Abstract:
A new human gene designated as &#34;CRIPTO&#34; gene has been identified and cloned. CRIPTO gene products and derivatives thereof have been obtained and various utilities of the same have been described. Association of CRIPTO gene with cancers, such as colorectal cancer and breast carcinoma, has been indicated.

Description:
BACKGROUND OF THE INVENTION 
     The present invention is related generally to the isolation and cloning of genes and obtaining products encoded by the gene. More particularly, the present invention is related to the isolation, cloning, sequencing and expression of the human CRIPTO gene and producing an isolated, substantially pure gene products including mRNA and recombinant CRIPTO protein. 
     &#34;CRIPTO&#34; is a new human gene which has never been previously described. The gene has been isolated, cloned and completely sequenced. FIG. 1 shows the nucleotide sequence of the CRIPTO cDNA and the amino acid sequence deduced therefrom. FIG. 1 also shows the amino acid sequence of the natural CRIPTO protein and FIG. 2 the amino acid sequence of the recombinant E. coli derived CRIPTO protein. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 (which consists of FIGS. 1A through 1C) shows the nucleotide sequence of the human CRIPTO gene cDNA and the corresponding amino acid sequence. 
     FIG. 2 shows the amino acid sequence of the human CRIPTO protein as it is recombinantly reproduced in E. coli. 
     FIG. 3 is a comparison of the amino acid sequence of the human CRIPTO gene to several prior art proteins. 
     FIG. 4 demonstrates the focus forming activity of the human CRIPTO gene when transfected into NIH 3T3 cells. 
     FIG. 5 is a Northern blot showing expression of the CRIPTO gene by various human colon tumor cell lines. 
     FIG. 6 is a Northern blot demonstrating that the CRIPTO gene is not expressed in normal human colon tissue. 
    
    
     DETAILED DESCRIPTION 
     Isolation and Characterization of Human CRIPTO cDNA 
     In screening 3×10 7  independent clones of a human teratocarcinoma NT2D1 cell line cDNA library that was expressed in λgt10 and that was originally derived from NT2D1 poly(A)+RNA to isolate a full-length glucose-6-phosphate dehydrogenase (G6Pd) cDNA, 16 different clones were identified (Persico et al., Nucleic Acid Research, 14:2511-2522, 1986). One of these clones exceeded the expected size for the G6PD mRNA. Restriction mapping and sequencing showed that the aberrant cDNA which was approximately 5 kb in length to be a composite of two separate coding entities. A nucleotide segment of 2.8 kb corresponded to G6PD while the remaining 2.2 kb fragment (16B6 cDNA) had no relationship to the G6PD gene. The 16B6 cDNA fragment was used to probe the same NT2D1 cDNA library to isolate a full-length cDNA. 
     From several positive clones, 10 clones were isolated and subcloned into pUC18 after EcoR1 digestion. Analysis by restriction enzyme mapping and agarose gel electrophoresis demonstrated that the size of the various cDNA inserts varied from about 0.9 Kb to 2.0 Kb. The two largest cDNA clones, p3B2 and p1C1, and the shortest p2B3, were sequenced by the Sanger method. The complete nucleotide sequence has been deposited in the EMBL Gene Data Bank. The open reading frame of 564 base pairs codes for a protein of 188 amino acids in length (FIG. 1). Proteolytic cleavage sites are present in this protein designated CRIPTO at V-A (amino acid residues 28-29 and 159-160), R-K (residues 111-112), K-K (residues 126-127) and R-T-T-T (residues 171-174). One potential asparagine glycosylation sequence (Asn-Arg-Thr) is present at residues 79-81. 
     Production and Purification of Recombinant CRIPTO Protein in E. Coli 
     The buffers are prepared as follows: 
     Buffer A: 25% sucrose, 10 mM Tris-HCl (pH 8.0), ImM EDTA, 150 mM NaCl and 10 μg lysozyme. 
     Buffer B: 10 mM Tris-HCl (pH 7.6), ImM EDTA and 0.5% Triton X-100. 
     Buffer C: 0.1% SDS. 0.05M Tris-HCl (pH 8.0), 0.ImM EDTA, 5 mM DTT and 0.20M NaCl. 
     1. Grow an inoculum of suitably transformed strain of bacteria in LB broth containing 100 μg/ml of ampicillin overnight at about 32° C. 
