Abstract:
A protein family includes four proteins that are bioactive against human tumor cell lines. The proteins are derived from eggs of the  Rana pipiens  frog, and are members of the superfamily of pancreatic ribonucleases.

Description:
BACKGROUND OF THE INVENTION 
     The invention relates to pharmaceuticals, and more particularly relates to pharmaceuticals for use in treating tumors in humans. 
     One object of the invention is to provide a family of pharmaceuticals for use in treating tumors in humans. 
     Another object is, in general, to improve upon known therapies for treatment of tumors in humans. 
     In accordance with the invention, there is a provided a family of highly-homologous and closely related proteins. The family is exemplified by four purified proteins, each having a) an amino acid sequence that is 114 amino acids long, b) an isoelectric point of approximately 10, and c) a molecular weight of approximately 13,000. Advantageously although not necessarily, the proteins are derived from frog eggs and preferably from the eggs of the  Rana pipiens  frog. 
     The above-referenced four purified proteins are glycoproteins, i.e. each contains glycans (carbohydrates) that are linked to the protein by covalent bonds. However, the glycan moieties are not necessary for the bioactivity of the proteins against human tumor cell lines. 
     U.S. Pat. Nos. 5,559,212 and 5,728,805 disclose two protein peaks coming off a cationic-exchange chromatography column. U.S. Pat. No. 5,559,212 describes the amino acid sequence and composition of the protein in the first (larger) peak, and the disclosure of U.S. Pat. No. 5,728,805 extends this description by describing the amino acid sequence and composition of the protein in the second (smaller) peak. A third protein peak also comes off the ion-exchange column, and this (still smaller) peak has now been shown to contain four proteins that are sufficiently similar to justify their designation as a family. Because the third protein peak is eluted after the first and second peaks, the proteins in that peak are more basic. 
     Tests have shown that each of the four proteins possesses bioactivity against two human carcinoma cell lines. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The invention will be better understood with reference to the following illustrative and non-limiting drawings, in which: 
     FIG. 1 is a flow chart showing how the four proteins are produced and purified; 
     FIG. 2 shows the results of the cationic exchange chromatography step and illustrates the fractions from which the four proteins are isolated and purified; 
     FIG. 3 shows the results of the reversed-phase high performance liquid chromatography step, and illustrates the fractions that contained the four proteins herein described; 
     FIG. 4 shows the amino acid sequence of the first of the four proteins herein described, namely the protein having SEQ ID NO: 1 as its amino acid sequence; 
     FIG. 5 shows the amino acid sequence of the second of the four proteins herein described, namely the protein having SEQ ID NO: 2 as its amino acid sequence; 
     FIG. 6 shows the amino acid sequence of the third of the four proteins herein described, namely the protein having SEQ ID NO: 3 as its amino acid sequence; 
     FIG. 7 shows the amino acid sequence of the fourth of the four proteins herein described, namely the protein having SEQ ID NO: 4 as its amino acid sequence; and 
     FIG. 8 illustrates the homology between the four proteins herein described. 
