Multiple protein differences distinguish clam leukemia cells from normal hemocytes: evidence for the involvement of p53 homologues

Comparative Biochemistry and Physiology Part C 129 Ž2001. 329᎐338

Multiple protein differences distinguish clam leukemia cells from normal hemocytes: evidence for the involvement of p53 homologues Raymond E. Stephens a,c,U , Charles W. Walker b, Carol L. Reinisch c a

b

Department of Physiology and Biophysics, Boston Uni¨ ersity School of Medicine, Boston, MA 02118, USA Marine Biomedical Research Group, Department of Zoology, Uni¨ ersity of New Hampshire, Durham, NH 03824, USA c Laboratory of Aquatic Biomedicine, Marine Biological Laboratory, Woods Hole, MA 02543, USA Received 12 February 2001; received in revised form 6 May 2001; accepted 7 May 2001

Abstract In coastal locations, marine invertebrates, primarily molluscs, develop fatal leukemias in their blood or hemolymph. In the clam Mya arenaria, non-adhesive, mitotic, spherical leukemia cells replace adhesive, motile, normal hemocytes as leukemia progresses. End-stage leukemia cells express a unique antigen, IE10, while normal cells express the 2A4 marker. The goals of this work were to further differentiate the normal and leukemia specific antigens relative to protein structure, determine if other protein distinctions exist, and examine p53 gene family expression in both cell types. Recognized by the monoclonal antibody 2A4, normal cells express a 185-kDa glycoprotein that may have multiple forms. Detected by the monoclonal antibody 1E10, leukemic cells express a very hydrophobic 252-kDa glycoprotein that is likely to be a transmembrane protein with spectrinrdystrophin-like characteristics. After normalization to the major cytoskeletal protein actin, sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals major distinguishing protein and glycoprotein differences between the two cell types. Most obvious is the near-absence of tubulin in the non-mitotic normal hemocytes. We have also characterized the expression of p53 gene family members in normal and end-stage leukemia cells, finding shifts in expression of the p53 gene homologues p73 and p97 coincident with leukemia-specific protein synthesis. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Leukemia; Cell-surface glycoproteins; p53; p73; p97; Tubulin; Mya arenaria

1. Introduction Leukemia in the soft shell clam Mya arenaria has been well documented among molluscan populations along the US East Coast ŽBrown et al., 1977.. In unaffected clams, circulating hemocytes are present in low density, resemble fibroU

Corresponding author: Tel: q1-508-2897373; fax: q1508-5401902. E-mail address: [email protected] ŽR.E. Stephens..

blasts, and are characteristically adhesive. In leukemic clams, the hemocytes are supplanted by a dense population of mitotic, non-adhesive cells, which are agranular, round and have a near-equal nuclearrcytoplasmic volume ratio. In severely affected clams, the disease invades the connective tissues and is lethal, particularly in summer months ŽCooper et al., 1982; Leavitt et al., 1990.. The etiology of leukemia in clams is unclear, but there is a reasonable correlation between occurrence and the presence of distinct environmental contaminants. For example, the highest

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incidence of the disease in Mya has been consistently found in New Bedford ŽMA. Harbor, a designated EPA Superfund site noted for its high levels of polychlorinated biphenyls ŽPCBs. and related industrial pollutants ŽReinisch et al., 1984.. On the other hand, clams from seemingly unpolluted sites may have the disease, albeit at lower frequency, leading to the suggestion that the disease is virally induced ŽOprandy et al., 1981; Oprandy and Chang, 1983.. In addition to morphologic and adhesive properties, other factors independently distinguish normal hemocytes from leukemia cells. Two murine monoclonal antibodies have been developed that allow immunologic classification. One, known as 1E10, detects a high molecular-weight antigen uniquely present on the cell surface of end-stage leukemia cells ŽMiosky et al., 1989.. The other, known as 2A4, detects a lower molecular-weight adhesion-related antigen on normal hemocytes and intermediate-stage cells ŽWhite et al., 1993.. These antibodies are invaluable for immunocytochemical screening and fluorescent cell sorting of normal and leukemia hemocytes from clams in environmentally impacted sites, but their use is limited antigenically to Mya. Although the 2A4 and 1E10 antigens of normal and leukemia cells were preliminarily Žbut ambiguously. identified by size ŽMiosky et al., 1989; White et al., 1993., further definition of these distinguishing cell surface proteins may lead to a better understanding of their function. We have now developed reproducible, high-resolution electrophoretic and immunoblotting techniques that permit quantification of these two key antigens. While doing so, we have discovered other diagnostic protein differences between the two extreme cell types, most significantly in the form of dramatic distinctions in the expression of tubulin and of members of the p53 gene family. The latter distinction is identical to that reported by Kelley et al. Ž2001. for p53 and p73, but our study reveals a new component, p97, as another potential reciprocal regulator of gene expression.

