Isolation of the Retinoblastoma cDNA from the Marine Flatfish Dab

Weymouth Laboratory, Barrack Road, The Nothe, Weymouth,. Dorset DT4 8UB, United Kingdom. We have isolated a dab (Limanda limanda) homologue of...
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Environ. Sci. Technol. 2005, 39, 9785-9790

Isolation of the Retinoblastoma cDNA from the Marine Flatfish Dab (Limanda limanda) and Evidence of Mutational Alterations in Liver Tumors FRANCES A. DU CORBIER,† GRANT D. STENTIFORD,‡ BRETT P. LYONS,‡ AND J E A N E T T E M . R O T C H E L L * ,† Department of Biology and Environmental Science, Centre for Environmental Research, University of Sussex, Falmer, Brighton BN1 9QJ, United Kingdom, and Centre for Environment, Fisheries and Aquaculture Science, Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB, United Kingdom

We have isolated a dab (Limanda limanda) homologue of the human retinoblastoma (Rb) tumor suppressor gene. The L. limanda partial Rb cDNA encodes a partial predicted protein of 753 amino acids. DNA sequence analysis with other vertebrate Rb sequences demonstrates that the L. limanda Rb cDNA is highly conserved in regions of functional importance. The sequence reported herein, combined with the high degree of conservation observed in critical domains, has also facilitated an investigation of the molecular etiology of environmentally induced liver tumor samples in a feral fish species. Mutational alterations were detected in liver adenoma samples, also in apparently “normal” regions of liver samples dissected from fish displaying adenoma, but not in normal liver samples from otherwise healthy feral fish. These results are the first reporting the appearance of Rb mutations in wildcaught fish and suggest that the molecular etiology of fish cancer appears to involve Rb-implicated tumorigenesis. The ecotoxicological relevance of the Rb mutations in feral fish liver tumors, in terms of future genome instability and possible development of a genotoxicity biomarker, is discussed.

Introduction Polycyclic aromatic hydrocarbons (PAHs) have been implicated in the etiology of carcinogenesis of fish liver. Sediment PAH levels have been correlated with the induction of detoxification enzymes such as cytochrome P450s (1), DNA adducts (2), and also incidence of liver tumors (3). The mechanism of PAH metabolism is well defined in mammalian models, and biotransformation is mediated by the cytochrome P450/epoxide hydrolase-containing detoxification systems to more reactive metabolites that can spontaneously form DNA adducts. These adducts can then result in mutations within specific regions of DNA, such as cancer genes. * Corresponding author phone: +44 1273 872862; fax: +44 1273 677196; e-mail: [email protected]. † University of Sussex. ‡ Centre for Environment, Fisheries and Aquaculture Science. 10.1021/es051367c CCC: $30.25 Published on Web 11/15/2005