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     Initially, and in accordance with the preferred embodiment shown in FIG. 1,  Rana pipiens  eggs are accumulated (step 10), homogenized in a weakly acidic buffer (step 20), repeatedly frozen and thawed (step 30), centrifuged and filtered (step, 40). The accumulation, homogenization, freezing, and thawing steps are described at column 3, lines 1 to 54. 103 g of thawed  Rana pipiens  eggs were combined at room temperature with 206 mL of 0.15 M sodium acetate buffer, pH 5.0. This mixture was then homogenized in a Waring Blender for 3 minutes at high speed. The resulting homogenate was repeatedly (four times) frozen to −20 °C. (±10° C.) and thawed. The thawed homogenate was then centrifuged at 10,000×g for 25 minutes, and the supernatent was then filtered through a filter paper (Whatman, No. 541) yielding 248 mL of clear extract, which had 1455 absorbance units of UV light having a wavelength of 280 nm. 
     The 248 mL of clear extract is then (step 50) subjected to cationic exchange chromatography, essentially as described in U.S. Pat. No. 5,728,805 at column 6, lines 50-64. Specifically, the clear extract was loaded on a chromatographic column packed with SP Sepharose FF (Pharmacia) resin to form a bed that is 2.5 cm in diameter and 6.7 cm long. The packed column was equilibrated in 0.15 M sodium acetate buffer, pH 5.0 and the chromatography was carried out at room temperature with the extract being loaded onto the column at a flow rate of 3 mL/min. The packed column was first washed with the equilibrating buffer and then (beginning at fraction 78) developed with a continuous (from 0 M to 0.46 M) sodium chloride gradient made in the equilibrating buffer. Eluate fractions of 4.8 mL were automatically collected while continuously monitoring the absorption of 280 nm UV light. The total volume of the gradient was 800 mL. 
     The eluate collected in the first 77 fractions is inactive, and was discarded. The protein in the first (largest) protein peak eluted in fractions 120 to 133 is described in U.S. Pat. No. 5,559,212, and the protein in the second (smaller) protein peak eluted in fractions 158 to 166 is described in U.S. Pat. No. 5,728,805. The proteins herein described are contained in the third (smallest) protein peak eluted in fractions 194 to 210. The total quantity of “third peak” material eluted in fractions 194 to 210 was 16.6 absorbance units of 280 nm UV light (about 1.14% of the material loaded onto the column), corresponding to about 25 mg. 
     The eluted “third peak” material from fractions 194 to 210 was then subjected to reversed-phase high performance liquid chromatography (step 60 in FIG.  1 ). The chromatography was conducted on a Zorbax SB300 C18 column having a diameter of 9.4 mm and a length of 250 mm. The column was attached to a Hewlett-Packard Series 1090 Liquid Chromatograph. 
     The eluted “third peak” material was loaded onto the column in 5 mg aliquots, at a temperature of 35° C. The column was equilibrated in 0.1% (v/v) trifluoroacetic acid (Buffer A) and developed with a gradient of acetonitrile/0.1% trifluoroacetic acid (Buffer B). The following elution method was employed after the loading: from the beginning of the chromatography to six minutes afterward, the slope of the gradient was increased from 20% Buffer B to 24.4% Buffer B. Thereafter, from minute 6 until minute 24, the slope of the gradient was increased from 24.4% Buffer B to 30% Buffer B. Protein peaks were collected manually. Equivalent peaks from 5 different runs (i.e. 5 aliquots of 5 mg each) were combined. 
     An exemplary chromatographic pattern is presented in FIG.  3 . The material eluted between 4.5 and 13.5 min of the run had no cytotoxic activity and was discarded. The proteins contained in the five major peaks eluted between 13.5 and 21 min proved to be bioactive as discussed below. These proteins were collected as indicated by the solid bars in FIG.  3  and were further purified. 