     2. Dilute the bacterial culture 100-fold in LB broth and grow at 32° C. until OD 600  reaches 0.2. 
     3. Shift the bacterial culture to 44° C. for 20 minutes and then to 42° C. for 4 hours until OD 600  reaches 1.7. 
     4. Spin 50 ml of the bacterial culture at 5,000 g for 10 minutes at 4° C. and resuspend the bacterial pellet in 10 ml LB broth at room temperature (RT). 
     5. To 10 ml of frozen buffer A add 10 ml of bacterial suspension and defrost at RT prior to incubation for 15 minutes at 37° C. 
     6. Spin at 27,000 g for 10 minutes at 4° C. 
     7. Resuspend the pellet in 3 ml of buffer B and spin at 15000 rpm for 15 minutes at 4° C. 
     8. Repeat step #7 three times, saving the supernatant each time. 
     9. Sonicate the final spheroplast suspension 6 times for 30 seconds at 40 watts. 
     10. Divide into 4 Eppendorff tubes and spin at RT in a microfuge at 12,000 g for 10 minutes. 
     11. Discard the supernatant and resuspend the pellet of inclusion bodies in 1 ml of IM urea. Incubate for 30 minutes at 37° C. Spin 10 minutes in microfuge at 12,000 g. 
     12. Repeat step #11 twice. 
     13. Each pellet of the inclusion bodies is then dissolved in 200 μl of Laemmli sample buffer and analyzed by SDS-PAGE. Alternatively, resuspend inclusion body pellets in 600 μl of buffer C to solubilize the recombinant CRIPTO protein. 
     Recombinant CRIPTO Protein Characterization 
     1. The CRIPTO cDNA is used to produce a recombinant CRIPTO protein in E. coli as described above. The amino acid sequence of the CRIPTO protein is shown in FIG. 2. The inclusion body pellets are resuspended in 600 μl of buffer C and incubated at 37° C. for 18 hours to achieve almost 100% solubilization of the CRIPTO protein. 
     2. A partial solubilization is achieved in either 0.1M Tris-HCl buffer (pH 8.0) containing 6M guanidine HCl, 10M reduced glutathione and IM oxidized glutathione or in 0.05M Tris-HCl buffer (pH 8.0) containing ImM EDTA, 0.1M NaCl, 8M urea diluted with nine volumes of 0.05M KH 2  PO 4  (pH 10.7), 1 mM EDTA (pH 8.0) and 0.05M NaCl. 
     3. Following solubilization and SDS-PAGE analysis, the CRIPTO protein has a Mr of about 20,000 to 22,000. 
     A deposit of the cloned cDNA of the CRIPTO gene has been made at the ATCC on Feb. 28, 1990 under accession number 61412. The deposit shall be viably maintained, replacing if it becomes non-viable during the life of the patent, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and upon issuance of the patent made available to the public without restriction in accordance with the provisions of the law. The Commissioner of Patents and Trademarks, upon request, shall have access to the deposit. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting. 
     The term &#34;substantially pure&#34; as used herein means as pure as can be obtained by standard isolation and purification techniques conventionally known to one of ordinary skill in the art. 
     The term &#34;a reactive amount&#34; as used herein means a quantity of the protein that would function in a manner desired in a particular application or utility of the protein. 
     As mentioned above, CRIPTO is transcribed into 2200 nucleotide long mRNA which is translated into a protein of 188 amino acid residues. Table 1 shows the expression of CRIPTO gene in humans and mice. The gene is active in teratocarcinoma cells, but inactive both in normal and other transformed cells and shut off when the teratocarcinoma cells are induced to differentiate by retinoic acid. 
     The amino acid sequence of CRIPTO protein was screened against a representative protein sequence database (Microgenie, Beckman). This search revealed that the CRIPTO protein is similar to several proteins, some of which are shown in FIG. 3. The similarity is restricted to a .sup.˜ 40 amino acid long, cysteine-rich, sequence known as the EGF-like segment. Besides the six cysteine residues in the characteristic spatial array, other amino acids are conserved among these proteins, e.g. the glycine, phenylalanine and tyrosine residues boxed in FIG. 1. 
     Transforming potential of the CRIPTO gene 
     It has been shown that certain oncogenes, such as K-FGF, c-sis, proto-dbl and c-erbB-2 can transform murine fibroblasts when their expression is driven by a strong promoter. Similarly, TGFα and EGF genes under the control of a strong promoter can induce transformation and tumorigenicity in fibroblasts. 