 2005 American Chemical Society

The genes most often implicated in vertebrate tumorigenesis are the ras oncogene and the p53 and retinoblastoma (Rb) tumor suppressor genes. When they are combined, the mutational activation or inactivation respectively of such cancer genes is observed in more than half of all human tumors (4-6). Ras genes encode proteins that play a central role in cell growth signaling cascades and are evolutionarily conserved (7). A large proportion and wide variety of experimentally induced or environmentally induced vertebrate tumors, including liver tumors in fish, possess mutant forms of ras (for a review, see ref 7). The p53 gene is an important negative regulator of cell cycle progression and is conserved across species including trout (Oncorhynchus mykiss) and medaka (Oryzias latipes) (8, 9). Conservation of specific functional domains suggests that the p53 protein plays similar functional roles in vertebrates as diverse as fish and humans (10). However, a preliminary investigation of carcinogen-induced tumors in the aquaria fish O. latipes has revealed no p53 mutations (9). Results, following an investigation of ultraviolet light inducibility of O. latipes p53, also suggest that the p53 protein has a different function in lower vertebrates compared with humans (11). Consequently, interest is now mounting in the role of alternate tumor suppressor genes, particularly Rb, in the development of tumors in fish. The retinoblastoma gene (Rb) was the first tumor suppressor gene to be characterized (12). In vertebrates, the Rb gene product is a nuclear phosphoprotein that regulates normal cell cycle progression (13). In humans, the loss of function of the Rb gene occurs by mutation or deletion and results in a diverse set of cancers including hepatocellular carcinoma (14). To date, Rb cDNAs have been isolated from two freshwater fish, trout (O. mykiss) and medaka (O. latipes), and recently from an estuarine species, mummichog (Fundulus heteroclitus) (15-17). Structural alterations in the coding region of the Rb gene in methylene-chloride-induced O. latipes liver tumors have also been reported (18) and include point mutations and a deletion. Such results suggest that the molecular etiology of fish hepatocellular carcinoma appears similar to that reported in humans. As such, Rb, in contrast to p53, may have an important role in fish tumorigenesis. After the experimental induction of liver tumors and subsequent evidence of Rb involvement in the tumorigenesis process using aquaria-held fish (18), the next step is to determine if this gene is similarly involved in the etiology of such tumors in feral fish. The marine flatfish dab (Limanda limanda) is currently used as a biomonitoring species in European coastal waters, and liver lesions have been observed in this species. Different prevalences occur according to the site sampled, with several sites in the U. K. National Marine Monitoring Program (NMMP) showing relatively high prevalence while other sites show negligible levels (19). This study describes the isolation of the Rb cDNA from L. limanda. The conservation of the Rb cDNA structure has also led us to investigate whether mutational alterations are involved in the etiology of environmentally induced tumors.

Materials and Methods Sample Collection. L. limanda were captured at NMMP sites in Cardigan Bay and Liverpool Bay during June and July of 2002 using 30 min tows of a standard Granton trawl. Upon landing, dab were immediately removed from the catch and placed into flow-through tanks containing aerated seawater. The sex, size (total length), and presence of external signs of disease were noted for each fish using methodology specified VOL. 39, NO. 24, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Degenerate PCR Primers and Specific PCR Primer Sets Used for the Amplification of the L. Limanda Rb cDNAa gene isolation: PCR primers 5′-ATCAAGGCTGARCCMASYCTG-3′ 5′-GTAGAAGACYATGATGGAGTC-3′ 5′-AGGCACAGTCAACTCTCAGG-3′ 5′-CCGCTCGAAGTCGTATGCAGGAGCAG-3′ 5′-ACCAGAGGTTTGAGAARACRTGC-3′ 5′-CCAACACCAACCAGGAGACCTT-3′

Rbfor Rbrev RbGSPfor RbGSPrev Rbfor2 RbGSPrev2 set

exon

1 2 3 4 5 6

11-16 15-17 17-19 19-21 20-22 21-23

mutation detection: gene-specific primers: forward primer 5′-CCTCCACAGACGCCAATCAGAGC-3′ 5′-CGGAAGAGAAGCGACTGTCTG-3′ 5′-TGGATGTGTTCAATCTCGCCGC-3′ 5′-TCTCAGGCTTCCTCTCACACTCC-3′ 5′-GCTGATGAGAGACCGTCACCTC-3′ 5′-GCGCTTCAAGACCATTGTCACGG-3′

28Sfor 28Srev RbEXPfor RbEXPrev a

degenerate forward primer degenerate reverse primer gene-specific forward primer gene-specific reverse primer degenerate forward primer gene-specific reverse primer reverse primer 5′-AAGTGATGTGTGGAATGTGG-3′ 5′-GCTCAGTGTGGGGTAGGCCTTG-3′ 5′-CTGGGAATCTGAGCAGGCGGCTG-3′ 5′-GCCGTGACAATGGTCTTGAAGCG-3′ 5′-GCCTGGTGGACGCATACTGTAGG-3′ 5′-CTGGAGCGAGGGGTCATGATAC-3′