     The major peak (denoted 2325p4 in FIG. 3) eluted at minutes 16 to 17 contained the protein (SEQ ID NO:1) most represented in the source. The small peak (denoted 2325p4a in FIG. 3) eluted at about 14 min contained a highly similar protein (SEQ ID NO:2) differing therefrom by only one amino acid residue. The peak eluted between minutes 14.5 and 15.5 contained a mixture of these two proteins. The remaining two peaks (denoted, respectively, 2325p6 and 2728 in FIG. 3) eluting from the column at 17 to 18.5 minutes and 19.5 to 20.5 minutes, respectively, contained two other proteins (SEQ ID NO:3 and SEQ ID NO:4, respectively). 
     All the isolated proteins were re-chromatographed (step 70 in FIG. 1) on the same column under identical conditions (patterns not shown) and lyophilized (step 80). The final yield of the individual proteins (from five runs) was as follows: SEQ ID NO:1, 5.7 mg (from the peak eluted from minute 14 to minute 16); SEQ ID NO:2, 1 mg (from the peak eluted at about 14 minutes); SEQ ID NO:3, 6.5 mg; SEQ ID NO:4, 1.3 mg. The peak that contained a mixture of the SEQ ID NO:1 and the SEQ ID NO:2 proteins yielded 4.6 mg. 
     The proteins isolated by the procedure described above were chemically and biologically characterized using amino acid sequencing and cytotoxicity studies. At the scale described above, the purification process was repeated several times to generate sufficient material. 
     FIGS. 4-7 show the amino acid sequences of the four proteins that were isolated and purified as discussed above. As can be seen in FIG. 8, the four proteins are so similar that they can be said to define a family. 95 residues (i.e. 83.3% of all 114 residues) are identical in all four proteins. The remaining 19 positions, namely positions 6, 9, 13, 27, 28, 32, 34, 35, 44, 51, 57, 63, 75, 80, 81, 83, 92, 95, and 111, are polymorphic. Only one residue is different between 2325p4 and 2325p4a (SEQ ID NO:1 and SEQ ID NO:2), so those proteins are 99.1% homologous. Other side-to-side comparisons revealed the following percentages of homology: 
     2325p6 vs 2728: 89.5% 
     2325p4 vs 2325p6: 87.7% 
     2325p4a vs 2728: 88.6% 
     2325p4 vs 2728: 87.7% 
     2325p4a vs 2325p6: 86.8%. 
     Version BLASTP of the BLAST (Basic Local Alignment Search Tool) program was used to check the relationships between the four proteins and 300,806 known protein sequences at the National Center for Biotechnology Information. This search revealed that each of the four proteins, and therefore the family in which they are included, belongs to the pancreatic ribonuclease A superfamily. 
     Tables 1-5 below show the amino acid compositions of the referenced four proteins; Tables 1-4 show mole percentages after hydrolysis, while Table 5 shows the number of amino acid residues per molecule. 
     Compositional analysis of these four proteins was carried out using a pre-column derivatization method with 6-Aminoqinolyl-N-Hydroxysuccinimidyl Carbamate. Derivatized amino acids were identified and quantified by a reversed phase chromatography on a C8 column (Zorbax, Eclipse XDB 2.1×150 mm) using a Hewlett-Packard Series 1090 Liquid Chromatograph. Prior to manual hydrolysis in 6.0 M hydrochloric acid (21 h at 105-110° C. in evacuated tubes) the protein samples were denatured, their disulfide bonds were reduced and the newly formed sulfhydryl groups were alkylated with 4-vinyl pyridine. The purpose of this sample preparation was to protect cysteine, which is largely destroyed during acid hydrolysis. The apparent cysteine content is usually somewhat lowered even in alkylated samples, because alkylation does not proceed to completion. Tryptophan, another amino acid destroyed by acid hydrolysis, was not determined. The values for serine, threonine, and methionine, which all undergo partial destruction during acid hydrolysis, were uncorrected. Also uncorrected were values for isoleucine (peptide bonds formed by this amino acid are more resistant to hydrolysis than are ordinary peptide bonds). 
     