2. Materials and methods 2.1. Clams, hemocytes and hemocyte lysates Specimens of the soft shell clam Mya arenaria were collected during winter months at low tide

from sand flats in New Bedford Harbor at Fairhaven, Massachusetts. Clams were also obtained periodically from Seabrook, NH, and from uncontaminated Cape Cod, MA, locations. Hemolymph was drawn from the blood sinus surrounding the heart using a syringe with a 22-gauge needle, then centrifuged at 1000 = g for 5 min at 4⬚C to recover hemocytes. The pelleted hemocytes were resuspended in cold seawater and recentrifuged to remove any interstitial serum. The seawater was drawn off and the hemocyte pellets were extracted with approximately 10 volumes of freshly prepared 0.5 or 1% NP-40 in 3 mM MgCl 2 , 30 mM Tris᎐HCl ŽpH 8., 0.1% 2-mercaptoethanol, and 2.5 mM phenylmethylsulfonyl fluoride ŽPMSF. for 10 min on ice. The suspensions were centrifuged at 10 000 = g for 10 min and the membranercytosol supernatants were removed. Lysates from ) 30 clams from the three locations were analyzed by various combinations of the methods to follow. 2.2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses Five-fold concentrated SDS sample buffer, containing fresh 1% 2-mercaptoethanol and 2.5 mM PMSF, was added to the lysates and they were immediately heated to 100⬚C for 3᎐5 min. Occasionally, additional SDS was added to a final concentration of 2% wrv andror dithiothreitol ŽDTT. was added to a final concentration of 10 mM. Samples were routinely analyzed on smallformat Ž1.5 mm thick, 6 cm long. 8% Tr2.5% C polyacrylamide gels by the Laemmli Ž1970. method. For more specialized analyses, large-format Ž10 cm long. 5᎐15% Tr1% C gradient gels or 8% Tr1% C uniform gels were used ŽStephens and Prior, 1995.. The gels were stained with coomassie blue by the equilibrium method of Fairbanks et al. Ž1971., or transferred to nitrocellulose by the 3 mM carbonater10 mM bicarbonater20% methanol renaturation method of Dunn Ž1985.. The transfers were carried out in Hoefer TE-series or Bio-Rad Mini Trans-Blot bath-type units at a voltage gradient of 10 Vrcm for 2 h ŽHoefer. or 20 Vrcm for 1 h ŽBio-Rad., with cooling sufficient to maintain the electrolyte temperature below ambient. To verify transfer and judge quality, the blots were routinely rinsed with distilled water and stained with 0.1% ponceau