Rb gene expression primers 5′-AATTGGCGTACGGAAGACC-3′ 5′-CATCTAGGCTAATCCGGCAC-3′ 5′-TCTCAGGCTTCCTCTCACACTCC-3′ 5′-GCCGTGACAATGGTCTTGAAGCG-3′

R ) A + G, M ) A + C, S ) C + G, and Y ) C + T.

by the International Council for the Exploration of the Sea (ICES) (20). Fish were sacrificed by a blow to the head, followed immediately by severing of the spinal chord. Upon opening of the body cavity, the liver was assessed for the presence of visible liver nodules according to the guidelines set out by Feist et al. (21). Liver nodules were then resected and fixed for 24 h in 10% neutral buffered formalin (NBF) before transfer to 70% industrial methylated spirit (IMS) for subsequent histological confirmation of the lesion type. In addition, samples of the same liver tumor and of surrounding nontumor tissue were resected and frozen immediately in liquid nitrogen and stored at -80 °C for the isolation of RNA and analysis of Rb mutations. Histopathology. Fixed samples were processed to wax in a vacuum infiltration processor using standard protocols (21). Sections were cut at 3-5 µm on a rotary microtome, and resulting tissue sections were mounted onto glass slides before staining with haematoxylin and eosin (H & E). Stained sections were analyzed by light microscopy (Eclipse E800, Nikon, Kingston upon Thames, U. K.), and the diagnosis of liver tumor type followed the ICES guidelines as detailed in (21) for tumors of the flatfish liver. Digital images of histological features were obtained using the Lucia Screen Measurement System (Nikon, U. K.). Isolation of Total RNA and First Strand cDNA Synthesis. Total RNA was extracted according to the supplier’s instructions (RNAeasy reagents, Qiagen Ltd., Crawley, U. K.). To remove any trace of genomic DNA contamination, total RNA (10 µg) was mixed with 5 µL of RNase free DNase (Promega U. K. Ltd., Southampton, U. K.), 10× reaction buffer, and nuclease-free dH2O to a total volume of 50 µL and incubated for 40 min at 37 °C. First strand cDNAs were synthesized using 1 µg of total RNA, oligo-dT primer and according to the supplier’s instructions (Invitrogen Ltd., Paisley, U. K.). After cDNA synthesis, 3 µL of the reaction was used as a template for subsequent polymerase chain reaction (PCR). Degenerate Reverse Transcriptase PCR. The Rb cDNA sequence was obtained using several primer pairs that generated three overlapping fragments. An initial Rb fragment was obtained using the following degenerate primer strategy. Sequences for the degenerate primers, used to yield a PCR product of 650 base pairs (bp), are included in Table 1. Amplification was performed with a BioRad iCycler in 50 µL reaction volumes using Invitrogen reagents. Amplification conditions were 35 cycles at 94 °C for 30 s, 46 °C for 30 s, 72 9786