       
         
               
             
               
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Amino Acid Composition of (2325p4) Protein 
               
               
                 SEQ ID NO: 1 
               
             
          
           
               
                   
                 Mole % 
                   
               
               
                   
                 (21 hour hydrolysis) 
               
             
          
           
               
                   
                 Amino Acid Residue 
                 Found 
                 From Sequence 
               
               
                   
                   
               
             
          
           
               
                   
                 Alanine 
                 2.68 
                 2.63 
               
               
                   
                 Arginine 
                 6.00 
                 6.14 
               
               
                   
                 Aspartic acid/Asparagine 
                 13.35 
                 12.28 
               
               
                   
                 Cysteine 1   
                 6.44 
                 7.02 
               
               
                   
                 Glutamic acid/Glutamine 
                 8.48 
                 7.89 
               
               
                   
                 Glycine 
                 3.80 
                 3.51 
               
               
                   
                 Histidine 
                 2.41 
                 2.63 
               
               
                   
                 Isoleucine 2   
                 6.05 
                 6.14 
               
               
                   
                 Leucine 
                 0.93 
                 0.88 
               
               
                   
                 Lysine 
                 10.43 
                 10.53 
               
               
                   
                 Methionine 3   
                 0.64 
                 0.88 
               
               
                   
                 Phenylalanine 
                 5.39 
                 5.26 
               
               
                   
                 Proline 
                 6.93 
                 6.14 
               
               
                   
                 Serine 3   
                 6.05 
                 6.14 
               
               
                   
                 Threonine 3   
                 13.38 
                 14.04 
               
               
                   
                 Tryptophan 
                 ND 
                 0.88 
               
               
                   
                 Tyrosine 
                 2.61 
                 2.63 
               
               
                   
                 Valine 
                 4.42 
                 4.39 
               
               
                   
                 Total 
                 99.99 
                 100.00 
               
               
                   
                   
               
               
                   
                 ND, not determined;  
               
               
                   
                   1 as a pyridylethyl derivative;  
               
               
                   
                   2 ucorrected for incomplete hydrolysis;  
               
               
                   
                   3 uncorrected for destruction.  
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Amino Acid Composition of (2325p4a) Protein 
               
               
                 SEQ ID NO: 2 
               
             
          
           
               
                   
                 Mole % 
                   
               
               
                   
                 (21 hour hydrolysis) 
               
             
          
           
               
                   
                 Amino Acid Residue 
                 Found 
                 From Sequence 
               
               
                   
                   
               
             
          
           
               
                   
                 Alanine 
                 2.86 
                 2.63 
               
               
                   
                 Arginine 
                 6.31 
                 6.14 
               
               
                   
                 Aspartic acid/Asparagine 
                 12.71 
                 12.28 
               
               
                   
                 Cysteine 1   
                 6.44 
                 7.02 
               
               
                   
                 Glutamic acid/Glutamine 
                 8.26 
                 7.89 
               
               
                   
                 Glycine 
                 4.32 
                 3.51 
               
               
                   
                 Histidine 
                 2.40 
                 2.63 
               
               
                   
                 Isoleucine 2   
                 5.14 
                 5.26 
               
               
                   
                 Leucine 
                 1.08 
                 0.88 
               
               
                   
                 Lysine 
                 9.90 
                 10.53 
               
               
                   
                 Methionine 3   
                 0.72 
                 0.88 
               
               
                   
                 Phenylalanine 
                 5.37 
                 5.26 
               
               
                   
                 Proline 
                 6.57 
                 6.14 
               
               
                   
                 Serine 3   
                 6.07 
                 6.14 
               
               
                   
                 Threonine 3   
                 13.96 
                 14.04 
               
               
                   
                 Tryptophan 
                 ND 
                 0.88 
               
               
                   
                 Tyrosine 
                 2.68 
                 2.63 
               
               
                   
                 Valine 
                 5.21 
                 5.26 
               
               
                   
                 Total 
                 100.00 
                 99.99 
               
               
                   
                   
               
               
                   
                 ND, not determined;  
               
               
                   
                   1 as a pyridylethyl derivative;  
               
               
                   
                   2 ucorrected for incomplete hydrolysis;  
               
               
                   
                   3 uncorrected for destruction.  
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Amino Acid Composition of (2325p6) Protein 
               
               
                 SEQ ID NO: 3 
               
             
          
           
               
                   
                 Mole % 
                   
               
               
                   
                 (21 hour hydrolysis) 
               
             
          
           
               
                   
                 Amino Acid Residue 
                 Found 
                 From Sequence 
               
               
                   
                   
               
             
          
           
               
                   
                 Alanine 
                 1.97 
                 1.75 
               
               
                   
                 Arginine 
                 4.50 
                 4.39 
               
               
                   
                 Aspartic acid/Asparagine 
                 14.37 
                 13.16 
               
               
                   
                 Cysteine 1   
                 6.23 
                 7.02 
               
               
                   