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S in 1% acetic acid ŽSalinovich and Montelaro, 1986.. After blocking with 1% BSA plus 5% nonfat dry milk in Tris-buffered saliner0.05% Tween-20 ŽTBST. and incubating for 1 h with primary antibody Žsee below., the immunoblots were developed with alkaline phosphatase-conjugated secondary antibody and the stable NBTrBCIP reagent ‘Western Blue’, both obtained from Promega. For glycoprotein analysis, blots were oxidized with 10 mM periodate, derivatized with biotin hydrazide, and the resulting biotinylated glycoproteins were visualized with alkaline phosphatase-conjugated streptavidin ŽBio-Rad Immuno-Blot 䊛 Kit for Glycoprotein Detection, 噛170-64900; based on Gershoni et al., 1984 and O’Shannessy et al., 1987.. Transmission and reflectance video densitometry were performed using the basic principles outlined by Haselgrove et al. Ž1985. and Stephens Ž1997.. Gel and immunoblot bands were quantified by determining the relative integrated density above background using the ‘fill’ feature of SigmaScan Pro 5.0 ŽSPSS Inc.. image analysis software, or by manual integration of scan profiles. 2.3. Amino acid composition and sequencing Samples of leukemia cell lysate were resolved by SDS-PAGE on large-format 5᎐15% Tr1% C gels, blotted to polyvinylidene difluoride ŽPVDF. membrane, lightly stained with 0.025% coomassie blue in methanolrwater Ž40:60., and destained with methanolrwater Ž1:1.. The 1E10 antigen band was excised from the blot and subjected to either acid hydrolysis for amino acid analysis, or to Edman degradation for N-terminal sequence determination ŽMatsudaira, 1987.. For internal sequence, the gels were briefly stained and destained; bands were excised from the gel, washed with 1:1 acetonitrilerwater, reduced, alkylated, and subjected to in-gel tryptic digestion. After extraction and reverse-phase HPLC separation, selected peptides were subjected to Edman degradation for direct internal sequence determination. In addition, microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry ŽLCrMSrMS. on a Finnigan LCQ quadrupole ion-trap mass spectrometer and database correlation was used to deduce internal sequence. Analytical procedures were performed by the Harvard Microchemistry Facility, Harvard

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University, Cambridge, MA. Sequences were evaluated via the Protein Information Resource ŽPIR; pir.georgetown.edu., using the Global and Domain Similarity Search and PatternrPeptide Match databases or by a BLAST search Žwww.ncbi.nlm.nih.govrblastr. with an e value of 10 000. 2.4. Primary antibodies The mouse monoclonal antibody ŽMAB. 1E10 is a ‘daughter’ clone of the leukemia-specific 1E11, characterized previously ŽMiosky et al., 1989.. Mouse MAB 2A4 is specific for normal hemocytes ŽWhite et al., 1993.. The rabbit polyclonal antibody Map53r73-23 was elicited against a 23aa synthetic peptide Ž ᎐ CACPGRDRKA DERGSLPPMVSGG᎐ ., the sequence of which includes part of the highly conserved domain V DNA binding region of p53rp73 from Mya arenaria ŽKelley et al., 2001.. A mouse MAB to chicken gizzard actin ŽN.350. was obtained from Amersham, while antibodies to chick brain ␣tubulin ŽDM-1A. and rat brain ␤-tubulin ŽTUB 2.1. were obtained from ICN. With the exception of MAB 2A4, which was used at dilutions of 1:25᎐1:200, other antibodies were routinely used at dilutions of 1:1000 or greater.

3. Results 3.1. Molecular weight identity of cell surface antigens recognized by MABs 1E10 and 2A4 These antigens, which immunologically distinguish adhesive normal cells from non-adhesive leukemia cells, have been reported with inconsistent molecular weights ŽMiosky et al., 1989; White et al., 1993.. To clarify this issue for analytical purposes, we resolved normal and leukemia cell lysates on large-pore Žlow cross-linked. gradient gels originally designed to separate and efficiently blot proteins of very high molecular weight ŽStephens and Prior, 1995.. Both types of cell lysate were run in parallel with established widerange molecular weight standards. After blotting and ponceau S staining, the standards were carefully marked, as were major reference bands in the lysates, and the blots were developed with the respective antibody. The numeric results of these analyses are illustrated in Fig. 1. Representative

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of standard molecular weight values, since their respective blot patterns for 1E10 and 2A4 were virtually identical to those of the present study. However, cross-reactive bands of lower molecular mass are observed Žcf. White et al., 1993., particularly at ; 155 kDa, and these may represent either natural degradation products of the original antigen, or precursors awaiting glycosylation Žsee below.. There is also some evidence of background aggregation ŽFig. 2E.. Thus, the 1E10 antigen is a 252-kDa entity that either does not easily unfold, or else readily refolds, sometimes running with a molecular weight of approximately 2r3 of its true value, due to a smaller retardation coefficient. This problem is easily corrected by adequate solubilization and complete reduction. The 2A4 antigen is maxiFig. 1. Molecular weight determination of the major 1E10 and 2A4 cross-reactive bands. The circles represent the mobilities of Bio-Rad Ž噛161-0317. broad-range molecular weight standards used to establish the least-squares calibration line. The diamond symbol is the intersection point for the mobility of the 1E10 antigen Ž Mr s 252 kDa.; the squares are the same for the major Ž185 kDa. and minor Ž155 kDa. 2A4 antigen. Average of three independent sample determinations Žexperimental range - "8 kDa..

