Isolation and characterization of the retinoblastoma protein from fish

Comparitive Biochemistry and Physiology Part B 130 Ž2001. 385᎐391

Isolation and characterization of the retinoblastoma protein from fish J.M. Rotchell a , B. Scogginsb, J.B. Blair b, G.K. Ostrander a,U a

Department of Biology and Di¨ ision of Comparati¨ e Medicine, Johns Hopkins Uni¨ ersity, 3400 North Charles Street, Baltimore, MD 21218, USA. b Department of Biochemistry and Molecular Biology, 246 Noble Research Center, Oklahoma State Uni¨ ersity, Stillwater, OK 74078, USA. Received 29 March 2001; received in revised form 4 July 2001; accepted 5 July 2001

Abstract The retinoblastoma Ž Rb . gene represents the first tumor suppressor gene characterized. The encoded protein, pRb, plays a crucial role in cell cycle control, preventing malignant cell proliferation. Recently, homologues of the Rb gene have been isolated in fish and the pocket domain, which is central to Rb function, was conserved. In our studies, using coelocanth Ž Latimeria chalumnae), rainbow trout Ž Oncorhynchus mykiss., medaka Ž Oryzias latipes. and English sole Ž Parophrys ¨ etulus., we have developed a simple protocol for the isolation of the Rb tumor suppressor protein and determined its’ tissue and cellular localization. Fish Rb proteins display apparent molecular weights in the range of 100᎐110 kDa, similar to the human pRb. The protein was detected in all tissues examined, consistent with the proteins’ universal role in cellular signalling. An interesting pattern of immunoreactive bands was detected in each of the cells’ two main compartments, suggesting differential proteolysis. Immuno-analysis of the pRb in trout liver tumor material revealed an additional Rb reactive product that was absent in normal liver cell extracts. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Fish; Medaka; Rainbow trout; English sole; Coelocanth; Retinoblastoma; Liver; Tumor; Hepatocyte; Antibody

1. Introduction The retinoblastoma gene Ž Rb. was the first tumor suppressor gene to be characterized ŽFriend et al., 1986.. Among humans, the loss of function of the Rb gene occurs by mutation and deletion.

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Corresponding author. Tel.:q1-410-516-8215; fax: q1410-516-4100. E-mail address: [email protected] ŽG.K. Ostrander..

Loss of function of the encoded gene product, pRb, results in a diverse set of cancers, including osteosarcoma ŽFriend et al., 1986., lung cancer ŽHarborn et al., 1988., breast cancer ŽT’Ang et al., 1988. and bladder cancer ŽIshikawa et al., 1991., as well as a high proportion of retinoblastomas ŽHorowitz et al., 1990.. Consequently, considerable research effort has been directed towards understanding the role of the Rb gene and its gene product, pRb, in various types of cancer. To date, Rb cDNAs have been isolated and

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J.M. Rotchell et al. r Comparati¨ e Biochemistry and Physiology Part B 130 (2001) 385᎐391