                 Glutamic acid/Glutamine 
                 9.37 
                 8.77 
               
               
                   
                 Glycine 
                 3.77 
                 3.51 
               
               
                   
                 Histidine 
                 2.32 
                 2.63 
               
               
                   
                 Isoleucine 2   
                 6.07 
                 6.14 
               
               
                   
                 Leucine 
                 0.95 
                 0.88 
               
               
                   
                 Lysine 
                 11.76 
                 12.28 
               
               
                   
                 Methionine 3   
                 0.74 
                 0.88 
               
               
                   
                 Phenylalanine 
                 5.38 
                 5.26 
               
               
                   
                 Proline 
                 7.56 
                 7.02 
               
               
                   
                 Serine 3   
                 7.44 
                 7.89 
               
               
                   
                 Threonine 3   
                 9.70 
                 9.65 
               
               
                   
                 Tryptophan 
                 ND 
                 0.88 
               
               
                   
                 Tyrosine 
                 2.68 
                 2.63 
               
               
                   
                 Valine 
                 5.20 
                 5.26 
               
               
                   
                 Total 
                 100.01 
                 100.00 
               
               
                   
                   
               
               
                   
                 ND, not determined;  
               
               
                   
                   1 as a pyridylethyl derivative;  
               
               
                   
                   2 ucorrected for incomplete hydrolysis;  
               
               
                   
                   3 uncorrected for destruction.  
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Amino Acid Composition of (2728) Protein 
               
               
                 SEQ ID NO: 4 
               
             
          
           
               
                   
                 Mole % 
                   
               
               
                   
                 (21 hour hydrolysis) 
               
             
          
           
               
                   
                 Amino Acid Residue 
                 Found 
                 From Sequence 
               
               
                   
                   
               
             
          
           
               
                   
                 Alanine 
                 2.74 
                 2.63 
               
               
                   
                 Arginine 
                 4.52 
                 4.39 
               
               
                   
                 Aspartic acid/Asparagine 
                 15.36 
                 14.91 
               
               
                   
                 Cysteine 1   
                 6.77 
                 7.02 
               
               
                   
                 Glutamic acid/Glutamine 
                 6.50 
                 6.14 
               
               
                   
                 Glycine 
                 3.76 
                 3.51 
               
               
                   
                 Histidine 
                 2.47 
                 2.63 
               
               
                   
                 Isoleucine 2   
                 6.19 
                 6.14 
               
               
                   
                 Leucine 
                 0.89 
                 0.88 
               
               
                   
                 Lysine 
                 11.96 
                 12.28 
               
               
                   
                 Methionine 3   
                 0.85 
                 0.88 
               
               
                   
                 Phenylalanine 
                 6.26 
                 6.14 
               
               
                   
                 Proline 
                 7.51 
                 7.02 
               
               
                   
                 Serine 3   
                 6.66 
                 7.02 
               
               
                   
                 Threonine 3   
                 9.50 
                 9.65 
               
               
                   
                 Tryptophan 
                 ND 
                 0.88 
               
               
                   
                 Tyrosine 
                 1.85 
                 1.75 
               
               
                   
                 Valine 
                 6.22 
                 6.14 
               
               
                   
                 Total 
                 100.01 
                 100.02 
               
               
                   
                   
               
               
                   
                 ND, not determined;  
               
               
                   
                   1 as a pyridylethyl derivative;  
               
               
                   
                   2 ucorrected for incomplete hydrolysis;  
               
               
                   
                   3 uncorrected for destruction.  
               