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°C for 45 s, and using 0.5 units of Pfx taq polymerase (Invitrogen Ltd.). Approximately 18 µL of PCR product was analyzed using agarose gel electrophoresis (1% agarose, Tris, boric acid, EDTA buffer), and bands were excised and purified (Qiagen Ltd.). cDNA was ligated into a TA cloning vector (Invitrogen), and recombinant plasmids were transformed and selected using kanamycin LB plates. Plasmid was purified for sequence analysis (by MWG Biotech, Ebersberg, Germany). The sequence obtained from the cloned Rb fragment subsequently served as a starting point for gene-specific primer design and isolation of further Rb fragments as follows. A second Rb fragment of 800 bp, encompassing the 3′ end of the gene, was isolated using the following gene-specific primers RbGSPfor and RbGSPrev (Table 1). A third Rb fragment of 1.8 kilobase (kb), encompassing the 5′ end of the gene, was isolated using a combination of degenerate and gene-specific primers, Rbfor2 and RbGSPrev2 (Table 1). Screening and Characterization of Mutations in L. limanda Samples. Normal, hepatocellular adenomata, and a putative pancreatic tumor sample were characterized histologically using criteria described above. Total RNA was extracted from normal liver (n ) 10) and neoplastic liver (n ) 10) tissues as described previously. The Rb cDNA, encompassing exons 11-23 (comprising the functionally important binding domains), was amplified using reverse transcriptase polymerase chain reaction (RT-PCR) and dab Rb-specific primers (Table 1). PCR was conducted using a thermal cycler for 35 cycles using the following parameters: denaturation at 94 °C, 20 s; annealing at 60 °C, 20 s; elongation at 72 °C for 20 s. Reactions were performed in 50 µL total volumes using Invitrogen reagents. PCR products generated were analyzed on a 0.8% agarose gel for the presence of large mutational alterations (such as deletions). PCR products were then sequenced directly to identify and characterize any specific DNA changes. RT-PCR Analysis of Rb Gene Expression. Total RNA and first strand cDNAs were prepared from normal, adenoma, and pancreatic tumor samples as described previously, and 2 µL was used as a template for each RT-PCR expression study reaction. 28S rRNA expression was analyzed as an internal control. Approximately 1 µL of dab 28S rRNA primers, 28Sfor and 28Srev, were used (Genbank Accession No. Z18681). Approximately 1.5 µL of dab Rb primers RbEXPfor and RbEXPrev were used. PCR amplification conditions

TABLE 2. Sample Details for Dab Assessed for Rb Expression and Mutationsa fish ID

CEFAS AHD referenceb

sex

length (cm)

sampling site

BEQUALM diagnostic category

associated lesionsc

1 2 3 4 5

Ra02050-2 Ra02050-72 Ra02051-11 Ra02051-26 Ra02051-29

M M F F F

19 20 22 25 24

Inner Cardigan Bay Liverpool Bay Liverpool Bay Liverpool Bay Liverpool Bay

HA HA HA HA PCC

NEC, MMA, INF, REG NEC, MMA, INF

a For diagnostic criteria for BEQUALM liver lesion categories, refer to ref 20. b Centre for Environment, Fisheries and Aquaculture Science (CEFAS) Aquatic Health Database (AHD) sample reference number. c Additional lesions observed within the “normal” portion of the sample. Key: HA, hepatocellular adenoma; PCC, pancreatic cell carcinoma; NEC, necrosis; MMA, melanomacrophage aggregates; INF, inflammation; REG, regenerating hepatocytes.

FIGURE 1. Liver tumors in dab (L. limanda). (A) Gross pathology of hepatocellular adenoma. (B) Histopathology of tumor shown in part A. Adenoma (asterisk) compresses surrounding parenchyma. Flattened blood vessels are present at the interface (arrows). Scale ) 500 µm. (C) Gross pathology of pancreatic cell carcinoma. (D) Histopathology of tumor shown in part C. Tumor is composed of putative pancreatic cells (asterisk). Some compression at periphery (arrows) in some regions. Multifocal nature of tumor led to appearance of scattered pancreatic cells in hepatocellular parenchyma. Scale ) 500 µm. were: 35 cycles of denaturation at 94 °C, 20 s; annealing at 60 °C, 20 s; elongation at 72 °C for 20 s. This yields a fragment of 310 bp for Rb and 175 bp for 28S rRNA. Results were summarized as relative levels of expression among tissues. RT-PCR reactions were electrophoresed through 0.8% agarose gels and photographed.

Results Histopathology of Liver Tumors. Histopathological liver lesions in dab were characaterized according to criteria set out in ref 21. Details of lesions present within the individual