sequenced from a variety of species including: human ŽFriend et al., 1986; Fung et al., 1987; Lee et al., 1987., mice ŽBernards et al., 1989., chicken ŽFeinstein et al., 1994; Boehmelt et al., 1994., salamander ŽTanaka et al., 1997., Xenopus ŽDestree et al., 1992., rainbow trout Ž Oncorhynchus mykiss. ŽGenBank Accession Number: AF102861; Brunelli and Thorgaard, 1999. and medaka Ž Oryzias latipes. ŽRotchell et al., 2001a,b.. We previously reported on the characterization of a medaka Rb homologue ŽGenBank Accession Number: AY008289.. Rb cDNAs span 3.8᎐4.8 kb and have single open reading frames that encode proteins of 928 Žhuman., 921 Žmouse., 919 Žchicken., 936 Žsalamander., 899 Žfrog., 910 Žtrout. and 909 Žmedaka. amino acids ŽFriend et al., 1986; Bernards et al., 1989; Feinstein et al., 1994; Tanaka et al., 1997; Destree et al., 1992; Brunelli and Thorgaard, 1999.. An additional smaller sized mRNA transcript, of approximately 2.8 kb, has also been identified in adult mouse testes ŽBernards et al., 1989., though its function is not known. Rb genes have also been identified in plants, further highlighting their universal importance across species ŽGenBank accession numbers: AB015221, Nicotiana tabacum; U52099, Zea mays.. In vertebrates, the principle encoded Rb gene product is a Mr 105,000, nuclear phosphoprotein that regulates normal cell cycle progression by binding with transcription factors and interacting with kinases ŽWeinberg, 1995; Knudson and Wang, 1997.. In addition to its cell cycle gatekeeping role, the retinoblastoma protein is also thought to play an important role in the regulation of cell apoptosis ŽHaas-Kogan et al., 1995.. A number of investigations using animal models have been conducted in order to determine the significance of Rb gene mutations, associated altered expression and corresponding cellular effects of the altered protein ŽLee et al., 1992; Jacks et al., 1992; Clarke et al., 1992.. There are limitations however associated with studying pRb, especially its role in retinoblastoma tumor formation, using rodent models since tumors cannot be induced with chemical carcinogens. Studies have since demonstrated that these tumors can be induced experimentally in fish using the compound methylazoxymethanol acetate ŽOstrander et al., 1992.. Thus, a unique opportunity may exist to examine the etiology of this form of malignancy using a fish model.

Described herein is a simple isolation method for the pRb protein from trout liver cells as well as a comparison of the results using affinity chromatography. The presence of the pRb in several species of evolutionarily primitive through advanced fish, including coelocanth, rainbow trout, medaka and English sole was investigated. The isolated pRb from two species, trout and medaka, were then analyzed using Western blot and immunotechniques to determine their size, tissue and cellular localization.

2. Materials and methods 2.1. Fish Medakas Ž Oryzias latipes. were from our breeding colony. Fish were maintained on a 12:12 Žlight:dark. illumination cycle in a 37᎐l aquaria with overhead floursecent lighting. Water temperature was maintained at 23 " 1⬚C and individual tank filtration and aeration was accomplished with biological sponge filters. Medakas were fed TetraMin Flake food and either live or frozen brine shrimp twice daily. Rainbow trout Ž Onchorynchus mykiss. were obtained from the Norfolk Fish Hatchery ŽMountain Home, AR. and maintained in a 900᎐l Living Stream Aquaria at 12 " 1⬚C on a 12:12 Žlight:dark. illumination cycle. Fish were fed Purina Trout Chow twice daily. English sole Ž Paropyhrys ¨ etulus) were collected from Puget Sound, WA in relatively pristine water near Bainbridge Island by otter trawl. Livers were immediately excised after capture, frozen, stored in liquid nitrogen, transferred to the laboratory, and stored at y80⬚C until analyzed. Frozen coelacanth Ž Latimeria chalumnae. liver tissue was obtained from Dr. George Brown, School of Fisheries, University of Washington. 2.2. Isolation of the trout retinoblastoma protein All procedures were carried out at 4⬚C unless specified otherwise. Rainbow trout livers were dissected and frozen immediately in liquid nitrogen. An aliquot of liver Žpooled from five fish. was homogenized in 5 volumes of lysis buffer Ž10 mM N-w2-hydroxyethylxpiperazine-N⬘w2-ethanesulfonic acidxŽHEPES., pH 7.5., 0.15 M NaCl, 10 mM ethylene glycol-bis-tetraacetic acid ŽEGTA., 1