             
          
         
       
     
     
       
         
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Amino Acid Composition of Four Proteins 
               
               
                 (as calculated from their amino acid sequences) 
               
             
          
           
               
                   
                 Number of Residues Per Molecule 
               
             
          
           
               
                   
                 Amino Acid 
                 (2325p4) 
                 (2325p4a) 
                 (2325p6) 
                 (2728) 
               
               
                   
                   
               
               
                   
                 Alanine 
                 3 
                 3 
                 2 
                 3 
               
               
                   
                 Arginine 
                 7 
                 7 
                 5 
                 5 
               
               
                   
                 Asparagine 
                 9 
                 9 
                 10  
                 10  
               
               
                   
                 Aspartic acid 
                 5 
                 5 
                 5 
                 7 
               
               
                   
                 Cysteine 
                 8 
                 8 
                 8 
                 8 
               
               
                   
                 Glutamine 
                 5 
                 5 
                 5 
                 5 
               
               
                   
                 Glutamic acid 
                 4 
                 4 
                 5 
                 2 
               
               
                   
                 Glycine 
                 4 
                 4 
                 4 
                 4 
               
               
                   
                 Histidine 
                 3 
                 3 
                 3 
                 3 
               
               
                   
                 Isoleucine 
                 7 
                 6 
                 7 
                 7 
               
               
                   
                 Leucine 
                 1 
                 1 
                 1 
                 1 
               
               
                   
                 Lysine 
                 12  
                 12  
                 14  
                 14  
               
               
                   
                 Methionine 
                 1 
                 1 
                 1 
                 1 
               
               
                   
                 Phenylalanine 
                 6 
                 6 
                 6 
                 7 
               
               
                   
                 Proline 
                 7 
                 7 
                 8 
                 8 
               
               
                   
                 Serine 
                 7 
                 7 
                 9 
                 8 
               
               
                   
                 Threonine 
                 16  
                 16  
                 11  
                 11  
               
               
                   
                 Tryptophan 
                 1 
                 1 
                 1 
                 1 
               
               
                   
                 Tyrosine 
                 3 
                 3 
                 3 
                 2 
               
               
                   
                 Valine 
                 5 
                 6 
                 6 
                 7 
               
               
                   
                 Total 
                 114  
                 114  
                 114  
                 114  
               
               
                   
                   
               
             
          
         
       
     
     As stated above, these proteins are glycoproteins, which are heterogeneous on polyacrylamide gel electrophoresis. For this reason, polyacrylamide gel electrophoresis can only produce a range of their apparent molecular weights. 
     An apparent molecular weight range of 13,000 to 20,000 was estimated by SDS-PAGE (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) using a 10%-20% gradient gel and a Tricine buffer system. This range represents the molecular weight of entire molecules of each protein, i.e. represents the molecular weight not only of the protein itself but of the glycans that are attached to it. 
     Molecular weight values of the protein moieties of each protein were calculated from its complete amino acid sequences (FIGS. 4-7) assuming that all eight cysteine residues were paired to form four disulfide bridges. 
     The isolectric point of each protein was also calculated from each sequence using the same assumption of cysteine pairing. Molecular weights and isoelectric points were calculated using the Protein Tools Version 5.04 computer program (1996), Window Chem Software, Inc. The values are presented in Table 6. 
     As can be seen from Table 6, the molecular weight of each of the proteins is approximately 13,000. The isoelectric point of each of these proteins is approximately 10, which agrees well with their behavior on a cationic exchange column (FIG.  2 ). 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Physical Parameters of Proteins 
               
               
                 (as calculated from their amino acid sequences) 
               
             
          
           
               
                   
                 Protein 
                 Molecular Weight 
                 Isoelectric Point 
               
               
                   
                   
               
             
          
           
               
                   
                 2325p4 
                 13077 
                 10.16 
               
               
                   
                 2325p4a 
                 13063 
                 10.16 
               
               
                   
                 2325p6 
                 13058 
                 9.95 
               
               
                   
                 2728 
                 12968 
                 10.10 
               
               
                   
                   
               
             
          
         
       