specimens are given in Table 2. Lesions in samples 1-4 were classified as hepatocellular adenoma due to their defined nodular form, thickened trabecular structure, relative lack of melanomacrophage aggregates, dilated blood vessels, and relative absence of atypical nuclear and cellular profiles (Figure 1). Lesion 5 was tentatively classified as the rare lesion, pancreatic cell carcinoma (Figure 1). In samples 1 and 2, nonspecific pathologies were also recorded in the nontumorous tissue. This included the presence of necrotic and inflammatory foci, melanomacrophage aggregates, and regenerating hepatocytes (see ref 21). Sample 1 harbored an VOL. 39, NO. 24, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Predicted amino acid sequence of the L. limanda Rb protein and comparison with those of the human (Homo sapiens) (12), medaka fish (O. latipes) (16), and the estuarine species mummichog (F. heteroclitus) (17) Rb proteins. Dashes represent gaps introduced to align the three sequences. Areas where all sequences match are represented by an asterisk. The functionally conserved binding domains A and B are boxed and highlighted. The triangles highlight sites of polymorphic variation. The closed circles highlight sites of mutational damage that change the amino acid residue. The open circles highlight sites of mutational damage that change the amino acid residue in medaka fish hepatocellular carcinoma samples (18).

TABLE 3. Summary of DNA Alterations in the L. limanda Rb cDNAa sample no.

DNA alteration

F45, F258, F257, F256 F45, F258, F257, F256

GfT GfT

sample no. 22N 21T, 2N, 26T, 49T, 22N, 25N, 50N 21T 1T, 21T, 2N, 26T, 49T, 22N, 25N 2N, 49T, 54N 1T, 21T a

normal liver samples position (in dab/human gene) exon 20 (codon 502/662) exon 20 (codon 528/688)

liver samples that displayed tumor cells DNA alteration position (in dab/human gene) CfA CfT GfA GfT AfT

exon 17 (codon 353/515) exon 17 (codon 358/520) exon 17 (codon 371/533) exon 20 (codon 528/688) exon 21 (codon 548/708)

putative consequence silent/polymorphic variation silent/polymorphic variation

putative consequence Pro f His silent/polymorphic variation Glu f Lys silent/polymorphic variation Met f Leu

Abbreviations: T, tumor sample; N, histologically “normal” cells (dissected in proximity to the same tissue that displayed a tumor).

infection by the microsporidian parasite Glugea stephani. Foci of cellular alteration (FCA) were not observed in the “normal” parenchyma of any of the specimens observed. Isolation and Sequencing of a cDNA Clone Encoding L. limanda Rb. To isolate the Rb gene, a combination of degenerate primers, gene-specific primers, and RT-PCR techniques were used to amplify an initial 650 bp fragment and two subsequent 800 bp and 1.8 kb fragments. The L. limanda partial Rb cDNA isolated encodes a 753 amino acid protein (Figure 2, GenBank Accession No. AY973250). Characterization of Mutations in Environmentally Induced L. limanda Tumors. The L. limanda tumor samples examined included several mutational alterations within the Rb cDNA sequence, including silent and missense mutations 9788

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(Table 3). No mutations were detected in the normal liver samples (Table 3). RT-PCR Analysis of Rb Gene Expression. Rb expression studies reveal that this gene is expressed in all tissues, with variations between individuals from low to medium expression in histologically normal tissues and comparatively high expression in the majority of tumor tissues (Figure 3).

Discussion Here, we report the L. limanda cDNA sequence for the cancer gene Rb. The partial Rb cDNA isolated encodes a 753 amino acid protein displaying a high identity with the human Rb, 58% (GenBank Accession No. M33647) (12), and fish counterparts, 76% with O. latipes (GenBank Accession No.