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␮grml aprotinin in phosphate buffered saline ŽPBS., 1 ␮grml phenylmethylsulfonyl fluoride ŽPMSF. in isopropanol. for 30 min. Homogenates were filtered using cheesecloth, centrifuged at 10 000 = g for 30 min and the resulting supernatant stored at y80 o C until analysis. Four affinity CNBr-activated Sepharose 4-B resins ŽAmersham Pharmacia Biotech. were prepared containing no ligand or one of the following three ligands: a synthesized peptide ŽGGELLCGEGG. containing the Rb binding LxCxE motif Žprepared by the Peptide Facility, Oklahoma State University., a commercial anti-human Rb monoclonal mouse antibody ŽRb-mAb-1, supplied by Oncogene Science ., and a blocking molecule ethanolamine. Human leukemia myeloma K562 cells were used as a positive control. Cells were harvested, washed using saline solution, pelleted and stored at y80 o C until lysed. The lysis was carried out using lysis buffer for 30 min. Cell debris was removed by centrifugation at 1000 = g for 15 min. Resin binding and washing strategies were as follows. Supernatant Ž50᎐100 mg mly1 protein. and resin Ž10:1 vrv. were incubated for 1 h with gentle mixing and then centrifuged briefly at low speed to remove non-bound protein. Resins were then subjected to several successive washing strategies using the following buffers: HEPES saline buffer Ž10 mM HEPES, pH 7.5; 0.15 M NaCl., 2% sodium dodecyl sulphate ŽSDS. at room temperature, HEPES saline buffer and ␤mercaptoethanol Ž1 mM.. The eluted fractions obtained after each washing step were retained and analyzed on Western blots. Protein concentrations were measured according to the Lowry method using bovine serum albumin as a standard ŽLowry et al., 1951.. 2.3. Western blot analysis Samples were diluted Ž2:3 vrv. in gel loading buffer Ž0.125 M Tris, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.01% bromophenol blue. and separated on a 9% SDSPAGE gel for 3 h at varying voltage. Commercial molecular weight standards ŽSigma. were also diluted Ž1:10 vrv. in loading buffer and run in parallel with samples. Proteins were electroblotted for 1.5 h at 25 V, 200 mA to a PVDF membrane ŽImmobilon-P, Millipore.. Following transfer, lanes containing molecular weight stan-

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dards and samples were stained with Coomassie brilliant blue or soaked overnight in blocking buffer Ž25 mM Tris, pH 8.0; 0.125 mM NaCl; 0.1% Tween 20; 4% bovine serum albumin. respectively. Membranes were immunolabelled with 250 ␮l of a 100 ␮g mly1 stock of monoclonal antibody against an epitope of amino acids 300᎐380 of the human pRb ŽOncogene Science, Rb-mAB-1., a secondary antibody Žalkaline phosphatase-conjugated rabbit anti-mouse IgG. ŽJackson ImmunoResearch Laboratories, Inc.. and detected with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium ŽSigma.. 2.4. Subcellular localization studies Homogenous fractions Ž) 99% purity. of rainbow trout hepatocytes were obtained by our previously described methods ŽOstrander et al., 1995.. Isolation of liver cell nuclei was conducted at 4⬚C. Minced trout liver Ž2᎐3 g. or enriched trout hepatocyte fractions Ž3᎐5 ml. were homogenized in five volumes of homginization solution Ž0.25 M sucrose᎐10 mM HEPES, pH 7.5., gravity filtered through cheesecloth and successively washed four times in 10 vols. of homogenization solution followed by centrifugation Ž500 = g ..

3. Results and discussion In this study we describe the isolation and localization of fish Rb proteins. To optimize isolation of the pRb from trout, cell extracts were separated using three resin-ligand complexes and resin alone. Immunoanalysis of Western blots incubated with a commercial monoclonal antibody Rb-mAB-1, revealed multiple immuno-reactive bands ŽFig. 1A.. Predominant product band sizes of approximately 105 kDa and 50 kDa were obtained. A range of additional products of approximately 28 kDa, 40 kDa and 50᎐60 kDa were also obtained. The pattern of immunoreactive products obtained was similar when using either the LxCxE motif ŽFig. 1A, lane 1. or the blocking agent ŽFig. 1A, lane 2. bound sepharose resins, suggesting that there is no actual isolation advantage in employing the conserved LxCxE motif liganded resin. The Rb-mAb-1 liganded resin appeared to bind more of the 50 kDa product compared with the former two resins ŽFig. 1A, lane 3.. The primary antibody used for the detection of pRb in