immunoblots are illustrated in another context below ŽFig. 2A,E.. The major band recognized by MAB 1E10 has an extrapolated molecular mass of 252 kDa, similar to but higher than that reported by Miosky et al. Ž1989.. In aged samples, or samples with insufficient SDS or reducing agent, a diffuse band or pair of bands of lower molecular mass Ž; 140᎐160 kDa. is frequently observed, in addition to the 252-kDa major band Žcf. Fig. 2A.. Such a pattern is identical to that reported by White et al. Ž1993., for which molecular masses of 150 and 100 kDa were incorrectly assigned to the major and minor bands, respectively. The lower molecular-weight band is not the result of proteolysis, since a single 252-kDa band can be obtained by adding SDS and DTT to the sample, re-boiling, and then re-running the SDS-PAGE separation. The major band recognized by MAB 2A4 has a molecular mass of 185 kDa, somewhat higher than the 130 kDa reported by White et al. Ž1993., but migrating on their gel system between the major and minor 1E10 antigenŽs., which we establish above as 252 and ; 150 kDa, respectively. This discrepancy is likely due to a misapplication

Fig. 2. Comparative antibody and glycoprotein staining for the antigens of 1E10 and 2A4. Lanes A᎐D were from a blot of a SDS-PAGE 5᎐15% Tr1% C gradient separation of leukemia cell lysate. Lanes E and F were from a normal hemocyte lysate. Lane A: immunostained with MAB 1E10 with no pre-processing Žarrows mark the ; 160r140-kDa minor bands.. Lane B: control, stained for glycoprotein without periodate oxidation Žaldehyde and background.. Lane C: stained for glycoprotein; the 1E10-positive band is reactive, as is one at ; 140 kDa Žtriangle.. Lane D: periodate-oxidized and biotin hydrazide-derivatized, then immunostained for 1E10 to demonstrate little loss of signal. Lanes E and F were from a parallel blot of normal cell lysate. Lane E: stained with MAB 2A4; arrow marks position of a consistent but minor band. Lane F: stained for glycoprotein; the 2A4-positive band is reactive. Note the near-absence of the prominent ; 140-kDa glycoprotein detected in leukemia cells, but the additional glycoprotein bands of lower molecular mass Ž; 95 and ; 60 kDa..

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mally a 185-kDa entity, but there may be lower molecular-weight forms as well. This clear immunologic assignment of molecular weight to the major antigen now allows us to identify these bands on protein-stained blots and in simple SDS-PAGE profiles. 3.2. The glycoprotein nature of the 1E10 and 2A4 antigens The relatively diffuse immunoblot bands routinely observed for both of these antigens, coupled with the fact that they are cell-surface expressed, suggests that they may be glycosylated. This possibility was explored by periodate oxidation to aldehyde and subsequent biotin hydrazide derivatization. Typical results for the 1E10 and 2A4 antigens are illustrated in Fig. 2. One major glycosylated band coincides with the 1E10-positive band on its sister blot ŽFig. 2, lanes A, D vs. C.. Periodate oxidation followed by derivatization Žlane D. diminishes the antibody signal minimally Žno more than equivalent buffer incubationrwashing alone., suggesting that the carbohydrate moiety per se is not the primary antigen. Processed without periodate oxidation Žlane B., terminal aldehydes and background can be determined. The 1E10 antigen band is totally unreactive without periodate oxidation, as is the other major glycoprotein Ž; 140 kDa.. Similarly, a distinct glycosylated band coincides with the major 2A4 antigen ŽFig. 2, lanes E vs. F.. In contrast to the fully reduced 1E10 antigen, there are additional but weak 2A4-positive bands of both lower and higher molecular weight. Note also the differences in carbohydrate staining for the normal cell lysate, which, most significantly, is nearly lacking the prominent ; 140-kDa glycoprotein band observed in the leukemia cell lysate ŽFig. 2, lane F vs. lane C., but contains an additional minor band at ; 95 and a major band at ; 60 kDa. 3.3. Sequence information on the 1E10 antigen Immunologically, the 1E10 antigen behaves as a single protein. The fact that it can be readily visualized by conventional staining on both gels Žsee Fig. 3. and blots affords the opportunity for its further characterization. Blotting to a PVDF membrane allows direct compositional and Nterminal sequence analysis, while excision from a

