Pancreatic cancer genes

The present invention provides the art with the DNA coding sequences of polynucleotides that are up- or down-regulated in cancer and dysplasia. These polynucleotides and encoded proteins or polypeptides can be used in the diagnosis or identification of cancer and dysplasia. Inhibitors of the up-regulated polynucleotides and proteins can decrease the abnormality of cancer and dysplasia. Enhancing the expression of down-regulated polynucleotides or introducing down-regulated proteins to cells can decrease the growth and/or abnormal characteristics of cancer and dysplasia.

TECHNICAL AREA OF THE INVENTION
 The invention relates to the area of diagnosis and treatment of pancreatic
 cancer and dysplasia. More specifically, it relates to polynucleotides
 which are differentially regulated in pancreatic cancer and dysplasia.
 BACKGROUND OF THE INVENTION
 Pancreatic cancer is the fifth leading cause of cancer death in the United
 States. According to the American Cancer Society, approximately 28,000
 people will die of pancreatic cancer in the United States in 1998. A high
 risk of developing pancreatic cancer, without a corresponding increase in
 the risk of developing other cancers, may be passed along in some
 families. Pancreatic cancer is most likely caused by an accumulation of
 mutations in specific cancer-causing genes. Pancreatic cancer is very
 aggressive and chemotherapeutic agents which may be active against other
 malignancies do not work effectively when used for pancreatic cancer.
 The majority of cells in the pancreas are in the exocrine glands, which
 produce pancreatic enzymes, and in the ducts that carry the pancreatic
 enzymes to the bile duct and to the small intestine. Cancers of the
 exocrine cells of the pancreas are usually adenocarcinomas. Pancreatic
 adenocarcinomas usually begin in the ducts of the pancreas, but may
 sometimes develop from the acinar cells. About 95% of cancers of the
 pancreas are adenocarcinomas. Less common cancers of the exocrine pancreas
 include adenosquamous carcinomas, squamous cell carcinomas, and giant cell
 carcinomas.
 Because pancreatic cancer is an aggressive cancer with very high mortality,
 there is a need in the art for genes that are up- or down-regulated in
 tumor progression. Such genes are useful for therapeutic purposes and for
 diagnosis of pancreatic as well as other cancers.
 SUMMARY OF THE INVENTION
 The invention provides isolated polynucleotides comprising coding regions
 or portions of genes whose expression is mis-regulated in cancer and
 dysplasia.
 The invention also provides isolated proteins and protein fragments whose
 expression is mis-regulated in cancer and dysplasia.
 The invention further provides an antibody preparation which specifically
 binds to a polypeptide the expression of which is mis-regulated in cancer
 and dysplasia.
 The invention provides a method for diagnosing cancer and dysplasia.
 The invention still further provides therapeutic compositions useful for
 treating cancer and dysplasia.
 These and other objects of the invention are provided by one or more of the
 embodiments described below. One embodiment of the invention provides
 isolated polynucleotides comprising at least twelve contiguous nucleotides
 selected from the group of polynucleotide sequences as shown in SEQ ID
 NOS:1-15.
 Another embodiment of the invention provides isolated polypeptides
 comprising at least six contiguous amino acids encoded by a polynucleotide
 selected from the group consisting of the polynucleotide sequences as
 shown in SEQ ID NOS:1-15.
 Even another embodiment of the invention provides an antibody preparation
 which specifically binds to a polypeptide comprising at least six
 contiguous amino acids encoded by a polynucleotide selected from the group
 of polynucleotide sequences as shown in SEQ ID NOS:1-15.
 Yet another embodiment of the invention provides isolated nucleotide probes
 consisting of a sequence selected from the group consisting of the
 polynucleotide sequences shown in SEQ ID NOS:1-15.
 Still another embodiment of the invention provides a method of diagnosing
 cancer. The amount of a polypeptide expressed from a polynucleotide having
 a sequence as shown in SEQ ID NO:12 in a test sample of tissue of a human
 suspected of being cancerous is determined. The amount of said polypeptide
 is also determined in a human tissue which is normal. The determined
 amounts are then compared. A test sample which contains less of the
 polypeptide than the normal tissue is identified as cancerous.