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Fig. 1. Immunodetection of trout and human Rb proteins. The trout and human protein extracts were mixed with ligand-resin complexes in separate experiments. The SDS eluted fraction was separated through 9% SDS-PAGE and transferred to a PVDF membrane. Blots were incubated with a commercial monoclonal antibody Rb-mAB-1 ŽOncogene Science., a secondary antibody, and developed as described in the methods. Ža. lane 1, using trout protein extract and resin with the LxCxE ligand; lane 2, resin with a blocking agent ligand; lane 3, resin with a commercial anti-human Rb monoclonal mouse antibody ŽRb-mAb-1. ligand. Žb. lane 1, using trout protein extract and sepharose resin alone Žno ligand.; lane 2, molecular weight marker ŽSigma.. Žc. lane 1, using human protein extract and sepharose resin alone Žno ligand.; lane 2, molecular weight marker ŽSigma..

immunoanalysis possesses 21% homology Ž17 out of 80 amino acids. across 5 species Žhuman, chicken, frog, salamander and trout. for this antibody epitope. Each of the three predominant bands ŽFig. 1B. were selected using non-liganded sepharose alone as a isolation media, suggesting that non-liganded sepharose affinity properties alone are sufficient for the isolation of pRb products from crude protein extract samples. Though we can never be certain that the antibodies will not react with unrelated proteins in fish, the apparent sizes of pRb immuno-reactive bands in trout samples ranged from 29 kDa and 105 kDa ŽFig. 1A.. The size of the predominant band in trout samples is consistent with pRb sizes of 105, 104᎐110, 95, 99᎐103 and 103 kDa reported for human ŽHu et al., 1991.Žalso shown in Fig. 1C., mouse ŽBernards et al., 1989., chicken ŽFeinstein et al., 1994., Xenopus ŽDestree et al.,

1992., and salamander ŽTanaka et al., 1997. respectively. The significantly smaller bands Ž- 50 kDa. may alternatively represent proteolytic degradation or metabolized products. Degraded pRb, with a fragment of approximately 60 kDa, has previously been observed in retinoic acid- or phorbol ester ŽTPA.-treated cells ŽFu et al., 1998.. A pRb cleavage product of approximately 68 kDa has also been observed during apoptosis in drugtreated human leukemic cells ŽAn and Dou, 1996.. The instability of pRb in mammalian models has thus been implicated in the loss of tumor suppressor function ŽChen et al., 1992. and also the initiation of the apoptotic process ŽJanicke et al., 1996.. It seems likely that the smaller fragments also represent proteolytic degradation or cleavage products in these fish samples, similarly to that reported for mammals. Immunoblotting of total protein extracts from

Fig. 2. SDS-PAGE analysis and immunodetection of Rb proteins in Ža. diverse fish species and Žb. tissue localization. Crude protein extracts were isolated and analyzed from whole medaka Ž2a, lane 1., coelocanth Žlane 2., trout Žlane 3., and English sole Žlane 4.. The analysis was repeated using protein extracts from dissected samples of medaka eggs Ž2b, lane1., eye Žlane 2., liver Žlane 3., viscera Žlane 4., brain Žlane 5., and muscle Žlane 6..

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Fig. 3. Immunodetection of Rb protein in normal and tumor samples isolated from trout liver. Crude protein extracts were isolated and analyzed from normal trout liver Žlanes 1 and 4.; liver displaying tumor Žlane 2.; and liver tumor cultured cells Žlane 3..

individual adult coelocanth, rainbow trout, medaka, and English sole revealed that pRb is present in each of the fish species examined ŽFig. 2A.. The size of the main immunoreactive band ranged from approximately 100 kDa in sole to approximately 110 kDa in coelocanth ŽFig. 2A.. Furthermore, pRb immunoreactive bands were present in each of the tissues examined ŽFig. 2B.,