Fig. 3. Coomassie blue-stained SDS-PAGE protein profiles of normal hemocyte and leukemia cell lysates, normalized to equal actin content. Upper panel: 8% Tr2.5% C conventional mini-gel. Lane A: lysate from pooled normal hemocytes. Lanes B᎐D: lysates from hemocytes of three individual leukemic clams taken from New Bedford Harbor. Arrows designate the 1E10 ŽB, upper arrow. and 2A4 ŽA, lower arrow. cross-reactive bands; N designates a high molecular weight protein prominent in normal cell populations. Lanes E᎐F: lysates of hemocytes from morphologically leukemic clams taken from an unpolluted Cape Cod area. The sample in lane E was IE10-negative, while that of lane F was 1E10-positive. Molecular mass standards Žleft edge. are 200, 116, 97, 66, 45 and 31 kDa, top to bottom. Lower panel: samples illustrated in lanes A᎐D, but resolved on an 8% Tr1% C gel with the high molecular-weight region magnified.

stained gel is useful for obtaining internal sequence information. Amino acid analysis, performed as a precedent to microsequencing, reveals that the 1E10 antigen is 5.3 mol.% in proline and 41 mol.% in the hydrophobic amino acids alanine, valine, leucine, isoleucine, and phenylalanine. Coupled with the known facts that the protein is both membraneassociated and surface-exposed ŽMiosky et al.,

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1989., these findings are consistent with a very hydrophobic, integral protein with low or no ␣helical content. Edman N-terminal analysis results in the sequence GTDQAARYIGA᎐. A global and domain similarity search of this sequence suggests a TM1 transmembrane domain Ž86% identity in a 7-aa overlap. and IMM immunoglobulin or AMT1 acyl carrier protein domains Ž70% identity in a 10-aa overlap for each.. A pattern search suggests a potential N-myristoylation site. Edman analysis of the most prominent internal peptide separated from an in-gel tryptic digest reveals the sequence ᎐TCQADGQLDGTSK᎐, which has only minimal similarity to any current database proteins. This sequence shows 67% identity in a 9-aa overlap to an FH6 complement factor H repeat, and 64% identify in an 11-aa overlap to the FH2 complement factor H repeat. A BLAST search indicates an IgM heavy-chain constant region domain similarity Ž63% identity in an 11-aa overlap.. The relative uniqueness of this peptide sequence renders it highly suitable for degenerate primer design, required for future cloning of the 1E10 gene. Four other internal peptides carry remarkably consistent information. Database correlation to mass spectra reveals the sequence ᎐LLDSYDLQR᎐ for a second peptide. This 9-amino acid peptide represents amino acids 1283᎐1291 in the spectrin alpha chain of Drosophila and has 100% identity in a 6-aa overlap to the SP12 spectrinrdystrophin repeat. Mass spectra correlation with partial Edman data from a third peptide yields the sequence ᎐DAPTYQLYV᎐. This sequence shows 86% identity in a 7-aa overlap to the procollagen alpha repeat ŽPARP. and 83% identity in a 6-aa overlap to the WW repeat homology, a family that includes dystrophin. Edman analysis of a fourth peptide, confirmed by mass spectra and evaluated via BLAST, yields the sequence ᎐ALDYYLLR᎐, while a fifth peptide yields the sequence ᎐QVANSIAQLVK᎐. Respectively, these show 87% identity in an 8-aa overlap and 72% identity in an 11-aa overlap to two different regions of mouse and human talin, a protein similar in function to spectrin and dystrophin. 3.4. Protein profiles of normal and leukemia cells Hemolymph from leukemia cells will typically

yield several orders of magnitude more cells than normal cell hemolymph Ž6 = 10 8rml vs. 1᎐6 = 10 6rml for normal.. However, even compensating for relative pellet volume, the former often contains more total protein than the latter. Consequently, we routinely normalize samples for actin content Žverified by antibody cross-reactivity ., which, coincidentally, also normalizes them for a prominent, unidentified 205-kDa protein. When such normalized lysates are run for survey purposes on either normal Ž2.5%. or low Ž1%. cross-linked 8% Žuniform. SDS-PAGE small-format gels, the 1E10 and 2A4 antigens are readily detected in a diagnostic fashion, as illustrated with a series of samples in Fig. 3. Furthermore, a characteristic 270-kDa protein ŽN. is detected mainly in normal cell lysates. Major differences in several other bands of molecular weight lower than the 2A4 antigen can also be detected. This is illustrated more quantitatively in Fig. 4, which shows corresponding densitometric scans obtained from a 5᎐15% gradient small-format gel, designed to expand the high molecular-weight region and compress the low Žcf. Fig. 1.. In this quantification, the equality between samples for both actin and the 205-kDa protein is more apparent Žalthough there is still approx. 1r3 less total protein in normal hemocytes.. Furthermore, it is evident that the 2A4 and 1E10 antigens are also present in amounts approximately equal to one another, assuming equal stain affinity. They represent 0.2᎐0.3% of the total protein in either sample. Present in a four-fold greater amount in normal cells is a very high molecular-weight protein ŽN., while at least five other proteins Žmarked between the gel lanes. clearly distinguish normal hemocytes from leukemia cells. Two of these can be identified immunologically as tubulin chains Žsee below., but direct scan integration is complicated by nearand co-migrating bands. These distinguishing cellular proteins do not correspond in size to any of the distinguishing serum proteins described earlier for normal versus leukemic clams ŽSunila and Dungan, 1992.. Given that sample normalization is performed, even approximately by pellet volume, the onedimensional SDS-PAGE profile can give a reasonable estimate of whether either of the two distinguishing cell-surface antigens is present. Moreover, judged by stain alone, such profiles also illustrate that these are not the only distinc-

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function of p53 and p73 Žcharacteristically found in clam leukemia cells. is undoubtedly related to their exclusion from the nucleus, where they normally have anti-proliferative function and induce gene expression related to DNA editing and repair andror apoptosis ŽKelley et al., 2001.. Consequently, we examined normal and leukemia cell lysates from several sources with respect to the expression of p53 gene family members. A revealing example, using an immunoblot from the samples in Fig. 3, is illustrated in Fig. 5 Župper panel.. When normalized for actin content, the samples all show approximately equal amounts of p53

Fig. 4. Comparative densitometry of representative normal and leukemia cell lysates. The samples illustrated in lanes A and B of Fig. 3 were resolved on a 5᎐15% Tr1% C small-format gradient gel, scanned, and plotted in parallel to illustrate major protein differences between them. The 1E10 and major 2A4 cross-reactive proteins are designated by arrows on the gels; the 205-kDa protein and actin are marked accordingly. Markers between gel lanes designate major quantitative protein differences. The positions of the normal cell protein N, the 205-kDa protein, the 1E10 and 2A4 antigens, actin, and the ␣ and ␤ chains of tubulin are noted on the scans.

tive protein differences between normal hemocytes and leukemia cells. 3.5. Differential expression of p53 family members in normal hemocytes and leukemic cells Mutations in the p53 gene have long been associated with aggressive tumors, and such a mutation has been reported in clam leukemia cells ŽBarker et al., 1997.. Additionally, lack of

Fig. 5. Differences in the expression of p53 gene family members and ␤-tubulin between normal hemocytes and leukemia cells. Upper panel: an immunoblot of the samples illustrated in Fig. 3 was probed with the antibody Map53r73-23, which is specific for a highly conserved DNA binding domain in the p53 gene family proteins in Mya ŽKelley et al., 2001.. Lane A: lysate from pooled normal hemocytes. Lanes B᎐D: lysates from hemocytes of three individual leukemic clams taken from New Bedford Harbor. Lanes E᎐F: lysates of hemocytes from morphologically leukemic clams taken from an unpolluted Cape Cod area: E was 1E10-negative, while F was 1E10-positive. Lower panel: replicate blot of samples described above, probed for ␤-tubulin with the monoclonal antibody TUB 2.1.

