygous deletions were apparent throughout exons 59 (data not shown).

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Fig. 3. Point mutation of the FHIT gene at exon 6. (A) Representative results of the PCRSSCP analysis. Arrowhead indicates abnormal band shift. N, normal lung tissue. (B) The mutation pattern of the FHIT gene detected by the sequencing analysis. AC, adenocarcinoma.
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No abnormal restriction patterns, such as genomic rearrangement or deletion, could be detected in any of the eight assessed adenocarcinomas by Southern blot analysis using the probe including the deletion sites for aberrant transcription (Figure 4
). Densitometric analysis showed no differences in the intensity of each band (data not shown). Six out of 10 adenocarcinomas (60% incidence) demonstrated markedly reduced expression of Fhit protein compared with normal lung tissue. Representative results are shown in Figure 5
.

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Fig. 4. Representative results of the Southern blot analysis of BamHI digested DNA. N, normal lung tissue.
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Fig. 5. Reduced expressions of Fhit protein detected by the western blot analysis. N, normal lung tissue.
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Discussion
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The present investigation of alterations of the FHIT gene in lung adenocarcinomas induced by BHP in rats demonstrated frequent aberrant transcripts due to absence, as well as one rare mutation of the FHIT gene, in association with frequent reduced expression of Fhit protein (see the summary of the results in Table II
). Abnormalities in products amplified from the FHIT gene transcripts have been found in 80% of SCLCs and 42% of NSCLCs in humans, suggesting the involvement of such abnormalities in lung carcinogenesis (8). The aberrant tumors show an absence of exons 46 or 48 in SCLCs; however, whereas the patterns vary, exon 5 is always missing in NSCLCs (8). In the present study, aberrant transcripts detected in rat lung adenocarcinomas were due to four patterns of absences, in which exon 5 was always lost, whereas exon 8 was also absent in four out of 15 aberrant transcripts (27% incidence). Our absence patterns are thus in accordance with the findings for human NSCLCs. The initial methionine codon exists in exon 5, and the highly conserved histidine triad is contained in exon 8 (7,30). These exons may, therefore, be critical for the activity of the FHIT gene product, and their loss may thus result in loss of the tumor suppressing function. On the other hand, wherease homozygous deletions within the FHIT gene have been detected in several cancer derived cell lines, DNA rearrangement was also found in such cell lines as well as primary lung cancers by Southern blot analysis with BamHI-digested DNAs (8,29). This suggests that the aberrant FHIT gene transcription could also result from DNA rearrangement, particularly in the region near exon 5 (8). We could not detect, however, DNA rearrangement or homozygous deletion throughout exons 59. Point mutations of FHIT are rare events in the pathogenesis of human cancers (8,31,32). As for primary tumors, only one gastric cancer has so far been reported to show a point mutation at codon 61 in exon 6 (31). In terms of lung cancers, whereas one lung cancer cell line has been shown to have a frameshift mutation at nt 15 of the open reading frame (8), no mutations have been found in primary SCLCs and NSCLCs (8). The present finding of only one mutation in one out of 25 adenocarcinomas is, therefore, in line with the human situation.
It has been reported that nearly 100% of SCLCs and 73% of NSCLCs do not express Fhit protein (33). Primary tumors and tumor cell lines with altered FHIT transcripts and LOH at the FHIT locus usually show loss or reduced expression (17,29,33). Inactivation might also be due to epigenetic mechanisms, such as hypermethylation of the 5' CpG island of the FHIT gene (34). The present study indicates a close relationship between aberrant transcripts and reduction of Fhit protein expression, because all assessed adenocarcinomas with decreased protein expression had an absence of regions. Involvement of the LOH and epigenetic alteration of the FHIT gene in this reduced expression remains to be clarified. Reduction of Fhit protein expression is observed more frequently in patients with a smoking history (9,17,33,35). Moreover, a marked decrease has been found in 5886% of squamous cell carcinomas, but 1057% in adenocarcinomas (1618). This suggests that the FHIT gene may be a target of chemical carcinogens such as nitroso-compounds found in tobacco smoke. Because our lung NSCLCs are induced by a nitrosamine, BHP, the presently used experimental model may be particularly useful to assess mechanisms whereby alterations of the FHIT gene and its product could be involved in lung carcinogenesis.
In conclusion, the alteration of the FHIT gene may play roles in rat lung carcinogenesis initiated with BHP. For extrapolation to the human situation, further investigations are now required to elucidate the underlying mechanisms in more detail.
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Notes
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1 To whom correspondence should be addressed
Email: ttujiuch{at}naramed-u.ac.jp 
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Acknowledgments
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The authors express their gratitude to Ms Yumi Horikawa, Mariko Okuda, Sachiko Nakai, Hiroko Masuda and Megumi Yamaguchi for their technical assistance. This work was supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health, Labor and Welfare of Japan and CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation (J.S.T.).
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Received May 1, 2001;
revised June 28, 2001;
accepted August 20, 2001.