 A further embodiment of the invention provides an additional method of
 diagnosing cancer. The amount of specific mRNA molecules in a test sample
 of tissue suspected of being cancerous and in a human tissue which is
 normal are determined. The mRNA molecules to be measured are complementary
 to the minus strand of a double-stranded polynucleotide sequence. The
 double-stranded polynucleotide sequence is shown in SEQ ID NO:12. The
 determined amounts of mRNA molecules are compared. A test sample of tissue
 which contains less of the mRNA molecules than the normal tissue is
 identified as cancerous.
 Another embodiment of the invention provides a therapeutic composition
 useful for reducing the growth rate of cancer cells. The composition is
 comprised of a polynucleotide comprising all or a portion of a nucleotide
 sequence which is operably linked to a promoter sequence and a
 pharmaceutically acceptable carrier. The polynucleotide comprising all or
 a portion of a nucleotide sequence comprises at least 18 contiguous
 nucleotides. The nucleotide sequence is shown in SEQ ID NO:12.
 Yet another embodiment of the invention provides a therapeutic composition
 useful for reducing the growth rate of cancer cells. The composition is
 comprised of a polypeptide comprising all or a portion of an amino acid
 sequence expressed from a polynucleotide sequence and a pharmaceutically
 acceptable carrier. The polynucleotide sequence is shown in SEQ ID NO:12.
 Another embodiment of the invention provides a method of diagnosing
 dysplasia and cancer. The amount of a polypeptide expressed from a
 polynucleotide having at least one of a sequence selected from the group
 consisting of the polynucleotide sequences shown in SEQ ID NOS:2, 5, and
 15 in a test sample of tissue suspected of being dysplastic or cancerous
 is determined. The amount of the polypeptide is also determined in a human
 tissue which is normal. The determined amounts are compared. A test sample
 of human tissue which contains more of at least one polypeptide than the
 normal tissue is identified as being dysplastic or cancerous.
 A further embodiment of the invention provides another method of diagnosing
 dysplasia. The amount of a polypeptide expressed from a polynucleotide
 having a sequence selected from the group consisting of the polynucleotide
 sequences shown in SEQ ID NOS:1, 3-4, 6-11, and 13-14 is determined in a
 test sample of tissue suspected of being dysplastic. The amount of said
 polypeptide is also determined in a human tissue which is normal. The two
 amounts are then compared. A test sample of human tissue which contains
 more of said polypeptide than the normal tissue is identified as being
 dysplastic.
 Another embodiment of the invention provides an additional method of
 diagnosing cancer.
 The amount of a polypeptide expressed from a polynucleotide having a
 sequence selected from the group consisting of the polynucleotide
 sequences shown in SEQ ID NOS:2, 5, and 15, is determined in a test sample
 of tissue suspected of containing cancer, and in a human tissue which is
 normal. The amount of a polypeptide expressed from a polynucleotide having
 a sequence selected from the group consisting of the polynucleotide
 sequences shown in SEQ ID NOS:1, 3-4, 6-11, and 13-14 is also determined
 in the test sample, and in the normal tissue. The determined amounts of
 said polypeptides are then compared. A test sample of tissue which
 contains more of the polypeptide expressed from a polynucleotide having a
 sequence selected from the group consisting of the polynucleotide
 sequences shown in SEQ ID NOS:2, 5, and 15, as compared to the normal
 tissue, and which contains substantially the same amount of a polypeptide
 expressed from a polynucleotide selected from the group as shown in SEQ ID
 NOS:1, 3-4, 6-11, and 13-14, as compared to the normal tissue, is
 identified as cancerous.
 Even another embodiment of the invention provides a method of diagnosing
 dysplasia and cancer. The amount of specific mRNA molecules is determined
 in a test sample of tissue suspected of being dysplastic or cancerous and
 in a human tissue which is normal. The mRNA molecules measured are
 complementary to the minus strand of a double-stranded polynucleotide
 sequence. The double-stranded polynucleotide sequence is selected from the
 group of polynucleotides as shown in SEQ ID NOS:2, 5, and 15. The
 determined amounts of mRNA molecules are compared. A test sample of human
 tissue which contains more of the mRNA molecules than the normal tissue is
 identified as being dysplastic or cancerous.
