Polypeptides whose expression is upregulated in liver tumor cells and cells from liver preneoplastic foci relative to expression in normal liver cells are disclosed as are polynucleotides that encode the polypeptides. In humans, the polynucleotide maps to a region of chromosome 15. The overexpression has also been confirmed in human liver, breast, colon and kidney cancer cell lines. It is believed that the polypeptides are overexpressed in tumor and preneoplastic cells in general.

SUMMARY OF THE INVENTION

The invention is summarized in that polypeptides of the invention are found in liver tumor cells and in cells from preneoplastic liver foci in human and non-human animals at levels higher than are found in regenerating or quiescent normal liver tissue. This finding has been confirmed in human breast, colon and kidney cancer cell lines. As a result of this differential overexpression, the polypeptides, as wells as polynucleotides that encode the polypeptides, are diagnostic markers for cancer in general, especially liver, breast, colon and kidney cancer, in a human or non-human animal.

In one aspect, the present invention relates to an isolated polypeptide containing an amino acid sequence of SEQ ID NO:2, an amino acid sequence that is at least 70% identical to SEQ ID NO:2 over the length of SEQ ID NO:2, an amino acid sequence of amino acid 22 to amino acid 439 of SEQ ID NO:2 (secreted portion of SEQ ID NO:2), an amino acid sequence that is at least about 68% identical to amino acid 22 to amino acid 439 of SEQ ID NO:2 over the length of amino acid 22 to amino acid 439 of SEQ ID NO:2, an amino acid sequence of SEQ ID NO:4, an amino acid sequence that is at least 70% identical to SEQ ID NO:4 over the length of SEQ ID NO:4, an amino acid sequence of amino acid 22 to amino acid 400 of SEQ ID NO:4 (secreted portion of SEQ ID NO:4), an amino acid sequence that is at least about 68% identical to amino acid 22 to amino acid 400 of SEQ ID NO:4 over the length of amino acid 22 to amino acid 400 of SEQ ID NO:4. The percentage identity of sequences is determined using the Blosum62 alignment method.

In another aspect, the invention also relates to an isolated nucleic acid containing a polynucleotide that encodes a polypeptide of the invention, to a complement of the polynucleotide, or to a polynucleotide that is at least about 80% identical, more preferably 90% identical, and still more preferably 95% identical to an aforementioned polynucleotide of the invention, using the Wilbur-Lipman DNA Alignment method. A polynucleotide that encodes a polypeptide of the invention can include but is not limited to SEQ ID NO:1 from nucleotide 25 to nucleotide 1341, which encodes SEQ ID NO:2, as well as SEQ ID NO:3 from nucleotide 1 to nucleotide 1200, which encodes SEQ ID NO:4. SEQ ID NO:3, predicted by the inventors to represent a coding region on human chromosome 15 (contig Hs15.sub.--10351), is 82.4% identical to the polypeptide-encoding portion of SEQ ID NO:1 using the Wilbur-Lipman DNA Alignment method.

In another aspect, a polynucleotide of the invention is engineered into a genetic construct downstream from a heterologous promoter not natively upstream of the polynucleotide that directs transcription of the polynucleotide. The genetic construct is introduced into a host cell that supports transcription of the polynucleotide and translation of the encoded polypeptide which can then be purified using methods known to those skilled in the art. Alternatively, the construct comprising a polynucleotide of the invention is provided in an in vitro transcription/translation system for producing the encoded polypeptide.

In yet another aspect, the present invention provides a host cell transfected with a genetic construct of the invention.

In still another aspect, the invention is an antibody that specifically binds to a polypeptide of the invention.

In yet another aspect, the invention is a method for identifying an agent that modulates the expression of a polypeptide of the invention (e.g., an inducer or suppressor). The method includes the steps of exposing a cell that contains a polynucleotide of the invention under the control of its native promoter, measuring the expression of the polynucleotide in the cell, and comparing the expression to that in a control cell that is not exposed to the test agent. A higher or lower than the expression in the control cell indicates that the agent can modulate the expression of the polynucleotide. The expression can be measured and compared at either the mRNA level or the protein level. Preferably, a liver, breast, colon or kidney cell (cancerous or normal) is used in the method. More preferably, a human or murine liver, breast, colon or kidney cell is used.

In still another aspect, the present invention is a method of diagnosing cancer or preneoplastic development in a tissue or organ of a human or non-human animal by measuring the expression of a polypeptide of the invention in cells of the tissue or organ obtained from a region suspected of cancer or preneoplastic development, and comparing the expression to a normal standard, wherein a higher than normal expression indicates cancer or preneoplastic development in the suspected region. A skilled artisan can readily establish a normal standard. For example, it can be the expression level in normal cells of the same tissue or organ in the same animal, or it can be an expression level range established by testing normal cells of the same tissue or organ of other animals of the same species. The expression can be measured and compared at either the mRNA level or protein level.

In a related aspect, the present invention is a method for identifying a candidate human or non-human animal for further cancer screening, where the method includes, in one embodiment, the step of determining the level of a polypeptide of the invention in a blood or blood-derived sample from the animal, whereby the animal is identified as a candidate for further cancer screening when the level exceeds either a normal range established by the same animal during a period that is tumor-free in the tissue or organ, or a normal range established by other animals of the same species that are tumor-free in the tissue or organ. In another embodiment, the method takes advantage of the expected secretion of the polypeptide and the development of antibodies to the polypeptide in a human or non-human animal that overexpresses the polypeptide in the cancerous or preneoplastic tissue or organ. The method includes the step of determining the level of an antibody to the polypeptide in a blood or blood-derived sample from the animal, whereby the animal is identified as a candidate for further cancer screening when the antibody level exceeds either a normal range established by the same animal during a period that is tumor-free in the tissue or organ, or a normal range established by other animals of the same species that are tumor-free in the tissue or organ. It is understood that individuals free of cancer or preneoplastic development in the tissue or organ may not develop an antibody to the polypeptide. Thus, the normal range for the level of the antibody can be zero.

In still another aspect, the invention relates to a kit suitable for use in a method for determining the level of a polypeptide or polynucleotide of the invention, where the kit contains at least one of an antibody specifically directed to an epitope on a polypeptide of the invention and a polynucleotide that hybridizes to a polynucleotide of the invention, as well as at least one control sample component for which the relative or absolute amount of the polynucleotide or polypeptide of the present invention is known, the control sample component being selected from liver cancer cells, preneoplastic liver cells, normal liver cells, breast cancer cells, normal breast cells, colon cancer cells, normal colon cells, kidney cancer cells, normal kidney cells, an extract of any of the foregoing cells, a blood sample from a human or non-human animal, and a blood-derived sample from a human or non-human animal.

It is an object of the present invention to provide a polynucleotide and a polypeptide that are differentially expressed in preneoplastic or cancer cells and normal regenerating or quiescent cells in a tissue or organ of a human and non-human animal.

DETAILED DESCRIPTION OF THE INVENTION

Liver cancer is generally studied in animal model systems, preferably in rodent systems, where certain strains are bred for high susceptibility to liver tumors. C3H/HeJ mice are highly susceptible to liver tumors after induction with diethylnitrosamine (DEN). To identify polynucleotide sequences or genes that show differential expression in liver tumor cells as compared to normal liver cells, gene expression differences between liver tumors and a regenerating liver were determined using representational difference analysis (RDA: Lisitsyn, et al., Science 259:946 (1993), incorporated by reference as if set forth herein in its entirety).

In this application, the applicants disclose polypeptides from murine animals (SEQ ID NO:2) and humans (SEQ ID NO:4) that are upregulated in cells and cell extracts from human and murine liver tumors and liver preneoplastic tissues, relative to quiescent and regenerating normal liver cells. The polypeptide is therefore given the name human or murine Cancer Related Gene-Liver 2 (CRG-L2). CRG-L2 overexpression was also found in human liver, breast, colon and kidney cancer cell lines. Thus, despite of its name indicative of liver origin, it is believed to be overexpressed in other types of cancer and preneoplastic cells in general, especially breast, colon and kidney cancer and preneoplastic cells.

Using the Blosum62 alignment method, the human and murine CRG-L2s are found to be 76% identical. It is expected that CRG-L2s from other animals, e.g., other mammals, are at least 70% identical to either the human or murine CRG-L2 if compared using the same alignment method. Hydrophobic sequences are present within the first 30 amino acids of SEQ ID NO:2 and SEQ ID NO:4. Based on information obtained from other proteins with leader sequences, the serine at amino acid position 21 of both SEQ ID NO:2 and SEQ ID NO:4 is believed to be the cleavage site of a leader sequence for the secretion of both of the CRG-L2s. Accordingly, it is believed that when amino acids 1 21 are cleaved, the remaining amino acid sequences of SEQ ID NO:2 and SEQ ID NO:4 can be secreted from cells. Corresponding leader sequences on other CRG-L2s can be readily identified by a skilled artisan. Depending on the variability of the leader sequences among CRG-L2s, the percentage of identity among the secreted sequences may be about 3% higher or lower than the overall 70% identity. Generally speaking, it is expected that the secreted portion of CRG-L2s in other animals, e.g., other mammalians, are at least about 68% identical to either the secreted portion of the human or that of the murine CRG-L2.

Also disclosed are polynucleotides that encode the polypeptides of the invention (e.g., the full length and the secreted CRG-L2s), which can include, without limitation, mRNA, single or double stranded DNA, cDNA and the like. In addition to the primary murine cDNA product disclosed as SEQ ID NO:1, two additional variant murine cDNAs that are believed to derive from alternative 3' untranslated regions were also obtained. The variant murine cDNA molecules differ from SEQ ID NO:1 in the 3' untranslated portion of the molecules, commencing respectively at nucleotide 1937 and at nucleotide 2342, as shown in the Sequence Listing. SEQ ID NO:3 discloses a sequence from human Chromosome 15 that encodes the human CRG-L2 of SEQ ID NO:4.

Further, the invention provides materials and methods for detecting expression (and changes in expression) of the polypeptides and of the polynucleotides that encode the polypeptides, thereby facilitating use as a diagnostic marker for cancer and preneoplastic development and as a system for assessing putative therapeutic agents. As described in detail in the example below, since the CRG-L2 either belongs or is similar to the family of cancer-testis antigens, it is expected that a patient will display an immune response to CRG-L2 when it is overexpressed in preneoplastic and cancerous tissues. Therefore, detecting or measuring the level of an antibody to CRG-L2 in a blood or blood-derived sample from a patient provides another diagnostic tool.

Structurally, the murine CRG-L2 protein (SEQ ID NO:2) contains 439 amino acids and has a predicted molecular weight of about 47.5 kDA. Using the Simple Modular Architecture Research Tool (available on the world wide web courtesy of the European Molecular Biology Laboratory--Heidelberg), it was determined that the murine CRG-L2 includes two collagen domains in the 5' region (corresponding to amino acids 29 88 and 89 149 of SEQ ID NO:2, respectively) and a large olfactomedin domain near the C-terminus (corresponding to amino acids 189 433 of SEQ ID NO:2). The human protein also contains two putative collagen domains and one olfactomedin domain at amino acids 27 85, 86 145, and 177 395 of SEQ ID NO:4, respectively. Olfactomedin-related proteins are secreted glycoproteins having conserved C terminal motifs. It is anticipated that CRG-L2 can be secreted into the blood and an increase in blood CRG-L2 level over normal levels is diagnostic of cancer and preneoplastic development. Preferably, the diagnostic blood CRG-L2 level is set to be at least about 5%, more preferably at least about 10%, and most preferably at least about 25% over a normal level.

The term "isolated nucleic acid" or "isolated polypeptide" used in the specification and claims of the present invention means a nucleic acid or polypeptide isolated from its natural environment or prepared using synthetic methods such as those known to one of ordinary skill in the art. Complete purification is not required in either case. Amino acid and nucleotide sequences that flank a polypeptide or polynucleotide that occurs in nature, respectively, can but need not be absent from the isolated form. The polypeptides and nucleic acids of the invention can be isolated and purified from normally associated material in conventional ways such that in the purified preparation the polypeptide or nucleic acid is the predominant species in the preparation. At the very least, the degree of purification is such that the extraneous material in the preparation does not interfere with use of the polypeptide or nucleic acid of the invention in the manner disclosed herein. The polypeptide or nucleic acid is preferably at least about 85% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.

Further, an isolated nucleic acid has a structure that is not identical to that of any naturally occurring polynucleotide or to that of any fragment of a naturally occurring genomic polynucleotide spanning more than three separate genes. An isolated nucleic acid also includes, without limitation, (a) a polynucleotide having a sequence of a naturally occurring genomic or extrachromosomal nucleic acid molecule but which is not flanked by the coding sequences that flank the sequence in its natural position; (b) a polynucleotide incorporated into a vector or into a prokaryote or eukaryote genome such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are polynucleotides present in mixtures of clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library. An isolated nucleic acid can be modified or unmodified DNA or RNA, whether fully or partially single-stranded or double-stranded or even triple-stranded. A nucleic acid can be chemically or enzymatically modified and can include so-called non-standard bases such as inosine.

The nucleotide sequences of the invention can be introduced into, and expressed in, host cells which can be prokaryotic (such as bacterial) cells or eukaryotic (such as yeast, insect, amphibian or mammalian) cells whereupon the transcription of polynucleotide and the properties of the encoded polypeptides can be assessed.

The disclosure of the CRG-L2 sequences that are upregulated in liver tumor and preneoplastic cells, and in human breast, colon and kidney cancer cell lines provides a means for identifying (in vivo or in vitro) candidates for further testing as preventive and therapeutic agents. For example, animal cells that contain a CRG-L2 sequence under the control of its native promoter can be exposed to a test agent and the effect of the test agent on the CRG-L2's expression at the mRNA or protein level relative to that of untreated controls can be measured. Alternatively, the level of expression can be assessed in biological samples taken directly from a human or non-human tissue. Presumably, an anti-tumor agent can bring down the mRNA and protein level in tumor cells. Accordingly, an agent that demonstrates such an activity is a good candidate for further testing for anti-tumor efficacy.

The presence and level of such a differentially expressed protein can be readily discerned using antibodies directed to an epitope on the protein using well known methods, such as an ELISA method. It is well within the skill of one of ordinary skill in the art to generate such antibodies. The presence and level of mRNA for the protein can be measured using methods for hybridizing nucleic acids (including, without limitation, RNA, DNA, and cDNA). Such methods are generally known to those skilled in the art, but are enabled by the disclosure herein of a tumor-specific sequence. Examples of such methods include but are not limited to RT-PCR amplification, Northern blot and Southern blot.

Given the disclosure herein of polynucleotides that encode CRG-L2 of human, murine and other animal species, one of ordinary skill in the art knows how to design primers for use in RT-PCR analysis and probes for Northern and Southern blot. The Example below describes a method of using RT-PCR to measure CRG-L2 mRNA level in liver tumor cells, liver preneoplastic cells and normal liver cells. The RT-PCR amplified a fragment of CRG-L2 cDNA (SEQ ID NO: 1) and its noted 3' end variants, and the mRNA level in liver tumor and preneoplastic cells was observed to be higher than that in normal liver cells. Accordingly, a suitable CRG-L2 sequence for amplifying or probing in analyzing differential CRG-L2 mRNA levels is one that corresponds to a fragment shared by all three CRG-L2 cDNA sequences. A CRG-L2 mRNA sequence that corresponds to a fragment unique to the longer 3' untranslated sequence variants could also be used to analyze differential CRG-L2 mRNA expression since Northern analysis has shown that all three mRNAs are differentially expressed in liver tumor and preneoplastic cells relative to normal liver cells.

A skilled artisan understands that the polynucleotides disclosed herein can contain additional nucleotides at the 5'-end, 3'-end or both that do not affect the function of the polynucleotides in terms of their uses contemplated herein. The additional nucleotides can but do not have to assist in the cloning, detection and purification procedures associated with the use of the polynucleotides. Similarly, a skilled artisan understands that the polypeptides disclosed herein can contain additional amino acid sequences at the N- or C-terminus or both that do not affect the function of the polypeptides. The additional amino acid sequences can but do not have to assist in purification, detection, or stabilization of the polypeptides.

Further, a skilled artisan understands that polynucleotide and polypeptide sequences presented herein can vary somewhat, whether as a result, e.g., of sequencing error or allelic variation or duplication, from the sequence presented while still retaining their essential nature, that is, higher expression level in tumor and preneoplastic cells relative to normal cells. Further, the polynucleotides of the invention include conservatively modified variants of the sequences presented herein, complementary sequences, and splice variants. In view of the known degeneracy in the genetic code, the proteins or polypeptides disclosed can also be encoded by a large number of other polynucleotide sequences, all of which are within the scope of the invention. Polynucleotide sequences that are at least 80% identical to the polynucleotide sequences that encode the polypeptide sequences disclosed herein can be used as hybridization probes for coding sequences and are thus within the scope of the present invention. The polynucleotides and polypeptides of the invention include, without limitation, polymorphic variants, alleles, mutants, and interspecies homologs that (1) are expressed at higher level in tumor and preneoplastic cells, especially in liver, breast, colon and kidney tumor and preneoplastic cells, (2) bind to antibodies raised against the coding region of the disclosed polypeptides, (3) specifically hybridize under stringent or moderately stringent hybridization conditions to a polynucleotide that encodes a polypeptide of the present invention, or (4) are amplified by primers that amplify a polynucleotide that encodes a polypeptide of the present invention.

Exemplary stringent hybridization conditions include 50% formamide, 5.times.SSC and 1% SDS incubated at 42.degree. C., or 5.times.SSC and 1% SDS incubated at 65.degree. C., followed by washing in 0.2.times.SSC and 0.1% SDS at 65.degree. C. Exemplary moderately stringent hybridization conditions include 40% formamide, 1M NaCl and 1% SDS incubated at 37.degree. C. followed by washing in 1.times.SSC at 45.degree. C. These conditions are merely exemplary as one skilled in the art is readily able to discern stringent from moderately stringent hybridization conditions.

Moreover, the sequences of the invention also encompass substitutions, additions and deletions of the sequences presented where the change affects one or a few amino acids in the presented polypeptide sequences, without substantial effect upon the activity of the polypeptide, i.e., differential expression in cancer cells and preneoplastic cells relative to normal cells.
 

Claim 1 of 4 Claims

1. An isolated polypeptide comprising amino acids 22 400 of SEQ ID NO:4.

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