     
     As stated above, all four proteins are glycoproteins, i.e. each contains glycans (carbohydrates) that are linked to the protein by covalent bonds. These glycan moieties are not essential for bioactivity. Glycan content apparently varies from one protein to another as well as from one molecule of a protein to another molecule of the same protein. As estimated using the Glycoprotein Carbohydrate Estimation Kit (Pierce), each protein contains at least several percent glycans. 
     Each protein is glycosylated at more than one site. Glycan moieties were found attached to asparagine residues (N-glycosylation). To date, two glycosylation sites have been determined for each protein. They are Asn 27 and 91 in proteins 2325p4, 2325p4a, and 2728 (SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4 respectively). In protein 2325p6 (SEQ ID NO:3) glycosylated residues are Asn 25 and 91. It appears that the size and the chemical nature of the glycan moieties vary from one molecule of a particular protein to another. 
     The herein-described proteins were tested for bioactivity against two human carcinoma cell lines, namely human submaxillary gland carcinoma (A-253) and human bladder carcinoma (T-24). The results of these tests are shown in Tables 7 and 8. Ranpirnase (known by its trademark ONCONASE and disclosed in U.S. Pat. No. 5,559,212) and bovine ribonuclease A served, respectively, as positive and negative controls. 
     As can be seen in Tables 7 and 8, each protein strongly inhibited growth of each cell line; A-253 human submaxillary gland carcinoma cells turned out to be more responsive. Antiproliferative/cytotoxic activities of the proteins were comparable to that of Ranpirnase. Ribonuclease A had only a negligible effect even when used at substantially higher concentrations. 
     
       
         
               
             
               
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Effect Of Proteins and Related Ribonucleases on Growth Of 
               
               
                 Human Submaxillary Gland Carcinoma (A-253) Cells 
               
             
          
           
               
                   
                 GROWTH INHIBITION 
                   
               
             
          
           
               
                   
                 PROTEIN 
                 MEAN % 
                 (SD) 
               
               
                   
                   
               
             
          
           
               
                   
                 2325p4 
                 97.1 
                 (0.13) 
               
               
                   
                 2325p4a 
                 88.0 
                 (0.56) 
               
               
                   
                 2325p6 
                 98.2 
                 (0.74) 
               
               
                   
                 2728 
                 90.6 
                 (4.99) 
               
               
                   
                 Ranpirnase 
                 85.5 
                 (4.91) 
               
               
                   
                 Ribonuclease A 
                 8.0 
                 (6.61) 
               
               
                   
                   
               
               
                   
                 Mean values of four experiments  
               
               
                   
                 Protein concentrations were 1.0 μg/mL except Ribonuclease A (which was 10 μg/mL).  
               
               
                   
                 SD = standard deviation.  
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Effect Of Proteins and Ranpirnase 
               
               
                 On Growth of Human Bladder Carcinoma (T-24) Cells 
               
             
          
           
               
                   
                 GROWTH INHIBITION 
                   
               
             
          
           
               
                   
                 PROTEIN 
                 MEAN % 
                 (SD) 
               
               
                   
                   
               
             
          
           
               
                   
                 2325p4 
                 63.9 
                 (11.9) 
               
               
                   
                 2325p4a 
                 68.4 
                 (10.2) 
               
               
                   
                 2325P6 
                 71.9 
                 (8.0) 
               
               
                   
                 2728 
                 61.5 
                 (8.7) 
               
               
                   
                 Ranpirnase 
                 68.5 
                 (9.5) 
               
               
                   
                   
               
               
                   
                 Mean values of four experiments  
               
               
                   
                 Protein concentrations were 2.0 μg/mL.  
               
               
                   
                 SD = standard deviation.  
               
             
          
         
       
     
     
       
         
           
             4 
           
           
             1 
             114 
             PRT 
             Rana pipiens 
           
            1
Lys Pro Lys Glu Asp Arg Glu Trp Glu Lys Phe Lys Thr Lys His Ile
  1               5                  10                  15
Thr Ser Gln Ser Val Ala Asp Phe Asn Cys Asn Arg Thr Met Asn Asp
             20                  25                  30
Pro Ala Tyr Thr Pro Asp Gly Gln Cys Lys Pro Ile Asn Thr Phe Ile
         35                  40                  45
His Ser Thr Thr Gly Pro Val Lys Glu Ile Cys Arg Arg Ala Thr Gly
     50                  55                  60
Arg Val Asn Lys Ser Ser Thr Gln Gln Phe Thr Leu Thr Thr Cys Lys
 65                  70                  75                  80
Asn Pro Ile Arg Cys Lys Tyr Ser Gln Ser Asn Thr Thr Asn Phe Ile
                 85                  90                  95
Cys Ile Thr Cys Arg Asp Asn Tyr Pro Val His Phe Val Lys Thr Gly
            100                 105                 110
Lys Cys 
           