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consistent with the proteins’ universal role in cellular signalling. However, the patterns of immunoreactive band sizes varied however among different tissue samples. The 100 kDa band predominates in egg, eye, liver and brain tissue extracts and the 50 kDa bands produced a stronger immunoreactive signal in the viscera and muscle samples ŽFig. 2B.. The actual cause and implications of this pattern of pRb product distribution throughout the fish tissues analyzed are not known, though it would appear that the rate of proteolytic degradation of the pRb is greater in the fish muscle and viscera samples. Altered patterns of predominant bands present were also observed for extracts derived from tumor material, compared with normal trout liver samples ŽFig. 3.. An additional band of approximately 35 kDa band ŽFig. 3, lane 2. was detected in tumor tissue. This extra band was absent in normal liver samples ŽFig. 3, lanes 1 and 4.. The extra immunoreactive band detected in the tumor sample may represent alternate metabolized or proteolytic degradation products of pRb proteins that, in turn, are encoded by mutated Rb genes. Our results suggest that fish pRb, as an altered form of the protein, may be involved in the development of carcinogenesis in the liver tumor samples investigated. Other research in our laboratory, involving a mutational analysis of the medaka Rb cDNA in chemically-induced liver tumor samples, confirmed the presence of inappropriate termination codons and, thus, potentially truncated Rb proteins ŽRotchell et al., 2001a..

Fig. 4. Cellular localization of pRb protein within trout liver cells. Lane 1, molecular weight marker. Immunodetection of Rb protein in total cell homogenate Žlanes 2᎐4., nuclear extracts Žlanes 5᎐7., and cytoplasmic extracts Žlanes 8᎐10..

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The cellular distribution of the pRb immunoreactive bands was also analyzed ŽFig. 4.. Two immunoreactive bands of approximately 47 kDa and 50 kDa were detected in total trout liver cell extracts Fig. 4, lanes 2᎐4.. On closer examination of the two main cell compartments, the nuclear and cytoplasm extracts contained the smaller sized immunoreactive band of 47 kDa ŽFig. 4, lanes 5᎐7. and the larger 50 kDa ŽFig. 4, lanes 8᎐10. products respectively. Immunoreactive bands may represent products of pRb proteolytic degradation. Recent studies have suggested that differential degradation of pRb by SPase; a relevant nuclear and cytoplasmic proteolytic enzyme, may play a role in regulation of the cell cycle ŽFu et al., 1998.. In summary, we have developed a simple protocol for isolating pRb from fish tissue. This protocol should benefit researchers interested in using fish as substitutes for rodent models when investigating various aspects of pRb production.

Acknowledgements These studies were funded by grants from the United States Biomedical Research and Development Command ŽDAMD17091᎐C᎐1079. and the National Institutes of Health ŽCA54950, CA58818 and RR07077. to GKO and JJB and a Research Assistantship from the Environmental Institute at Oklahoma State University ŽBS.. References An, B., Dou, Q.P., 1996. Cleavage of retinoblastoma protein during apoptosis: an interleukin 1␤-converting enzyme-like protease as candidate. Cancer Res. 56, 438᎐442. Bernards, R., Schackleford, G.M., Gerber, M.R., Horowitz, J.M., Friend, S.H., Schartl, M., Bogemann, E., Rapaport, J.M., McGee, T., Dryja, R.P., Weinberg, R.A., 1989. Structure and expression of the murine retinoblastoma gene and characterization of its encoded protein. Proc. Natl. Acad. Sci. USA 86, 6474᎐6478. Boehmelt, G., Ulrich, E., Kurzbauer, R., Mellitzer, G., Bird, A., Zenke, M., 1994. Structure and expression of the chicken retinoblastoma gene. Cell. Growth Differ. 5, 221᎐230. Brunelli, J.P., Thorgaard, G.H., 1999. Sequence, expression and genetic mapping of a rainbow trout retinoblastoma cDNA. Gene 226, 175᎐180.

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