FIGURE 3. Expression analysis of the Rb gene in tissues recovered from adult dab. Key to samples: lanes 1-8, all normal liver tissues from different individuals; lane 9, histologically normal sample dissected from pancreatic tissue displaying a malignant tumor in the near vicinity; lane 10, pancreatic malignant tumor tissue; lanes 11, 12, 14, and 15, all hepatocellular adenoma benign tissues from different individuals; lanes 13 and 16-18, all histologically normal samples dissected from liver tissues displaying hepatocellular adenoma in the near vicinity. AY008289) (16), 78% with F. heteroclitus (GenBank Accession No. AAS80140), and 73% with O. mykiss (GenBank Accession No. AAD13390) (15). Many of the human pRb structural motifs are observed with high homology. The most conserved regions include two binding domains (shown boxed in Figure 2), which incorporate an LxCxE motif binding loci (chain A, amino acids 487-614, is 78% similar to human; chain B, amino acids 223-413, 61% similar). Other conserved structural regions of functional importance are a leucine zipper (amino acids 504-525), a nuclear targeting site (amino acids 697-713) (22), CDK2 kinase phosphorylation sites (amino acids 188, 205, and 638) (15), and a myc binding domain (proline-rich area amino acids 618-671). Several single nucleotide polymorphism (SNP) variants were detected within the coding region of the dab Rb gene. Polymorphisms and variants within the Rb gene have been reported in a number of human (23-25) and fish samples (18), including missense variants (26). High polymorphic variation is considered an indication of genomic instability. In many human cancers, SNPs, including several in the Rb gene (27), are important factors in determining individual susceptibility and may potentially explain population differences in liver tumor incidences in fish that are observed in the field. Other factors identified in mammalian models that are involved in tumorigenesis, such as methylation status, have yet to be determined in any fish species and are the subject of our future studies. Sequence analysis of dab tumor samples has revealed three point mutations and suggests that Rb involvement may also be important in fish tumorigenesis. The mutations detected are within the structurally conserved binding domains of the deduced Rb protein and within a similar exonic range (exons 17-21) as those detected previously in chemically induced medaka fish hepatocellular carcinoma samples (18). Mutational spectra, obtained using fish, have been linked with classes of environmental inducing agents and are available in the literature. Of those observed in this study, G to T is the predominant difference observed and can be caused by hydrocarbons such as benzo[a]pyrene (28), but in these instances where the amino acid residue is not affected, they may represent polymorphic variants rather

than environmentally induced lesions; A to T lesions are also linked with hydrocarbons (25); G to A lesions are caused by nitroso and alkylating compounds (29, 30); C to A lesions are oxidative damage related (31). Mutations that disable the Rb pathway are common in mammalian cancers (6). The spectra of mutations in the human Rb gene are well documented (http://rb1-lsdb.dlohmann.de/). There are several similarities between the pattern of mutational alterations in the human and fish Rb gene worthy of mention. First, exons encompassing the two conserved binding domains, that is, exons 12-18 and 20-23, are preferentially altered. Second, no mutations were detected in exons 24-27 of the L. limanda Rb gene, consistent with human and medaka tumor samples and suggesting that mutations at the 3′-terminal end of the Rb gene may not be oncogenic. Changes in Rb gene expression are also associated with the development of mammalian cancers. The differences in dab Rb expression among histologically normal individuals may reflect the extent to which those specific tissues were undergoing mitotic division. This is consistent with the normal role of the Rb protein acting as a negative regulator of the normal cell growth cycle and attempting to limit propagation of potentially deleterious mutations. Likewise, the comparatively high levels of Rb expression in many of the tumor samples may correlate with the high mitotic growth occurring in neoplastic cells that have undertaken the first stages of carcinogenesis and unregulated cell growth. In terms of ecotoxicological relevance, there are two important points. First, disruption of cell cycle regulating genes, such as Rb, might predispose individuals to environmentally (either chemical-contaminant-induced or UVinduced) cancers. This is a consequence of Rb mutational damage reducing the ability of cells to repair chemically or UV-induced DNA damage and may lead to a mutator phenotype within fish populations. This may, in part, explain why some populations of dab, such as those at Cardigan Bay, U. K., are more susceptible to contaminant-induced neoplasia. Second, if Rb mutations are a common early event in fish tumorigenesis, preceding histopathogical cellular damage, then their detection could potentially lend itself as a biomarker of contaminant-induced genotoxic damage. VOL. 39, NO. 24, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Acknowledgments This work was funded by a Natural Environment Research Council funded studentship to Frances Du Corbier.

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Received for review July 14, 2005. Revised manuscript received October 18, 2005. Accepted October 20, 2005. ES051367C