             2 
             114 
             PRT 
             Rana pipiens 
           
            2
Lys Pro Lys Glu Asp Arg Glu Trp Glu Lys Phe Lys Thr Lys His Ile
  1               5                  10                  15
Thr Ser Gln Ser Val Ala Asp Phe Asn Cys Asn Arg Thr Met Asn Asp
             20                  25                  30
Pro Ala Tyr Thr Pro Asp Gly Gln Cys Lys Pro Val Asn Thr Phe Ile
         35                  40                  45
His Ser Thr Thr Gly Pro Val Lys Glu Ile Cys Arg Arg Ala Thr Gly
     50                  55                  60
Arg Val Asn Lys Ser Ser Thr Gln Gln Phe Thr Leu Thr Thr Cys Lys
 65                  70                  75                  80
Asn Pro Ile Arg Cys Lys Tyr Ser Gln Ser Asn Thr Thr Asn Phe Ile
                 85                  90                  95
Cys Ile Thr Cys Arg Asp Asn Tyr Pro Val His Phe Val Lys Thr Gly
            100                 105                 110
Lys Cys 
           
             3 
             114 
             PRT 
             Rana pipiens 
           
            3
Lys Pro Lys Glu Asp Lys Glu Trp Glu Lys Phe Lys Val Lys His Ile
  1               5                  10                  15
Thr Ser Gln Ser Val Ala Asp Phe Asn Cys Thr Ser Thr Met Asn Asn
             20                  25                  30
Pro Asp Phe Thr Pro Asp Gly Gln Cys Lys Pro Ile Asn Thr Phe Ile
         35                  40                  45
His Ser Asn Thr Gly Pro Val Lys Glu Ile Cys Arg Arg Ala Ser Gly
     50                  55                  60
Arg Val Asn Lys Ser Ser Thr Gln Gln Phe Pro Leu Thr Thr Cys Lys
 65                  70                  75                  80
Asn Pro Lys Arg Cys Lys Tyr Ser Gln Ser Asn Glu Thr Asn Tyr Ile
                 85                  90                  95
Cys Ile Thr Cys Arg Asp Asn Tyr Pro Val His Phe Val Lys Ile Gly
            100                 105                 110
Lys Cys 
           
             4 
             114 
             PRT 
             Rana pipiens 
           
            4
Lys Pro Lys Glu Asp Lys Glu Trp Val Lys Phe Lys Ala Lys His Ile
  1               5                  10                  15
Thr Ser Gln Ser Val Ala Asp Phe Asn Cys Asn Lys Thr Met Asn Asp
             20                  25                  30
Pro Asp Phe Thr Pro Asp Gly Gln Cys Lys Pro Val Asn Thr Phe Ile
         35                  40                  45
His Ser Asn Thr Gly Pro Val Lys Asp Ile Cys Arg Arg Ala Ser Gly
     50                  55                  60
Arg Val Asn Lys Ser Ser Thr Gln Gln Phe Pro Leu Thr Thr Cys Asn
 65                  70                  75                  80
Lys Pro Ile Arg Cys Lys Tyr Ser Gln Ser Asn Thr Thr Asn Phe Ile
                 85                  90                  95
Cys Ile Thr Cys Arg Asp Asn Tyr Pro Val His Phe Val Lys Ile Gly
            100                 105                 110
Lys Cys 
         
       
     
     Although preferred embodiments of the invention have been described above, the scope of the invention is limited only by the following claims: