WO1999009205A2 - Diagnosis of clear cell type renal carcinoma - Google Patents

Abstract

Method for detecting clear cell type renal carcinoma in a mammal comprising determining the concentration of Na,K-ATPase's β-subunit in a biological sample. Alternatively the concentration of N-linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof in Na,K-ATPase's β-subunit in the biological sample is determined and correlated with the presence of clear cell type renal carcinoma in the mammal. The method further includes detecting the Na,K-ATPase's spatial distribution in the tissue section by detecting the biological agent's spatial distribution in the tissue and correlating the Na,K-ATPase's spatial distribution in the tissue section to the presence of clear cell type renal carcinoma in the mammal. In addition, a kit for detecting clear cell type renal carcinoma is disclosed. The kit includes an agent capable of cleaving N-linked sugar residues, lactosamine residues, sialic acid, or combinations thereof and a biological agent which binds to Na,K-ATPase.

Description

DIAGNOSIS OF CLEAR CELL TYPE RENAL CARCINOMA

The present application claims the benefit of U.S. Provisional Patent Application Serial No. 60/056,232, filed August 21, 1997, which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to methods for detecting clear cell type renal carcinoma.

BACKGROUND OF THE INVENTION

Na.K-ATPase

Na,K-ATPase catalyzes the movement of sodium and potassium ions across the plasma membrane and is present in all tissues of higher organisms. It is most abundant in the kidney, where it is responsible for reabsorbing sodium ions from the glomerular filtrate (Lingrel et al., "Structure-Function Studies of the Na,K-ATPase," Kidney Int., 45:S32-S39 (1994) ("Lingrel")). In renal tubules, Na,K-ATPase transports Na⁺ ions across the basolateral membrane into the interstitial fluid, creating an osmotic pressure that moves water from the tubular lumen into the interstitial space. Since Na⁺ gradients are crucial for efficient functioning of other Na⁺-coupled transport systems, Na,K- ATPase is a key enzyme and is implicated in many general and specialized kidney functions (Katz, "Role of Na-K-ATPase in Kidney Function," in Skou et al., eds., The Na⁺, K⁺- pump: Part B: Cellular Aspects, New York: Alan R. Liss, Inc., pp. 207-232 (1988)). The Na,K- ATPase is an oligomeric transmembrane protein consisting of two non- covalently linked subunits: the α-subunit (approximately 112 kDa) and a smaller, glycosylated β-subunit (approximately 55 kDa). At least three α isoforms exist, and these exhibit a tissue-specific distribution and differences in functional properties (Sweadner, "Isozymes of the Na,K-ATPase," Biochem. Biophys. Acta. 988:185-220 (1989)). The predominant isoform in the kidney is αl . Three isoforms of the β-subunit have also been described (Lingrel et al., "Molecular Genetics of Na,K- ATPase," Progress in Nucleic Acids Research and Molecular Biology, 38:37-89, (1990) and Mercer, "Structure of the Na,K-ATPase," Int. Rev. CytoL 137C:139-168 (1993) ("Mercer")), βl is expressed in all tissues including kidney (Lingrel). Neural tissues express β2 (Martin-Vasallo et al., "Identification of a Putative Isoform of the Na,K- ATPase β-Subunit Primary Structure and Tissue-Specific Expression," J. Biol. Chem.. 264:4613-4618 (1989)), and β3 is expressed in Xenopus (Good et al., "A Nervous System-Specific Isotype of the Beta Subunit of Na,K-ATPase Expressed During Early Development of Xenopus Laevis," Proc. Natl. Acad. Sci. USA. 87:9088-9092 (1990)). β2 isoforms have been shown to exhibit characteristics of adhesion molecules (Gloor et al., "The Adhesion Molecule on Glia (AMOG) is a Homologue of the β Subunit of the Na,K- ATPase," J. Cell Biol., 110:165-174 (1990)).

The βl-subunit from human (Kawakami et al., "Molecular Cloning and Sequence Analysis of Human Na,K-ATPase β-Subunit," Nucleic Acids Res.. 14:2833-2844 (1986)) and non-mammalian species has been cloned and characterized (Mercer). The βl subunits consist of 302 to 305 amino acids with a corresponding molecular weight of 35 kDa. The βl-subunit has a single transmembrane segment, a large carboxy-terminal extracellular domain, and a short amino-terminal cytoplasmic domain (Dzhandzhugazyan et al, "Asymmetric Orientation of Amino Groups in the α-Subunit and the β-Subunit of (Na⁺+K⁺)-ATPase in Tight Right-Side-Out Vesicles of Basolateral Membranes from Outer Medulla," Biochim. Biophvs. Acta. 817:165-173 (1985) and Ohta et al., "Structure of the Extra-Membranous Domain of the β-Subunit of (Na,K)- ATPase Revealed by the Sequences of its Tryptic Peptides," FEBS, 204:297-301 (1986)). The extracellular domain contains three potential N-linked glycosylation sites (Asn-X-Ser/Thr). Isolation and characterization of glycosylated peptide fragments and in vitro expression of β- subunit cDNA have demonstrated that all of the glycosylation sites are utilized in mammalian β-subunits (Ovchinnikov et al., "Pig Kidney Na,K-ATPase. Primary Structure and Spatial Organization," FEBS Lett.. 201 :237-245 (1986) and Gilmore- Hebert et al., "In Vitro Expression of the Alpha and Beta Subunits of the Na,K-ATPase," in The Na⁺. K⁺-pump. New York: Alan R. Liss, Inc., pp. 71-76 (1988)).

The significance of the β-subunit in sodium pump function remains obscure. It has been suggested that the β-subunit facilitates insertion of the αβ-complex into the cell membrane (Noguchi et al., "Primary Structure of the Beta Subunit of Torpedo Californica," FEBS Lett.. 196:315-320 (1986)). It may also be involved in maturation of the enzyme and localization to the plasma membrane (Geering, "Posttranslational Modifications and Intracellular Transport of Sodium Pumps: Importance of Subunit Assembly," The Sodium Pump: Structure, Mechanism, and Regulation. New York: The Rockefeller University Press, pp. 32-43 (1991)). Coexpression studies of α- and β- subunits have indicated that the β-subunit has a stabilizing effect on the α-subunit (Ackermann et al., "Mutual Dependence of Na,K-ATPase α- and β-Subunits for Correct Posttranslational Processing and Intracellular Transport," FEBS Lett., 269: 105- 108

(1990) ("Ackermann"). Both α- and β-subunits have been shown to be essential for the expression of a functional enzyme in yeast and Xenopus oocyte expression systems (Geering, "The Functional Role of the β-Subunit in the Maturation and Intracellular Transport of Na,K- ATPase," FEBS, 285:189-193 (1991) ("Geering")). However, functioning of the α-subunit independent of the β-subunit has also been reported (Blanco et al., "The α-Subunit of the Na,K-ATPase has Catalytic Activity Independent of the β- Subunit," J. Biol. Chem.. 269:23420-23425 (1994)), making the precise role of β-subunit in Na,K- ATPase function unclear.

Renal Cell Carcinoma

Renal cell carcinoma ("RCC") is the most common malignant tumor of the kidney. It represents almost 3% of adult malignant diseases and is the third most common urologic cancer. Each year there are about 25,000 cases of kidney cancer in the United States with an estimated 10,600 deaths annually (Ries et al., eds., National Institute of Health Publication No. 91-2789, Bethesda, Maryland: National Cancer Institute (1991)). Symptoms of RCC rarely occur before metastatic spread, and, once RCC has metastasized, 5 year survival rates are less than 10% (Maldazys et al., "Prognostic Factors in Metastatic Renal Carcinoma," J. Urol.. 136:376-379 (1986)). RCCs metastasize early, particularly to lungs, lymph nodes, bones, brain, and liver (Garnick, "Advanced Renal Cell Cancer," Kidney International. 20:127-136 (1981)). Several tumor characteristics have been proposed as means to define tumor behavior. Pathological staging methods are widely used to predict prognosis (Weiss et al., "Adult Renal Epithelial Neoplasms," Anatomical Pathology. 103:624-635 (1994) and Skinner et al., "Diagnosis and Management of Renal Cell Carcinoma, a Clinical and Pathologic Study of 309 Cases," Cancer. 28:1165-1177 (1971) ("Skinner")). Histopathological classification, based on the tumor cell types (e.g., clear-cell, chromophilic (papillary), oncocytoma, and chromophobe) (Motzer et al., "Renal-Cell Carcinoma," New Eng. J. Med„ 335:865-875 (1996) and Thoenes et al., "Histopathology and Classification of Renal Tumors (Adenomas, Oncocytomas and Carcinomas): The Basic Cytological and Histopathological Elements and Their Use for Diagnostics," Pathol. Res. Pract, 181 :125- 143 (1986)), nuclear grading systems (Skinner; Fuhrman et al., "Prognostic Significance of Morphologic Parameters in Renal Cell Carcinoma," Am. J. Surg. Pathol., 6:655-663 (1982); and Novelli et al., "Renal Cell Carcinoma: Comparison of Morphological and Flow Cytometric Parameters of Primary Tumor and Invasive Tumor Lying Within the Renal Vein," J. Pathol., 167:229-233 (1992)), and DNA ploidy (Ljungberg et al., "DNA Content and Prognosis in Renal Cell Carcinoma. A Comparison Between Primary Tumor Tumor Metastases," Cancer, 57:2346-2350 (1986)) are also being used to attempt to predict tumor behavior. These approaches have been only marginally successful. A diagnostic marker for RCC has not yet been defined.