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but no p73 is detectable in normal hemocyte lysates ŽFig. 5, lane A.. Rather, a protein with a molecular mass of ; 102 kDa is observed. This most likely corresponds to p97, a putative p53 gene family member identified in embryos and adult tissues of Spisula solidissima Žsurf clam. by the p53r73 antibody ŽJessen-Eller et al., submitted; Stephens, unpublished.. In leukemia cell lysates, this p97 homologue is not detected, whereas p73 is prominent. The proteins p53 and p73 appear to be proportionate among the individual leukemic clam lysates ŽFig. 5, lanes B᎐D.. Interestingly, in a case where cells were judged leukemic by both abundance and cell morphology, the preparation tested negative for the 1E10 antigen, both immunocytochemically and by Western blotting. The lysate from these 1E10negative cells showed a p53rp97 pattern ŽFig. 5, lane E. characteristic of normal hemocytes, whereas that from 1E10-positive cells, obtained from a clam from the same location, showed a mixed p53rp73rp97 pattern ŽFig. 5, lane F.. We interpret the former as representing a cell population that has attained the characteristic leukemia cell morphology, but has yet to express either the 1E10 cell-surface antigen or p73. The 1E10-positive cells, on the other hand, may also be intermediate, since they still express p97. This is consistent with the fact that the corresponding protein profiles ŽFig. 3E,F. appear intermediate as well when compared with normal hemocytes versus end-stage leukemia cells ŽFig. 3A vs. B᎐D..

3.6. Tubulin differences between cell types

Using replicate blots, the sample set described above was also probed with monoclonal antibodies to tubulin, illustrated here for the ␤ subunit ŽFig. 5, lower panel.. As might be expected from the fact that leukemia cells are mitotic whereas normal hemocytes are not, there is a ) 20-fold greater amount of tubulin in the leukemia cells. Consistent with the above p73rp97 observations, cells not yet expressing the1E10 antigen Žlane E. have nearly undetectable tubulin, while those judged as late intermediate Žlane F. have an intermediate amount of tubulin Ž; 1r3. compared to end-stage leukemia cells Žlanes B᎐D..

4. Discussion The dual characterization of cell surface proteins, coupled with the differential appearance of p53 gene family members, reveals coincident shifts in expression, and perhaps function, as malignant cells replace normal hemocytes. As leukemia cells develop, they progressively lose the 2A4 antigen ŽWhite et al., 1993.. In end-stage leukemia, the cells are 1E10- and p73-positive, but p97-negative. In contrast, normal hemocytes are both 2A4and p97-positive, but p73 negative. Similar reciprocal expression is observed in two other major glycoproteins, and in numerous medium and low molecular-weight proteins, but with the exception of tubulin, none of these has been specifically identified or localized. The initial identification of 1E10 and 2A4 antigens utilized Triton X-100 lysates of membrane preparations made by nitrogen cavitation ŽMiosky et al., 1989; White et al., 1993.. The direct membranercytosol solubilization method used here gives the same basic immunologic distinction, but allows further insight into the total amount of antigen present in the respective cell type populations, and reveals major differences in glycoproteins and cytosolic proteins as well. Accurate size identification of these two antigens and normalization for the major cytoskeletal protein, actin, provide a basis for quantification and efficient rapid screening, whether by the customary 1E10r2A4 antibodies, or by simple SDS-PAGE protein profile comparison. Primary structural analysis suggests that the 1E10 antigen is a large, very hydrophobic, integral glycoprotein with a potential myristoylation site at its N-terminus. Coupled with this, the spectrinrdystrophinrtalin domain homologies suggest a membrane protein with cytoskeletal connections. These properties would enable the protein to provide, via its cytoplasmic portion, a wellanchored membrane ‘skeleton’ for the unusually round, non-adhesive cells in which it uniquely occurs. At the same time, it would allow communication with the extracellular environment. Homologies with immunoglobulin heavy chains and with complement factor H further suggest a potential role of the protein in primitive immunity or cell recognition. Alternatively or additionally, the 1E10 antigen may be related to the Ig superfamily of cell adhesion molecules ŽCAMs..

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Because of limited quantity, band multiplicity, and possible co-migrating bands, we did not attempt direct sequence analysis of the 2A4 antigen. A better approach for this protein will be molecular cloning. It is interesting to speculate, however, that the 2A4 antigen may serve a similar transmembrane communication function in normal hemocytes, perhaps mediating adhesion. It may be significant that the characteristically nonadhesive leukemia cells express instead a prominent ; 140-kDa glycoprotein. Based on immunologic distinction, the 2A4 and 1E10 antigens are unlikely to be directly related as precursor and product, but this cannot be strictly ruled out until we know more about the genes that encode them. Rather, they appear to be reciprocally expressed, with the former decreasing markedly before the latter appears. In this respect, a re-examination of the immunocytochemical results of White et al. Ž1993. is revealing. That study illustrated a mitotic leukemia cell with a single ‘capped’ region of 2A4 antigen at the pole of only one daughter cell, while some interphase cells in the population showed similar but diminished single ‘caps’ of this antigen. This progressive phenomenon suggests membrane protein Ž2A4. internalization. Furthermore, these workers showed that the 2A4 antigen in permeabilized normal cells is also localized to internal membranes resembling ERrGolgi, but no such pattern is observed in leukemia cells bearing 2A4 caps. Earlier, it was shown that a different antigen Ž4A9. present on leukemia cells also occurred on a sub-population of circulating hemocytes ŽSmolowitz et al., 1989.. In light of these observations, it would be of particular interest to perform double-label immunocytochemistry on mid- and end-stage leukemia cells to determine the spatiotemporal distribution of these three distinct antigens. Normalized to actin, there is considerably less tubulin in normal hemocytes, a likely consequence of the fact that they are terminally differentiated and no longer undergo mitosis. The relatively high amount of actin in leukemia cells is somewhat surprising, since these cells are mitotic and spherical, in contrast to normal hemocytes, which are motile and assemble extensive pseudopods of actin filament bundles. Perhaps these two cell types maintain relatively constant pools of actin from which to assemble microfilaments for limited or differential deployment.

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Thus, some of the unidentified cytosolic and glycoproteins that distinguish the two cell types may be critical in determining cytoskeletal dynamics and the interaction of the resultant cytoskeleton with the membrane. For example, leukemia cells ᎏ normally spherical ᎏ can assemble long, well-defined ‘microspikes’ under certain in vitro conditions Že.g. suspension in buffered seawater.. Although expression of p53 is not measurably different in the two cell types, other members of this gene family differ significantly: p97 is found predominantly in normal hemocytes, while p73 appears exclusively in leukemia cells. The latter result was observed earlier ŽKelley et al., 2001., but the former was not. It is likely that we can now detect p97 in normal hemocyte lysates, since the blotting conditions used here were designed to improve transfer and renaturation. Yet to be characterized fully, p97 occurs in oocytes and developing embryos of the surf clam, Spisula solidissima, but degrades in embryos exposed in vitro to PCBs ŽJessen-Eller et al., submitted.. The appearance of p73 in leukemia cells may be highly significant, since p73 in other systems has been postulated as a potential p53 antagonist ŽMorrison and Kinosita, 2000; Pozniak et al., 2000.. Leukemia is prevalent in a variety of other bivalve genera ŽFarley, 1969a,b., but the 2A4 and 1E10 monoclonal antibodies only detect their respective antigens in hemocytes from the soft shell clam Mya arenaria. However, cell lysates analyzed simply by SDS-PAGE protein profiles or by broad-range p53 gene family antibodies will extend our explorations with Mya to other species impacted by environmental contamination. Shifts in the ratio of p97rp73rp53 may precede cell transformation, and thus these proteins may serve as useful biomarkers of gene activation. Furthermore, parallels between clam and certain human leukemias ŽKelley et al., 2001. suggest that fundamental new data may be obtained from this model system in terms of the basic mechanisms of environmentally induced cancers and leukemias.

Acknowledgements This study was supported by a program grant ‘Activities to Promote Research Collaborations’ from the National Cancer Institute and by USPHS research grants GM 20644 to RES, HD 28204 to CWW, and CA 44307 to CLR. We thank L. Levy

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