     To investigate whether the human CRIPTO gene has these properties, its cDNA was introduced into an expression vector in which transcription is controlled by the RSV long terminal repeats (LTR) (Gorman et al, 1982). The construct was transfected into NIH3T3 cells and its focus-forming activity was monitored (FIG. 4). In this experiment, the CRIPTO cDNA induced foci of transformed cells at an efficiency of 600 focus-forming units per pmol of DNA. 
     When CRIPTO cDNA was placed into a retroviral expression vector plasmid and transfected into mouse NIH-3T3 fibroblasts and into mouse NOG-8 mammary epithelial cells, in both of these cell types overexpression of this gene resulted in the in vitro transformation of these cells. (Tables 2 and 3). 
     In addition, substantially pure, isolated, recombinant CRIPTO protein (rCRIPTO) was obtained from a baculo virus expression vector in which the CRIPTO cDNA had been integrated. The availability of the CRIPTO cDNA and rCRIPTO protein now make it possible to detect cells or tissues expressing the CRIPTO gene. Various utilities of the CRIPTO cDNA and CRIPTO protein are now described. 
     Application and utilities of the CRIPTO cDNA and CRIPTO protein 
     Since the mRNA for the CRIPTO gene is expressed in approximately 60% to 70% of human colon tumor cell lines and at an equal frequency in primary human colon tumors, but not in normal human colon tissue (see Northern blot FIGS. 5 and 6), therefore, expression of CRIPTO mRNA and CRIPTO protein in a tissue would be a major tumor specific marker for the diagnosis and eventual prognosis of different types of cancer such as colorectal cancer. In addition, the CRIPTO gene maps to human chromosome 3 potentially at a region where deletions frequently occur and where such deletions have been found to be associated with a subset of primary human breast tumors and with a majority of small cell lung carcinomas. Hence, a loss of heterozygosity for this gene and/or a loss of or a reduction in CRIPTO mRNA expression due to deletions of one or both alleles of the CRIPTO gene may serve as adjunct tumor specific markers for other types of human cancer. Additionally, it has been experimentally demonstrated that introduction and subsequent overexpression of the human CRIPTO gene in a retroviral expression vector can lead to the in vitro transformation as detected by focus-forming activity or by anchorage-independent growth in soft agar of mouse NIH-3T3 fibroblast cells and of mouse NOG-8 mammary epithelial cells (Tables 2 and 3) indicating a role of this gene in the neoplastic process. Based on these facts, the availability of the CRIPTO cDNA and recombinant CRIPTO protein now allows the following applications: 
     1. The molecularly cloned, full-length human CRIPTO cDNA can be nick-translated, isotopically labeled, for example, with  32  P nucleotides and subsequently used as a probe for the analysis of Southern blots containing endonuclease digested DNA preparations to ascertain if there are amplifications, rearrangements, deletions or restriction fragment length polymorphisms of the CRIPTO gene in normal versus tumor tissue. 
     2. The labeled nick-translated CRIPTO cDNA can also be utilized for the analysis of Northern blots that contain poly(A)+RNA to determine the relative levels of CRIPTO mRNA expression in various normal and pathologic tissue samples. 
     3. The CRIPTO cDNA can be cloned into an SP6/T7 pGEM expression vector and the like and can then be used to generate a corresponding cRNA antisense riboprobe. This antisense riboprobe could then be labeled with  35  S nucleotides and utilized as a suitable probe for in situ RNA:RNA hybridization for histologic localization in normal or pathologic cells expressing CRIPTO mRNA. 
     4. CRIPTO sense oligonucleotides can be chemically synthesized and can be used as appropriate probes in a polymerase chain reaction (PCR) for potential detection of low levels of CRIPTO mRNA and for amplification of CRIPTO genomic sequences for subsequent isolation and cloning. 
     5. The CRIPTO cDNA can be utilized to generate either expression vector plasmids for transfection or to generate replication defective recombinant ecotropic or amphotropic retroviral expression vectors for infection into cells for determining whether overexpression of this gene in vitro might lead to malignant transformation or might alter the growth or differentiation properties of different mammalian cell types. 
     6. The CRIPTO cDNA when placed in an appropriate expression vector plasmid or in a comparable retroviral expression vector in the opposite orientation can be used to generate antisense mRNA. Such antisense expression vectors can then be used to transfect or to infect normal and malignant cells in vitro in order to determine whether endogenous CRIPTO expression is important in maintaining the proliferation, differentiation or transformation of these cells. 
     7. Nonderivatized or thio-derivatized CRIPTO antisense oligonucleotides can be chemically synthesized and used to treat cells in vitro similarly as described in #6 above. Additionally, antisense CRIPTO oligonucleotides can be incorporated into liposomes for site-directed delivery in vivo to tumors when appropriate tumor-specific monoclonal antibodies are also incorporated into these same vesicles. 
     8. The CRIPTO cDNA can be placed into various bacterial, yeast, insect baculo virus or mammalian expression vectors in order to obtain sufficient quantities of a potentially biologically active, recombinant CRIPTO protein. 
     9. A recombinant CRIPTO protein can be used to generate a panel of polyclonal (in rabbits, sheep, goat or pigs) and mouse monoclonal antibodies such that these immunological reagents can be used to screen for CRIPTO protein expression in normal and pathologic human and animal tissue samples by immunocytochemistry, by Western blot analysis, by enzyme-linked immune substrate assay (ELISA), by radioimmunoassay (RIA) and the like. 
     10. Since the CRIPTO protein is a member of the epidermal growth factor (EGF) supergene family that contains a variety of peptide mitogens and growth inhibitors, a biologically active recombinant CRIPTO protein can be used to determine if this peptide has any growth regulatory activity on a variety of normal and tumor cells in vitro. 
     11. Additionally, a recombinant CRIPTO protein can be iodinated and can be utilized to identify and characterize specific cell surface receptors for this potential growth modulatory peptide using conventional chemical cross-linking techniques. 
     It is noted that the methodologies for the above noted utilities are well known to one of ordinary skill in the art and no novel techniques are seen involved in making such usages. A composition of matter, in accordance with the present invention, comprises a reactive amount of the rCRIPTO protein in a sterile, non-toxic carrier or vehicle. 
     It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. 
     
                       TABLE 1______________________________________EXPRESSION OF CRIPTO GENE IN HUMANS AND MICE                Total Poly (A).sup.+                RNA   RNA______________________________________Organs and tissuesPlacenta (human)       --      --Testis (mouse)         --      --Cell linesHL60 (undifferentiated human myeloid                  --      NDcells)JEG (human choriocarcinoma cells)                  --      --PA-1 (human neuroblastoma cells)                  --      NDCa-Ma (human mammary carcinoma cells)                  --      NDHuman T lymphocyte     --      NDHeLa                   --      --NA43 (human fibroblasts)                  ND      --NT2D1 (undifferentiated human teratocar-                  +       +cinoma cells)ΔNT2D1 (differentiated human teratocar-                  --      --cinoma cells)Term placenta fibroblasts                  ND      --Term placenta primary culture                  ND      --F9 (undifferentiated mouse teratocarcinoma                  +       NDcells)ΔF9 (differentiated mouse teratocarcinoma                  --      --cells)NIH3T3 (mouse fibroblasts)                  --      ND______________________________________ ND, Not determined 
    
     
                       TABLE 2______________________________________Anchorage-Independent Growth of Mouse NOG-8 MammaryEpithelial Cells Transfected with a Human cripto cDNA in aRSV Expression Vector PlasmidClone             Total number of colonies/dish______________________________________NOG-8 (parental nontransfected)              10 ± 5.sup.a (-)2E                  1690 ± 80 (+++)2L                 925 ± 70 (++)2F                175 ± 25 (+)2H                166 ± 10 (+)______________________________________ .sup.a 2 × 10.sup.4 cells were seeded in 0.3% soft agar over a 0.8% agar overlay in 35 mm tissue culture dishes. Cultures were maintained for 14 days prior to staining of the cells with nitroblue tetrazolium. Colonies greater than 50 μm were scored and counted on an Artek colony counter. Results are the average from four separate dishes ± S.D. Numbers in parenthesis represent relative amounts of cripto mRNA as detected in cells following Northern blot hybridization with a labeled human cripto cDNA insert. 
    
     
                       TABLE 3______________________________________Focus-Forming Activity of Human cripto cDNA in a RSVExpression Vector Plasmid after Transfection into MouseNIH-3T3 CellsClone              Total number of foci/dish______________________________________NIH-3T3 (parental nontransfected)              .sup.  5 ± 2.sup.aClone γ9     82 ± 5______________________________________ .sup.a 2 × 10.sup.3 cells were seeded in 35 mm dishes and maintaine for 2 weeks prior to staining with crystal violet.