The present invention is directed to overcoming the above noted deficiencies in detecting clear cell type renal carcinoma.

SUMMARY OF THE INVENTION

The present invention relates to a method for detecting clear cell type renal carcinoma in a mammal. The method includes providing a biological sample from the mammal and determining concentration of Na,K-ATPase's β-subunit in the biological sample. The concentration of Na,K- ATPase' s β-subunit in the biological sample is then correlated with the presence of clear cell type renal carcinoma in the mammal.

The present invention also relates to another method for detecting clear cell type renal carcinoma in a mammal. A biological sample from the mammal is provided and contacted with an agent capable of cleaving N-linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof from Na,K-ATPase's β-subunit under conditions effective to cleave N-linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof from Na,K-ATPase's β-subunit in normal kidney tissue. The concentration of N-linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof in Na,K-ATPase's β-subunit in the biological sample is then determined and correlated with the presence of clear cell type renal carcinoma in the mammal. The present invention relates to yet another method for detecting clear cell type renal carcinoma in a mammal. A tissue section from the mammal and a biological agent which binds to Na,K- ATPase are provided. The tissue section is contacted with the biological agent under conditions effective to permit binding of the biological agent to Na,K-ATPase in the tissue section. The method further includes detecting the Na,K- ATPase's spatial distribution in the tissue section by detecting the biological agent's spatial distribution in the tissue and correlating the Na,K-ATPase's spatial distribution in the tissue section to the presence of clear cell type renal carcinoma in the mammal.

The present invention, in another aspect thereof, relates to a kit for detecting clear cell type renal carcinoma. The kit includes an agent capable of cleaving N-linked sugar residues, lactosamine residues, sialic acid, or combinations thereof from Na,K-ATPase's β-subunit and a biological agent which binds to Na,K-ATPase.

The methods of the present invention can be used individually for the detection of clear cell type renal carcinoma. Alternatively, these methods can be used in combination to confirm the diagnosis of this disease.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 A-1H are photographs showing the immunohistochemical localization of α- and β-subunits in normal and clear RCC tissues, α-subunits are localized to proximal tubules (Figure 1A), distal tubules (Figure 1C), and collecting ducts (Figure E). β- subunits are localized in proximal tubules (Figure IB), distal tubules (Figure ID), and collecting ducts (Figure IF). Figures 1G and 1H show the α- and β- subunit localization in clear-cell RCC tissue. In these figures Gl denotes glomerulus. The magnification is 600X.

Figures 2A-2D are laser scanning confocal micrographs of α- and β-subunits in normal and RCC tissues. Figures 2A and 2B show that the α-subunit is localized in normal distal tubule and RCC tissue, respectively. Figures 2C and 2D show the β-subunit localization in normal distal tubule and RCC tissue, respectively. Note the basolateral localization of α- and β-subunit (arrows) compared to the diffused staining pattern in RCC tissues. The bar in the figure represents 5μ. Figure 3A and 3B are immunoblot analyses of the β-subunit of Na,K-ATPase in clear-cell RCC specimens. In Figure 3 A, total cell lysates (100 μg protein) were separated on a SDS-PAGE, transferred to nitrocellulose, immunoblotted with anti-β- subunit antibody, and visualized by [ I]protein A. NK refers to normal kidney lysate. Patient #1-14 designates RCC lysates from patients #1-14. Figure 3B shows blots from two gels that were quantified by phosphorlmager and expressed as % of the levels expressed in normal kidney.

Figures 4A and 4B are an immunoblot analysis of the α-subunit of Na,K- ATPase. In Figure 4A, total cell lysates (100 μg protein) were separated on a SDS-PAGE, transferred to nitrocellulose immunoblotted with anti-α-subunit antibody, and visualized by [¹²⁵I] protein A. NK refers to normal kidney lysate. Patient #1-14 designates RCC lysates from patients #1-14. Figure 4B shows blots from two gels were quantified by phosphorlmager and that were expressed as % of the levels expressed in normal kidney. Figures 5 A and 5B are immunoblot analysis of the β-subunit of Na,K- ATPase in non clear-cell RCC specimens. In Figure 5A, total cell lysates (100 μg protein) were separated on a SDS-PAGE, and protein bands visualized as described in the Figure 3 legend. NK refers to normal kidney lysate. Patient #15, 16, and #17, 18 represent oncocytoma and chromophilic carcinoma, respectively. Patient #19 represents chromophobe carcinoma. Figure 5B shows blots from two gels that were quantified by phosphorlmager and expressed as % of the levels expressed in normal kidney.

Figure 6 shows the Na,K- ATPase activity in RCC tissues. Total membranes were prepared from RCC and autologous normal kidney tissues and assayed for Na,K- ATPase activity as described in Example 6. Percentage of Na,K- ATPase activity in RCC membranes compared to autologous normal kidney membranes is plotted. Data represent the average of two determinations, each done in triplicate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for detecting clear cell type renal carcinoma in a mammal, such as a human. The method includes providing a biological sample from the mammal and determining the concentration of Na,K-ATPase's β-subunit in the biological sample. The concentration of Na,K-ATPase's β-subunit in the biological sample is then correlated with the presence of clear cell type renal carcinoma in the mammal.

Biological samples that can be used in the practice of the present invention include tissue samples from, for example, a kidney of the mammal. In the case where clear cell type renal carcinoma is suspected to be present in the mammal's kidney (for example, by observing necrotic lesions or the like), the kidney tissue sample is preferably taken from the suspect region. Typically, the tissue sample is isolated (e.g., by biopsy) and homogenized, for example, by sonication. Alternatively or additionally, the tissue sample can be minced or otherwise finely divided and resuspended in lysis buffer. Suitable lysis buffers include those having a pH of between about 7 and about 8 and containing about 10-500 mM NaCl, about 5-150 mM Tris, about 0.1-2 mM of a chelating agent (e.g., ethylenediaminetetraacetic acid ("EDTA")), about 0.2-10% of a surfactant (e.g., sodium dodecyl sulfate ("SDS")), and one or more protease inhibitors. Preferred lysis buffers are those having a pH of from about 7.2 to about 7.6 and containing about 50-200 mM NaCl, about 10-50 mM Tris, about 0.3-1 mM of EDTA, about 1-4 % of SDS, and one or more protease inhibitors. Lysis is advantageously carried out for from about 10 minutes to about 5 hours at a temperature of from about 2°C to about 5°C. After lysis, the biological sample can be advantageously homogenized, for example, by sonication. The liquid can then be separated from the solids, preferably by centrifugation, for example for from about 5 minutes to about an hour, at a rotor speed of from about 5000 rpm to about 25,000 rpm.

Once the biological sample is prepared, for example, as described above, the concentration of Na,K-ATPase's β-subunit is determined. The units used to specify the concentration is not critical to the practice of the present invention. For example, concentration, as used herein, may be expressed in moles per unit volume, moles per unit weight, moles per total volume, moles per total weight, weight per unit volume, weight per unit weight, weight per total volume, or weight per total weight. In addition, it is not necessary to specify a numerical value for the concentration to practice the present invention. For example, where the concentration of Na,K-ATPase's β-subunit is related mathematically to absorption, fluorescence, radioactivity, or some other physical property of the biological sample, concentration can be expressed in terms of these physical quantities rather than in terms of the more traditional units of concentration. Although the present invention is not limited to a particular method of determining the concentration of Na,K-ATPase's β-subunit in the biological sample, the concentration is preferably determined immunologically. Briefly, a biological agent which recognizes or otherwise binds to the β-subunit of Na,K- ATPase is contacted with the biological sample, preferably, in a suitable buffer and under conditions that permit binding of the biological agent to the β-subunit of Na,K- ATPase. The concentration of the β-subunit of Na,K- ATPase in the biological sample is detected by detection of the biological agent. As indicated above, the method of the present invention can be used to screen patients for clear cell type renal carcinoma. It can also be used to confirm suspected cases of clear cell type renal carcinoma. Still alternatively, it can be used to identify the recurrence of this disease or to monitor the effects of a particular treatment regimen.

Biological agents suitable for detecting clear cell type renal carcinoma include antibodies, such as monoclonal or polyclonal antibodies. In addition, antibody fragments, half-antibodies, hybrid derivatives, probes, and other molecular constructs may be utilized. These biological agents, such as antibodies, binding portions thereof, probes, or ligands, can bind to any antigenic portion or portions of the β-subunit of Na,K- ATPase.

Antibodies which can be used in the method of the present invention include monoclonal antibodies raised against the β-subunit of sheep Na,K- ATPase. One such antibody is M17-P5-F11, which is commercially available from Affinity BioReagents, Inc., Golden, Colorado. Its preparation is described in detail in Sun et al., Biochim. Biophys. Acta., 1207:236-248 (1994) ("Sun") which is hereby incorporated by reference. Other monoclonal antibodies which recognize or otherwise bind to the β-subunit of Na,K- ATPase can also be used in the practice of the present invention. Monoclonal antibody production may be effected by techniques which are well-known in the art. Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a mammal (e.g., mouse) which has been previously immunized with the antigen of interest either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, Nature 256:495 (1975), which is hereby incorporated by reference. Mammalian lymphocytes are immunized by in vivo immunization of the animal

(e.g., a mouse) with the β-subunit of Na,K- ATPase. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Following the last antigen boost, the animals are sacrificed and spleen cells removed. Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol ("PEG") or other fusing agents (See Milstein and Kohler, Eur. J. Immunol. 6:51 1 (1976), which is hereby incorporated by reference). This immortal cell line, which is preferably murine, but may also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described.

Procedures for raising polyclonal antibodies are also well known. Typically, such antibodies can be raised by administering β-subunit of Na,K- ATPase subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The antigens can be injected at a total volume of 100 μl per site at six different sites. Each injected material will contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Ultimately, the rabbits are euthenized with pentobarbital 150 mg/Kg IV. This and other procedures for raising polyclonal antibodies are disclosed in E. Harlow, et. al., editors, Antibodies: A Laboratory Manual (1988), which is hereby incorporated by reference. In addition to utilizing whole antibodies, the method of the present invention encompass use of binding portions of such antibodies. Such binding portions include Fab fragments, F(ab')₂ fragments, and Fv fragments. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in J. Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118 (N.Y. Academic Press 1983), which is hereby incorporated by reference.

Alternatively, the processes of the present invention can utilize probes or ligands found either in nature or prepared synthetically by recombinant DNA procedures or other biological or molecular procedures. Suitable probes or ligands are molecules which bind to the Na,K-ATPase's β-subunit antigens identified by the monoclonal antibodies of the present invention. Other suitable probes or ligands are molecules which bind to Na,K- ATPase's β-subunit. Such probes or ligands can be, for example, proteins, peptides, lectins, or nucleic acid probes.

The antibodies described above or made according to the above protocol can be used alone or as a component in a mixture with other antibodies or other biological agents to detect clear cell type renal carcinoma.

To facilitate detection, it is advantageous to use a labeled biological agent. Examples of labels useful for detection of increased levels of Na,K-ATPase's β-subunit in accordance with the present invention are radiolabels such as ¹³¹I, ^{ι n}In, ¹²³I, "mTc, ³²P, ¹²⁵1, ³H, ¹⁴C, and ¹⁸⁸Rh, fluorescent labels such as fluorescein and rhodamine, nuclear magnetic resonance active labels, positron emitting isotopes, chemiluminescers such as luciferin, and enzymatic markers such as peroxidase or phosphatase.

The biological agent can be labeled with such reagents using techniques known in the art. For example, see Wensel and Meares, Radioimmunoimaging and Radioimmunotherapy, Elsevier, New York (1983), which is hereby incorporated by reference, for techniques relating to the radiolabeling of antibodies. See also, D. Colcher et al., "Use of Monoclonal Antibodies as Radiopharmaceuticals for the Localization of Human Carcinoma Xenografts in Athymic Mice", Meth. Enzymol. 121 : 802-816 (1986), which is hereby incorporated by reference. A radiolabeled biological agent can be used for diagnostic tests. The specific activity of a tagged biological agent, such as a tagged antibody, binding portion thereof, probe, or ligand, depends upon the half-life, the isotopic purity of the radioactive label, and how the label is incorporated into the biological agent. Table 1 lists several commonly-used isotopes, their specific activities and half-lives. In immunoassay tests, the higher the specific activity, in general, the better the sensitivity.

TABLE 1

Specific Activity of Pure

Isotope Isotope (Curies/mole) Half-Life

14C 6.25 x lO¹ 5720 years 3H 2.01 x lO⁴ 12.5 years

35S 1.50 x 10° 87 days 1251 2.18 x lO⁶ 60 days 32P 3.16 x lO⁶ 14.3 days 1311 1.62 x lO⁷ 8.1 days

Procedures for labeling biological agents with the radioactive isotopes listed in Table 1 are generally known in the art. Tritium labeling procedures are described in U.S. Patent No. 4,302,438, which is hereby incorporated by reference. Iodinating, tritium labeling, and ³⁵S labeling procedures especially adapted for murine monoclonal antibodies are described by Goding, J.W. (supra, pp 124-126) and the references cited therein, which are hereby incorporated by reference. Other procedures for iodinating biological agents, such as antibodies, binding portions thereof, probes, or ligands, are described by Hunter and Greenwood, Nature 144:945 (1962), David et al., Biochemistry 13:1014-1021 (1974), and U.S. Patent Nos. 3,867,517 and 4,376,110, which are hereby incorporated by reference. Radiolabeling elements which are useful in detecting antibodies and other biological agents include ¹²³I, ¹³¹I, ^mIn, and ^99mTc, for example. Procedures for iodinating biological agents are described by Greenwood, F. et al., Biochem. J. 89:114-123 (1963); Marchalonis, J., Biochem. J. 113:299-305 (1969); and Morrison, M. et al., Immunochemistry, 289-297 (1971), which are hereby incorporated by reference. Procedures for ^99mTc-labeling are described by Rhodes, B. et al. in Burchiel, S. et al. (eds.), Tumor Imaging: The Radioimmunochemical Detection of Cancer, New York: Masson 111-123 (1982) and the references cited therein, which are hereby incorporated by reference. Procedures suitable for ⁿ 'in-labeling biological agents are described by Hnatowich, D.J. et al., J. Immul. Methods, 65:147-157 (1983), Hnatowich, D. et al., J. Applied Radiation, 35:554-557 (1984), and Buckley, R. G. et al., F.E.B.S. 166:202-204 (1984), which are hereby incorporated by reference.

Fluorophore and chromophore labeled biological agents can be prepared from standard moieties known in the art. Since antibodies and other proteins absorb light having wavelengths up to about 310 nm, the fluorescent moieties should be selected to have substantial absorption at wavelengths above 310 nm and preferably above 400 nm. A variety of suitable fluorescers and chromophores are described by Stryer, Science, 162:526 (1968) and Brand, L. et al., Annual Review of Biochemistry. 41 :843-868 (1972), which are hereby incorporated by reference. The biological agents can be labeled with fluorescent chromophore groups by conventional procedures such as those disclosed in U.S. Patent Nos. 3,940,475, 4,289,747, and 4,376,110, which are hereby incorporated by reference.

One group of fluorescers having a number of the desirable properties described above are the xanthene dyes, which include the fluoresceins derived from 3,6-dihydroxy-9-henylxanthhydrol and resamines and rhodamines derived from 3,6-diamino-9-phenylxanthydrol and lissanime rhodamine B. The rhodamine and fluorescein derivatives of 9-o-carboxyphenylxanthhydrol have a 9-o-carboxyphenyl group. Fluorescein compounds having reactive coupling groups such as amino and isothiocyanate groups such as fluorescein isothiocyanate and fluorescamine are readily available. Another group of fluorescent compounds are the naphthylamines, having an amino group in the α or β position.

Biological agents can be labeled with fluorchromes or chromophores by the procedures described by Goding, J. (supra, pp 208-249). The biological agents can be labeled with an indicating group containing the NMR-active ¹⁹F atom or a plurality of such atoms inasmuch as (i) substantially all of naturally abundant fluorine atoms are the ¹⁹F isotope and, thus, substantially all fluorine-containing compounds are NMR-active and (ii) many chemically active polyfluorinated compounds such as trifluoracetic anhydride are commercially available at relatively low cost.

Once the concentration of the Na,K-ATPase's β-subunit in the biological sample is determined, it is correlated with the presence of clear cell type renal carcinoma. This correlation is carried out by comparing the concentration of Na,K-ATPase's β-subunit in the biological sample to the concentration of Na,K-ATPase's β-subunit in normal kidney tissue. Lower concentrations of Na,K-ATPase's β-subunit in the mammal's biological sample relative to normal kidney tissue is indicative of the presence of clear cell type renal carcinoma in the mammal providing the biological sample. To establish that one concentration is lower than another, absolute numbers can be calculated for the mammal's biological sample and for the normal kidney tissue. Alternatively, the biological sample's Na,K-ATPase's β-subunit concentration can be compared with that of normal kidney tissue by comparing some property that varies with concentration (e.g., the intensities produced in immunoblotting). Preferably, the concentration of Na,K-ATPase's β-subunit in the mammal's biological sample is substantially lower than that in normal kidney tissue. For example, it is preferred that the concentration of Na,K-ATPase's β-subunit in the mammal's biological sample be less than about 50%, more preferably, less than about 25%, and most preferably, less than about 10% of the concentration of Na,K-ATPase's β- subunit in normal kidney tissue.

As one skilled in the art will recognize, the concentration of Na,K-ATPase's β- subunit in normal kidney tissue can be determined by the same methods described above for determining the concentration of Na,K-ATPase's β-subunit in the subject mammal's biological sample. Suitable sources of kidney tissue for establishing the concentration of Na,K-ATPase's β-subunit in normal kidney tissue include the kidneys of mammals that are free of clear cell type renal carcinoma, a kidney that is free of clear cell type renal carcinoma in a mammal whose other kidney is infected with the disease, and uninfected regions of a kidney that is infected with clear cell type renal carcinoma. As one skilled in the art will also note, it is preferred that the normal kidney tissue be from the same type of mammal as the mammal whose biological sample is being tested using the method of the present invention. The present invention also relates to another method for detecting clear cell type renal carcinoma in a mammal.

First, a biological sample is provided from the mammal. Suitable biological samples include those described above with regard to the method involving a comparison of concentrations of Na,K- ATPase' s β-subunit. Preferably, the biological sample is the lysate of a lysed and homogenized kidney tissue sample. Preferably, the biological sample is denatured, such as by contacting it with (preferably, boiling it with) 10-200 mM dithiotritol ("DTT"). The biological sample is then contacted with an agent capable of cleaving N- linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof from Na,K-ATPase's β-subunit. Typically, the agent capable of cleaving N-linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof from Na,K- ATPase's β-subunit will be those that are capable of cleaving N-linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof, non-specifically, from any protein. For example, the biological sample can be contacted with a glycosidase which is capable of cleaving N-linked sugar residues from Na,K- ATPase's β-subunit. Suitable glycosidases include endoglycosidases, such as endoglycosidase-F/N- glycosidase-F ("Endo-F/N"). Alternatively or additionally, the biological sample can be contacted with a galactosidase, such as endo-b-galactosidase which cleave lactosamine residues from Na,K-ATPase's β-subunit. Still alternatively or additionally, the biological sample can be contacted with a neuraminidase which cleave sialic acid residues from Na,K- ATPase's β-subunit. Use of an enzyme which cleaves more than one of these residues is also contemplated.

The contacting is carried out under conditions that are effective to cleave N-linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof from Na,K-ATPase's β-subunit in normal kidney tissue. Typically, these conditions are the same as those which would be employed to cleave N-linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof, non-specifically, from any and all proteins in the sample. For example, the agent and the biological sample can be contacted in an appropriate buffer, such as citrate buffer, preferably citrate buffer having a concentration of from about 25 mM to about 400 mM, at a temperature of from about 20° C to about 60° C, preferably at about 35°C to about 40 °C, and for a period of time from about 2 hours to about 30 hours.

The concentrations of N-linked sugar residues, lactosamine residues, and sialic acid residues (or combinations of these) in Na,K- ATPase's β-subunit are then determined. Although this can be done in a variety of ways, it is preferred that the determination be carried out electrophoretically, such as by SDS-PAGE, which indicates the degree of, for example, glycosylation in the Na,K-ATPase's β-subunit in the biological sample. From this value, one can establish the concentration of (in this example) of N-linked sugar residues in Na,K- ATPase's β-subunit in the biological sample. Of course, as indicated above, one need not determine the concentration of, e.g., N-linked sugar residues in the sample's Na,K-ATPase β-subunits using traditional units of concentration (e.g., moles per unit volume, etc.). For example, the present invention can be practiced by determining the concentration in terms of a physical property of the biological sample that correlates to the concentration in traditional units.

Once the concentration of the N-linked sugar residues, lactosamine residues, and/or sialic acid residues in the sample's Na,K- ATPase β-subunits are determined, the concentration is correlated with the presence of clear cell type renal carcinoma. This correlation is carried out by comparing the concentration of N-linked sugar residues, lactosamine residues, and/or sialic acid residues in the biological sample's Na,K-ATPase β-subunits to the concentration of these residues in a normal kidney tissue sample's Na,K- ATPase β-subunits. Higher concentrations of N-linked sugar residues, lactosamine residues, and/or sialic acid residues in the biological sample's Na,K- ATPase β-subunits following treatment with the cleaving agent relative to normal kidney tissue is indicative of the presence of clear cell type renal carcinoma in the mammal providing the biological sample. To establish that one concentration is higher than another, absolute concentration values (using traditional units) can be calculated for the mammal's biological sample and for the normal kidney tissue. Alternatively, the concentration of N-linked sugar residues, lactosamine residues, and/or sialic acid residues in the biological sample's Na,K- ATPase β-subunits can be compared with that of normal kidney tissue by comparing some property that varies with concentration (e.g., mobilities on SDS-PAGE). Preferably, the cleaving step is carried out so that, after the cleaving step, the concentration of N-linked sugar residues, lactosamine residues, and/or sialic acid residues in the Na,K- ATPase β- subunits of the mammal's biological sample is substantially higher than that in normal kidney tissue which has been cleaved under the same conditions. For example, it is preferred that the concentration of N-linked sugar residues, lactosamine residues, and/or sialic acid residues in the Na,K- ATPase β-subunits of mammal's biological sample after cleaving be greater than about 110%, more preferably, greater than about 125%, and most preferably, greater than about 150% of the concentration of these residues, after cleavage, in normal kidney tissue.

As one skilled in the art will recognize, the concentration of N-linked sugar residues, lactosamine residues, and/or sialic acid residues, subsequent to cleavage, in the Na,K- ATPase β-subunits of normal kidney tissue can be determined by the same methods described above for determining the concentration of these residues, subsequent to cleavage, in the Na,K- ATPase β-subunits of a biological sample from the subject mammal. Suitable sources of kidney tissue for establishing the concentration of N-linked sugar residues, lactosamine residues, and/or sialic acid residues subsequent to cleavage in the Na,K- ATPase β-subunits of normal kidney tissue include those discussed above with regard to the method involving the concentration of Na,K- ATPase's β-subunit.

The present invention relates to yet another method of detecting clear cell type renal carcinoma in mammals. In this method, a tissue section from the mammal is provided by standard histological methods. Briefly, for example, a tissue sample is removed from the mammal, preferably from the mammal's kidney, for example by biopsy or in the course of an operation where direct access to the kidney is available. The tissue sample can then be resectioned, preferably in from about 1 μm to about 20 μm thick samples, and the resectioned samples are snap frozen, preferably in liquid nitrogen. The cryostat sections can then be fixed in a cold inert solvent, such as methanol at from about -30°C to -10°C, and then rehydrated, preferably in a suitable buffer, such as in phosphate buffered saline ("PBS"), preferably, containing from 0.5 to 8% bovine serum albumin ("BSA") to produce the desired tissue section. Further details regarding the preparation of tissue sections for use in the practice of this method of the present invention can be found, for example, in Finstad et al., "Specificity Analysis of Mouse Monoclonal

Antibodies Defining Cell Surface Antigens of Human Renal Cancer," Proc. Natl. Acad. Sci. USA, 82:2955-2959 (1985), which is hereby incorporated by reference.

The tissue section is then contacted with a biological agent which binds to Na,K- ATPase. This biological agent can be an antibody, such as a polyclonal antibody raised against the β-subunit of sheep Na,K- ATPase. Methods for making such a polyclonal antibody are discussed above, and more detail is available in Abbott et al., "The Epitope for the Inhibitory Antibody M7-PB-E9 Contains Ser-646 and Asp-652 of Sheep Na,K- ATPase α-Subunit," Biochemistry, 32:3511-3518 (1993) ("Abbott"), which is hereby incorporated by reference. Alternatively the biological agent can be one that binds exclusively to the β-subunit of Na,K- ATPase, such as the ones discussed in detail above in the context of method which involved Na,K- ATPase's β-subunit concentration. Alternatively, the biological agent can be one that binds exclusively to the α-subunit of Na,K-ATPase. For example, an antibody, preferably a monoclonal antibody which binds to the α-subunit of Na,K- ATPase can be used in the method of the present invention. One such antibody is mouse monoclonal antibody M7-PB-E9, which is commercially available from Affinity BioReagents, Inc., Golden, Colorado. Its preparation is described in detail in Sun, which is hereby incorporated by reference. Other monoclonal antibodies which recognize or otherwise bind to the α- or β-subunits of Na,K- ATPase can also be used in the practice of the present invention. These can be readily prepared by the methods set forth above

The tissue section and biological agent are contacted under conditions effective to permit binding of the biological agent to Na,K- ATPase in the tissue section. For example, such contacting can be effected by incubating the biological agent and the tissue section, preferably for from about 1 to about 3 hours at a temperature of from about 15°C to about 25°C, more preferably at about room temperature. After incubation is complete, the tissue sections are washed extensively, for example, with a buffer, such as PBS containing from about 0.2 to about 5 % BSA.

The spatial distribution of the Na,K- ATPase in the tissue section is then detected by detecting the biological agent's spatial distribution in the tissue. Preferably the biological agent is labeled, such as described above, with a label which permits the spatial distribution of the biological agent to be determined by detecting the label. A variety of techniques, such as, confocal microscopy, immunohistochemistry, and immunofluorescence can be used to image the spatial distribution of the labeled biological agent, and, thus, the spatial distribution of the Na,K- ATPase.

The spatial distribution of the Na,K- ATPase in the tissue section is then correlated to the presence of clear cell type renal carcinoma in the mammal. This can be carried out by determining whether the spatial distribution of the Na,K- ATPase shows a well-defined epithelial phenotype. In normal kidney tissue sections, an intense staining of both α- and β-subunits is observed. In contrast, tissue sections infected with clear cell type renal carcinoma show predominantly a diffuse staining pattern of these proteins in most of the tumor areas. Moreover, correlation of Na,K- ATPase's spatial distribution with the presence of clear cell type renal carcinoma can be based on the staining patterns of the tubular epithelia. In normal kidney tissue sections, Na,K-ATPase is localized to the basolateral plasma membrane in kidney tubular epithelia. However, in clear cell type renal carcinoma infected sections, these proteins are distributed diffusely on the membrane surface and intracellularly.

The three methods of the present invention for the detection of clear cell type renal carcinoma can be used individually. Alternatively, they can be used in combination with one another, either two or three at a time. When used together they can make use of the same sample or, alternatively, they can be carried out on separate samples from the subject matter.

In addition, the present invention relates to a kit for detecting clear cell type renal carcinoma in a mammal. The kit includes an agent capable of cleaving N-linked sugar residues, lactosamine residues, sialic acid, or combinations thereof from Na,K- ATPase's β-subunit and a biological agent which binds to Na,K- ATPase, such as a biological agent which binds to Na,K-ATPase's α-subunit, Na,K-ATPase's β-subunit, or combinations thereof. In a preferred embodiment, the kit also includes a means to detect the biological agent. The present invention is further illustrated by the following examples.

EXAMPLES

Example 1 - Materials and Methods Patients and tumor specimens. A total of 19 patients diagnosed with RCC who underwent radical nephrectomy at New York Hospital or University of California, Los

Angeles were included in this study. The age of the patients ranged from 34 to 74.

Specimens were histopathologically evaluated by the Department of Pathology (Table 2).

From each specimen, samples from an area of grossly normal appearing kidney, which included both cortex and medulla, were utilized as normal kidney samples in the analyses.

TABLE 2

Patient # Pathology Tumor Guide Na,K- ATPase Levels beta-subunit alpha-subunit

% of normal kidney

1 Clear cell III 3.9 136.2

2 Clear cell II 6.8 105.8

3 Clear cell II 0.8 20.5

4 Clear cell II 3.7 158.7

5 Clear cell IV 1.9 54.9

6 Clear cell II 2.4 228.2

7 Clear cell I 1.7 31.9

8 Clear cell II 6.9 218.8

9 Clear cell I 5.9 72.9

10 Clear cell III 12.6 111.5

11 Clear cell II 5.2 102.1

12 Clear cell I 7.8 70.2

13 Clear cell III 4.3 38.9

14 Clear cell III* 2.2 31.5

15 Oncocytoma II 11.1 59.53

16 Oncocytoma NA 41.79 48.9

17 Chromophilic IV* 50.03 33.06

18 Chromophilic III 9.7 32.55

19 Chromophobe II 85.02 88.87

Antibodies. Mouse monoclonal antibodies raised against sheep α- (M7-PB-E9) and β-subunit (M17-P5-F11) and rabbit polyclonal antisera (#517) against sheep Na,K- ATPase have been characterized and described previously (Sun and Abbott, which are hereby incorporated by reference). These antibodies recognize epitopes that are common in human, sheep, and dog (Sun and Abbott, which are hereby incorporated by reference). Secondary antibody, fluorescein isothiocyanate ("FITC") conjugated donkey anti-mouse IgG was obtained from Jackson Immunoresearch Laboratories (West Grove, Pennsylvania). Immunohistochemistry, immunofluorescencc, and laser scanning confocal microscopy. Cryostat sections or cells in tissue culture were fixed in cold methanol for 30 min and processed for immunohistochemistry (Liu et al., "Monoclonal Antibodies to the Extracellular Domain of Prostate Specific Membrane Antigen Also React with Tumor Vascular Endothelium," Cancer Res., 57:3629-3634 (1997), which is hereby incorporated by reference) or immunofluorescence localization as described earlier (Rajasekaran et al., "Catenins and Zolula Occludens-1 Form a Complex During Early Stages in the Assembly of Tight Junctions," J. Cell Biol.. 132:451-463 (1996) ("Rajasekaran"), which is hereby incorporated by reference). Tissue sections were incubated with antibodies against the α- and β-subunits of Na,K- ATPase, washed extensively in phosphate buffered saline containing 1% bovine serum albumin and labeled with FITC conjugated secondary antibody and propidium iodide. After extensive washing, the sections were mounted using Vectashield (Vector Labs). Confocal microscopy was performed as described earlier (Rajasekaran, which is hereby incorporated by reference). Briefly, to simultaneously detect FITC-labeled antigens and propidium iodide, samples were excited were at 540 nm with an argon laser, and the light emitted between 525 and 540 nm was recorded for FITC and above 630 nm for propidium iodide. 30-40 horizontal (X,Y) confocal sections were obtained and used to generate three dimensional images using the Image Space software (version 3.1) from Molecular Dynamics (Sunnyvale, California) on an Iris Indigo work station (Silicon Graphics, Mountain View, California).

Preparation of cell lysate, gel electrophoresis and immunoblot analysis. Tissues were minced and resuspended in a lysis buffer (95 mM NaCl, 25 mM Tris, pH 7.4, 0.5 mM EDTA, 2% SDS, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml each of antipain, leupeptin, and pepstatin) and briefly homogenized in a Dounce homogenizer and sonicated twice for 15 seconds using a microtip (Heat Systems Ultrasonics, Inc., New York). The lysates were spun in a microfuge at 14,000 rpm for 15 min, and the supernatant used for SDS-PAGE and immunoblot analysis. The protein concentration of the supernatant was determined using BioRad DC reagent following manufacturer's instructions. Since the normal kidney ("NK") lysates from all patients showed similar protein levels of α- and β-subunits, an aliquot of NK lysate containing equal amounts of protein from each patient was pooled. Pooled NK lysate (lOOμg protein) and individual RCC lysates (lOOμg) were used for immunoblot analysis. Immunoblots were carried out as described earlier (Rajasekaran, which is hereby incorporated by reference). Bands -ye were visualized by [ I]-protein A, autoradiographed, and quantified using phosphorlmager (Molecular Dynamics, Sunnyvale, California) analysis. Glycosidase treatment. Tissue lysates were treated with glycosidases following manufacturer's instructions. Briefly, the lysates were denatured in 50 mM dithiotritol ("DTT") and boiled for 3 min. After cooling, trasylol (20 μg), Triton X-100 (0.5% final concentration), and citrate buffer (100 mM, pH 5.6) were added. Endo-glycosidase F/N, (Boehringer Mannheim, Indianapolis, IN) was added and the reactions were incubated for at least 16 hrs at 37°C. The samples were then subjected to SDS-PAGE and immunoblot analysis as described above.

Na,K- ATPase activity. Total membranes for Na,K- ATPase were prepared as described earlier (Sun, which is hereby incorporated by reference). Briefly, 200-300 mg of tissue from normal and cancer tissues were minced, resuspended in two to three volumes of homogenization buffer (0.25 M Sucrose, 25 mM EDTA, 25 mM imidazole (pH 7.2), 0.5 mM phenylmethylsulfonyl fluoride, 5 μg/ml each of antipain, leupeptin, and pepstatin), and sheared in a polytron followed by homogenization (30 strokes) with a tight fitting Dounce homogenizer. The resulting homogenate was spun at 8,000 X g in a TLS- 55 rotor (Beckman). The pellet was discarded, and the supernatant was spun at 48,000g for 45 min using a TLS-55 rotor. The pellet was resuspended in homogenization buffer and utilized to determine the Na,K- ATPase activity. The protein concentrations of the membrane preparations were determined using the method described in Lowry et al., "Protein Measurement with the Folin Phenol Reagent," J. Bio. Chem.. 193:265-275 (1951), which is hereby incorporated by reference, using BSA as the standard. The Na,K- ATPase activity was determined using a coupled-enzyme, spectrophotometric assay (Schwartz et al., "Possible Involvement of Cardiac Na,K-Adenosine Triphotase in the Mechanisms of Action of Cardiac Glycosides," J. Pharmacol. Exp. Ther.. 168:31-41 (1969), which is hereby incorporated by reference), which consisted of a reaction mixture of 30 mM histidine, 5 mM MgCl₂, 100 mM NaCl, 10 mM KC1, 1 mM EDTA, 3.6 mM NADH, 1 mM phosphenolpyruvate with 10.5 Units of pyruvate kinase and 15 Units of lactate dehydrogenase, pH 7.2. The ATPase activity of the membrane preparation was determined by first monitoring (10 min, 37°C) any NADH oxidative activity of the membrane preparation in the absence of ATP, followed by that occurring upon the addition of 5 mM ATP. The Na⁺ pump activity was calculated as the difference in the ATPase activity in the absence of and presence of 10 mM ouabain, after a 20 min incubation of the membranes in the reaction mixture at 37°C. To rule out the possible interference of mitochondrial ATPase, the ouabain sensitive ATPase activity was also determined in presence of 5 mM NaN₃, a mitochondrial ATPase inhibitor. The activities are calculated as μmoles of ATP hydrolyzed/mg protein/h.

Example 2 — Na,K- ATPase Consistent with their known epitope specificity, monoclonal antibodies raised against sheep α-and β-subunits (Sun and Abbott, which are hereby incorporated by reference) reacted with human α-and β-subunits of Na,K- ATPase as determined by immunohistochemical, immunofluorescence, and immunoblot analyses of both cultured human cells and fresh tissue samples (Figures 1 A-1H, 2A-2D, 3A-3B, and 4A-4B). Immunoblot analysis with anti-α-subunit (M7-PB-E9) and anti-β-subunit (M17-P5-F11) antibodies revealed bands corresponding to α- (1 10 kDa) (Figures 4A-4B) and β- (54-68 kDa) (Figures 3A-3B) subunits of Na,K- ATPase. To further confirm the identity of the glycosylated β-subunit band, tissue lysates were treated with N-glycosidase, which removes all N-linked sugar moieties of glycoproteins. Immunoblot analysis revealed a shift in the molecular mass of the β-subunit from ~58kDa to ~35kDa which corresponds to the molecular mass of the core protein (data not shown). In addition, immunohistochemical and immunofluorescence analyses of normal kidney tissue sections showed that these antibodies distinctly stained kidney tubular epithelia (Figures 1 A-1H and 2A-2D). These studies confirmed that antibodies raised against sheep α-subunit (M7-PB-E9) and β-subunit (M17-P5-F11) react with human Na,K-ATPase α- and β- subunits, respectively, and were suitable to be utilized in this study.

Example 3 — Immunohistochemical Localization of α-and β-Subunits in Normal Kidney and Clear-Cell RCC Immunohistochemical analysis of normal kidney tissue sections with anti-α- subunit (Figures 1A, 1C, IE, and IG) and anti-β-subunit (Figures IB, ID, IF, and IH) antibodies revealed a distinct staining of proximal tubules (Figures 1 A and IB), distal tubules (Figures 1C and ID) and collecting ducts (Figures IE and IF) indicating that both α- and β-subunits of Na,K-ATPase show a similar distribution in normal kidney tubules. However, tissue sections from clear-cell RCC (Figures IG and IH) did not show a well- differentiated epithelial phenotype. In contrast to the intense staining of both α- and β- subunits observed in normal kidney tissue sections, clear-cell RCC tissue sections revealed predominantly a diffuse staining pattern of these proteins in most of the tumor areas (Figures IG and IH). Some localized areas of intense staining of both the α- and β- subunit were observed in most of the RCC tissue sections.

Na,K-ATPase is localized to the basolateral plasma membrane in kidney tubular epithelia (Aimers et al., "Distribution of Transport Proteins Over Animal Cell

Membranes," J. Membrane Biol., 77:169-186 (1984), which is hereby incorporated by reference). High-resolution laser scanning confocal microscopy revealed that both α- subunit (Figure 2A) and β-subunit (Figure 2C) were distinctly localized to the basolateral plasma membrane in tubular epithelia. However, in clear-cell RCC, these proteins were distributed diffusely on the membrane surface and intracellularly (Figures 2B and 2D).

Example 4 — Determination of the Levels of α- and β-Subunit of Na.K- ATPase in

Normal Kidney and RCC Samples

To determine the protein levels of α- and β-subunit of Na,K- ATPase in clear-cell RCC we utilized an immunoblot analysis. The β-subunit detected in normal kidney lysate has a broad band due to glycosylation and is intense due to its abundance (Figure 3A, lane

NK). Although the amount of protein loaded onto gels for each sample was similar (as revealed by a coommassie blue stained protein gel (data not shown)), the levels of the β- subunit in all the patient's clear-cell RCC lysates were substantially lower than that of control samples (Figure 3 A, lanes 1-14). Quantification of the immunoblots revealed the levels of β-subunit in clear-cell RCC lysates were reduced by 90-95% as compared to NK lysate (Figure 3B).

In contrast to the consistently low protein levels of β-subunit in clear-cell RCC lysates, the α-subunit protein levels did not reveal a uniform pattern (Figures 4A and 4B). The α-subunit levels in 9 RCC specimens were either normal or higher as compared to the levels of the NK lysate (Figure 4B). Five patients showed levels below normal (30-

50%).

Immunoblot analysis using a polyclonal antiserum against β-subunit revealed a similar reduction in the β-subunit levels (data not shown) indicating that the diminished levels of β-subunit in clear-cell RCC is authentic and not due to the failure of the monoclonal antibody to recognize the epitope of the β-subunit protein. Furthermore,

RCC lysates subjected to N-glycosidase treatment also revealed reduced β-subunit levels (data not shown) ruling out the possibility that altered glycosylation of β-subunit in RCC may have changed the specificity of the monoclonal antibodies used in this study.

Example 5 - Determination of the Levels of β-Subunit in Non Clear-Cell RCC To test whether β-subunit reduction occurs specifically in clear-cell RCC, the levels of β-subunit in RCC lysates of two specimens each of oncocytoma and chromophilic carcinoma and one specimen of chromophobe carcinoma were tested. As shown in Figures 5A and 5B, the β-subunit level was variable in these specimens compared to the consistently reduced levels observed in clear-cell RCC (Figures 3A and 3B). The β-subunit level was below the normal level in the oncocytoma and chromophilic carcinoma specimens while the chromophobe carcinoma specimen contained 85% of the β-subunit present in NK lysate. These results suggest that reduction of β-subunit levels occurs most consistently in clear-cell RCC.

Example 6 - Determination of Na,K- ATPase Activity in Clear-Cell RCC

Since the α-subunit is the catalytic subunit, a test was performed to determine whether the reduced levels of β-subunit are accompanied by an altered Na,K- ATPase activity in clear-cell RCC. To test the Na,K- ATPase activity, total membrane fractions were isolated from the RCC and autologous normal kidney tissues obtained from four patients (patients #9, 11, 13, and 14), and their activity was determined. As shown in

Figures 3 A and 3B, the levels of β-subunit in the RCC lysates of these patients were -5% of the levels found in the NK lysate. In contrast the α-subunit levels in the RCC lysates of these patients (#9, 11, 13, and 14) were 73, 102, 39, and 32% respectively, relative to NK lysate (Figure 4B). All four patients showed significant reduction in the levels of Na,K- ATPase activity in RCC membranes irrespective of their α-subunit levels. RCC membranes of patient #9 had 49% of the Na,K- ATPase activity compared to NK membranes (Figure 6), while RCC membranes from three other patients had only 25 to 35% of Na,K- ATPase activity compared to autologous NK membranes (Figure 6). These results indicate that the reduction in the levels of β-subunit correlates with a reduction in the activity of the enzyme. Example 7 — Discussion

A monoclonal antibody against the β-subunit of Na,K- ATPase (M17-P5-F11) consistently revealed reduced levels of β-subunit in 100% of the tested lysates of clear- cell RCC specimens. The β-subunit level was uniformly lower in clear-cell RCC specimens of tumor grades I, II, III or IV (Table 2). Reduced β-subunit levels in low- grade tumors (Table 2, patients #7, 9, and 12) indicate that reduction in the levels of β- subunit likely occurred early in the development of the tumor. Immunoblot analysis of a limited number of non clear-cell RCC specimens studied did not reveal a consistent pattern of β-subunit expression (Figures 5 A and 5B). Since these forms of RCC are less common, at present, an adequate sample size regarding β-subunit levels in these tumors was not obtained. Therefore, it remains to be elucidated whether significantly reduced level of β-subunit is specifically a clear-cell RCC associated event.

The majority of clear-cell RCC specimens exhibit a deletion of one or both copies of chromosome 3p (Presti et al., "Histopathological, Cytogenetic, and Molecular Characterization of Renal Cortical Tumors," Cancer Res., 51 :1544-1552 (1991), which is hereby incorporated by reference), while human αl and βl subunits of Na,K- ATPase have been mapped to chromosome 1 (Yang-Feng et al., "Chromosomal Localization of Human Na,K-ATPase α- and β-Subunit Genes," Genomics, 2:128-138 (1988), which is hereby incorporated by reference). Therefore, it is unlikely that the loss of alleles from chromosome 3 explains the reduced protein levels of β-subunit of Na,K- ATPase.

Nevertheless, loss of β-subunit specific regulator present on chromosome 3p leading to reduced levels in clear-cell type RCC cannot be ruled out at this time.

The β-subunit of Na,K- ATPase has been suggested to be essential for the stability of the α-subunit (Geering, which is hereby incorporated by reference). Soon after synthesis, both α- and β-subunits appear to assemble in the endoplasmic reticulum ("ER") and are transported to the plasma membrane (Tamkun et al., "The Na'K-ATPase of Chick Sensory Neurons," J. Biol. Chem., 261 :1009-1019 (1986), which is hereby incorporated "by reference). In the absence of the β-subunit, the α-subunit is retained and further degraded in the ER (Ackermann, which is hereby incorporated by reference). Normal or elevated levels of α-subunit in nine clear-cell RCC lysates in spite of significantly reduced levels of β-subunit suggests that alternate mechanism(s) may exist in clear-cell RCC cells to maintain normal or increased levels of the α-subunit. In addition, a large excess of α- over the β-subunit in clear-cell RCC suggests that α- and β-subunits can be regulated independently of each other in this disease. Differential regulation of α- and β- subunits has been reported in rat alveolar cells (Ridge et al., "Differential Expression of Na-K- ATPase Isoforms in Rat Alveolar Epithelial Cells," Am J. PhvsioL. 273 :L246-255 (1997), which is hereby incorporated by reference).

Although the Na,K-ATPase catalytic activity resides in the α-subunit, reduced β- subunit levels in clear-cell RCC distinctly showed an effect on the Na,K- ATPase activity in their membranes. Due to the limited availability of the tissues, ATPase activity tests were not performed in all the clear-cell RCC specimens. However, the four RCC specimens tested clearly showed reduced Na,K- ATPase activity in their RCC membranes (Figure 6). A diminished Na,K-ATPase activity in spite of normal or similar levels of a α-subunit may be explained by the presence of an inactive form of the α-subunit in RCC membranes. Immunoblot analysis does not differentiate between active and inactive forms. Our results on the level of β-subunit and reduced ATPase activity suggest that in vivo the β-subunit levels may regulate the activity of the enzyme. For example, in spite of comparable α-subunit levels in RCC and normal kidney lysates in patients #9 and 11 (Figures 4A and 4B) the reduced Na,K-ATPase activity in these tissues strongly indicates that the β-subunit plays a significant role for the enzyme activity in kidney membranes. To our knowledge, this study shows for the first time that in a human disease a dramatic reduction of β-subunit levels is accompanied by reduced enzyme activity.

The significance of reduced levels of the β-subunit of Na,K- ATPase and the diminished enzyme activity in clear-cell RCC is not known at present. Loss of functional efficiency of Na,K-ATPase during carcinogenesis has been proposed in Racker, "Why Do Tumor Cells Have a High Aerobic Glycolysis?," J. Cell. Phvsiol.. 89:697-700 (1976), which is hereby incorporated by reference, while Davies et al., "Inhibition of the Na,K- ATPase Pump During Induction of Experimental Colon Cancer," Cancer Biochem. Biophys.. 12:81-94 (1991), which is hereby incorporated by reference, has shown a reduction in the total Na,K- ATPase activity during early development of malignant colon carcinoma. The observed reduction of Na,K- ATPase activity in low grade clear-cell RCC (Figure 6) suggests a possible role in the development or progression of clear-cell RCC. In addition, significant reductions of β-subunit of Na,K- ATPase in clear-cell RCC suggest that this protein may have additional roles in the kidney epithelia. Based on the glycosylation pattern of the β-subunit, a role in cell adhesion has been suggested (Treuheit et al., "Structures of the Complex Glycans Found on Theb-Subunit of (Na,K)- ATPase," J. Biol. Chem.. 268:13914-13919 (1993), which is hereby incorporated by reference). If the β-subunit of Na,K- ATPase does have a role in cell adhesion, then loss of this protein in clear-cell RCC might contribute or reflect the loss of the polarized phenotype and development of invasive behavior of clear-cell renal-cell carcinoma.

Example 8 - Kit for Detecting Clear Cell Type Renal Carcinoma This example describes an illustrative kit for detecting clear cell type renal carcinoma in a mammal. The illustrative kit contains a tissue solubilization buffer (e.g., 95 mM NaCl, 25 mM Tris, pH 7.4, 0.5 mM EDTA, and 2% SDS); monoclonal antibody M7-PB-E9 or another antibody raised against an α-subunit of Na,K- ATPase (such as a lamb α-subunit of Na,K- ATPase); monoclonal antibody M17-P5-F11 or another antibody raised against an β-subunit of Na,K- ATPase (such as a lamb β-subunit of Na,K- ATPase); endoglycosidase F/N (available, for example, from Boehringer Mannheim); and a glycosidase reaction buffer (e.g., 1 M DTT and 200 mM citrate buffer pH 5.6 and trasyltol 10 mg/ml). At the time of the kit is used, protease inhibitors, such as 1 mM PMSF and 5 μM each of pepsatatin, leupeptin, and antipain, are added to the tissue solubilization buffer. These protease inhibitors can be included in the kit.

The kit can be used according to the following illustrative procedure. First, a cancerous tissue sample from the mammal is cleaned in autoclaved PBS. It is then minced into small pieces, and 1 ml of the solubilization buffer is added for every 100-200 mg of tissue sample. The sample is transferred to a Dounce homogenizer, briefly homogenized, and then sonicated in the cold until the tissue is completely homogenized. The sample is then centrifuged in a microfuge, for example at 14,000 rpm. The supernatant is then used for analysis after the concentration of protein is determined by any suitable method. The procedure is repeated for a normal tissue sample from the mammal to produce a normal tissue lysate. Duplicate aliquots of cancerous tissue lysate and normal tissue lysate, each equivalent to 80 μg of protein, are prepared for endoglycosidase F/N digestion. The volume of the lysate (i.e., the supernatant) is adjusted to 20 μl with the glycosidase reaction buffer. The sample is boiled for 3 minutes and cooled. After cooling, 1 μl of 20% Triton X-100, 2 μl of trasylol (10 mg/ml), and 23 μl of citrate buffer are added. 2 μl of endoglycosidase F/N is then added to one of the normal tissue lysates and to one of the cancerous tissue lysates. The samples are then incubated at 37°C for 10-12 hours. After incubating the sample, 3 μl of 1 M Tris pH 8.0 are added to the samples. They are then run on a 10% SDS-PAGE by standard methods. The gel is then transferred to a nitrocellulose membrane.

Immunoblot analysis is conducted using a 1 :500 dilution of the antibody against Na,K- ATPase's β-subunit and following any conventional procedure. [ I]-Protein A can be advantageously used to visualize the bands.

Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose and variations can be made by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims

WHAT IS CLAIMED:

1. A method for detecting clear cell type renal carcinoma in a mammal comprising: providing a biological sample from the mammal; determining concentration of Na,K- ATPase's ╬▓-subunit in the biological sample; and correlating the concentration of Na,K-ATPase's ╬▓-subunit in the biological sample with the presence of clear cell type renal carcinoma in the mammal.

2. A method according to claim 1, wherein said correlating comprises: comparing the concentration of the Na,K- ATPase's ╬▓-subunit in the biological sample with the concentration of Na,K- ATPase's ╬▓-subunit from normal kidney tissue, where a lower concentration of Na,K- ATPase's ╬▓-subunit in the biological sample relative to normal kidney tissue is indicative of clear cell type renal carcinoma in the mammal.

3. A method according to claim 1, wherein the biological sample is a tissue sample.

4. A method according to claim 3, wherein the tissue sample is a homogenized tissue sample and wherein said providing comprises: isolating the tissue sample from the mammal; and homogenizing the tissue sample.

5. A method according to claim 3, wherein the tissue sample is a kidney tissue sample.

6. A method according to claim 1, wherein said determining comprises: providing a biological agent which binds to Na,K-ATPase's ╬▓-subunit; contacting the biological sample with the biological agent under conditions effective to permit binding of the biological agent to the Na,K- ATPase's ╬▓-subunit in the biological sample; determining the concentration of Na,K- ATPase's ╬▓-subunit in the biological sample by determining the amount of biological agent bound to Na,K- ATPase's ╬▓-subunit in the sample.

7. A method according to claim 6, wherein the biological agent is selected from the group consisting of an antibody or a binding portion thereof.

8. A method according to claim 7, wherein the biological agent is an antibody selected from the group consisting of a polyclonal antibody and a monoclonal antibody.

9. A method according to claim 7, wherein the biological agent is a binding portion of an antibody selected from the group consisting of a Fab fragment, a F(ab') fragment, and a Fv fragment.

10. A method according to claim 6, wherein the biological agent is a labeled biological agent.

11. A method according to claim 10, wherein the labeled biological agent is labeled with a label selected from the group consisting of a fluorescent label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

12. A method for detecting clear cell type renal carcinoma in a mammal comprising: providing a biological sample from the mammal; contacting the biological sample with an agent capable of cleaving N- linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof from Na,K- ATPase's ╬▓-subunit under conditions effective to cleave N-linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof from Na,K- ATPase's ╬▓-subunit in normal kidney tissue; determining concentration of N-linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof in Na,K- ATPase's ╬▓-subunit in the biological sample; and correlating the concentration of N-linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof in Na,K- ATPase's ╬▓-subunit in the biological sample with the presence of clear cell type renal carcinoma in the mammal.

13. A method according to claim 12, wherein said correlating comprises: comparing the concentration N-linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof in Na,K- ATPase's ╬▓-subunit in the biological sample with the concentration of N-linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof in Na,K- ATPase's ╬▓-subunit from normal kidney tissue, where a higher concentration of N-linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof in Na,K- ATPase's ╬▓- subunit in the biological sample relative to normal kidney tissue is indicative of clear cell type renal carcinoma.

14. A method according to claim 12, wherein the biological sample is a tissue sample.

15. A method according to claim 14, wherein the tissue sample is a homogenized tissue sample and wherein said providing comprises: isolating the tissue sample from the mammal; and homogenizing the tissue sample.

16. A method according to claim 14, wherein the tissue sample is a kidney tissue sample.

17. A method according to claim 12, wherein said determining is carried out electrophoretically.

18. A method according to claim 12, wherein the agent capable of cleaving N- linked sugar residues, lactosamine residues, sialic acid residues, or combinations thereof from Na,K- ATPase's ╬▓-subunit is selected from a glycosidase, a galactosidase, a neuraiminidase, and combinations thereof.

19. A method according to claim 12 further comprising: determining concentration of Na,K- ATPase's ╬▓-subunit in the biological sample; and correlating the concentration of Na,K-ATPase's ╬▓-subunit in the biological sample with the presence of clear cell type renal carcinoma in the mammal.

20. A method of detecting clear cell type renal carcinoma in a mammal comprising: providing a tissue section from the mammal; providing a biological agent which binds to Na,K-ATPase; contacting the tissue section with the biological agent under conditions effective to permit binding of the biological agent to Na,K- ATPase in the tissue section; detecting the Na,K- ATPase's spatial distribution in the tissue section by detecting the biological agent's spatial distribution in the tissue; and correlating the Na,K-ATPase's spatial distribution in the tissue section to the presence of clear cell type renal carcinoma in the mammal.

21. A method according to claim 20, wherein the biological agent which binds to Na,K- ATPase is a biological agent which binds Na,K- ATPase's ╬▒-subunit.

22. A method according to claim 20, wherein the biological agent which binds to Na,K- ATPase is a biological agent which binds Na,K- ATPase's ╬▓-subunit.

23. A method according to claim 20, wherein the tissue section is a kidney tissue section.

24. A method according to claim 20, wherein the biological agent is selected from the group consisting of an antibody or a binding portion thereof.

25. A method according to claim 24, wherein the biological agent is an antibody selected from the group consisting of a polyclonal antibody and a monoclonal antibody.

26. A method according to claim 24, wherein the biological agent is a binding portion of an antibody selected from the group consisting of a Fab fragment, a F(ab')₂ fragment, and a Fv fragment.

27. A method according to claim 20, wherein the biological agent is a labeled biological agent.

28. A method according to claim 27, wherein the labeled biological agent is labeled with a label selected from the group consisting of a fluorescent label, a radioactive label, a nuclear magnetic resonance active label, a luminescent label, and a chromophore label.

29. A kit for detecting clear cell type renal carcinoma comprising: an agent capable of cleaving N-linked sugar residues, lactosamine residues, sialic acid, or combinations thereof from Na,K- ATPase's ╬▓-subunits; and a biological agent which binds to Na,K-ATPase.

30. A kit according to claim 29 further comprising: a means to detect the biological agent.

31. A kit according to claim 29, wherein the biological agent which binds to Na,K-ATPase is a biological which binds to Na,K-ATPase's ╬▒-subunit, Na,K- ATPase's ╬▓- subunit, or combinations thereof.