 Yet another embodiment of the invention provides a method of diagnosing
 dysplasia. The amounts of specific mRNA molecules in a test sample of
 human tissue suspected of being dysplastic and in a human tissue which is
 normal are determined. The mRNA molecules are complementary to the minus
 strand of a double-stranded polynucleotide sequence. The double-stranded
 polynucleotide sequence is selected from the group of polynucleotides as
 shown in SEQ ID NOS:1, 3-4, 6-11, and 13-14. The determined amounts of
 mRNA molecules are then compared. A test sample of human tissue which
 contains more of the mRNA molecules than the normal tissue is identified
 as being dysplastic.
 Still another embodiment of the invention provides a method of diagnosing
 cancer. The amounts of a first set of specific mRNA molecules in a test
 sample of tissue of a human suspected of being cancerous and in a human
 tissue which is normal are determined. The mRNA molecules are
 complementary to the minus strand of a double-stranded polynucleotide
 sequence. The double-stranded polynucleotide sequence is selected from the
 group of polynucleotide sequences as shown in SEQ ID NOS:1, 3-4, 6-11, and
 13-14. In addition, the amounts of a second set of specific mRNA molecules
 in a test sample of tissue of a human suspected of being cancerous and in
 a human tissue which is normal are determined. The mRNA molecules are
 complementary to the minus strand of a double-stranded polynucleotide
 sequence. The double-stranded polynucleotide sequence is selected from the
 group of polynucleotide sequences as shown in SEQ ID NOS2, 5, and 15. The
 determined amounts of the first and second sets of mRNA molecules are
 compared. A test sample of human tissue which contains more of the second
 set of mRNA molecules than the normal tissue, and which contains
 substantially the same amount of the first set of mRNA molecules, as
 compared to the normal tissue, is identified as cancerous.
 Yet another embodiment of the invention provides a therapeutic composition
 useful for decreasing the amount of translation of an mRNA molecule in a
 cell. The composition comprises an antisense polynucleotide complementary
 to the plus strand of a double-stranded polynucleotide. The
 double-stranded polynucleotide is selected from the group consisting of
 polynucleotides comprising a nucleotide sequence as shown in SEQ ID
 NOS:1-11, and 13-15, wherein said antisense polynucleotide binds to an
 mRNA molecule. The composition also includes a pharmaceutically acceptable
 carrier.
 A further embodiment of the invention provides a therapeutic composition
 useful for reducing the expression of a polypeptide. The composition
 comprises an antibody which specifically binds to a polypeptide expressed
 from a polynucleotide selected from the group consisting of
 polynucleotides comprising a nucleotide sequence as shown in SEQ ID
 NOS:1-11 and 13-15. The composition also includes a pharmaceutically
 acceptable carrier.
 Another embodiment of the invention provides a therapeutic composition
 useful for reducing the translation from an mRNA molecule. The composition
 comprises a ribozyme which binds to an mRNA molecule, wherein a portion of
 said ribozyme is complementary to the plus strand of a double-stranded
 polynucleotide. The polynucleotide is selected from the group consisting
 of the polynucleotides comprising a sequence as shown in SEQ ID NOS:1-11,
 and 13-15. The composition also comprises a pharmaceutically acceptable
 carrier.
 The present invention provides the art with useful polynucleotides which
 represent expressed sequences of genes. Expression of the genes is
 mis-regulated in cancer. The invention also provides the art with
 diagnostic methods based on the overand under-expression of the genes and
 the polypeptides encoded by the genes in cancer and dysplastic cells.
 Inhibitors of the over-expressed polynucleotides and polypeptides can be
 used to reduce the growth of cancer cells and dysplastic cells. The
 polynucleotides and polypeptides which are under-expressed in cancer and
 dysplasia can be delivered therapeutically to reduce the abnormal
 characteristics of cancer cells and dysplastic cells.
 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 Polynucleotides that are mis-regulated in cancer and dysplasia are
 disclosed. The mis-regulated polynucleotide sequences are shown in SEQ ID
 NOS:1-15. The polynucleotides are mis-regulated as follows:
 SEQ ID NO:12 is down-regulated in cancer;
 SEQ ID NOS:2, 5, and 15 are up-regulated in cancer and dysplasia; and
 SEQ ID NOS:1, 3-4, 6-11, and 13-14 are up-regulated in dysplasia only.
 Polynucleotides that are differentially regulated in cancer or dysplasia or
 both can be useful in the diagnosis and treatment of these diseases.
 Dysplasia is an atypical proliferation of epithelial or mesenchymal cells
 that may represent an early stage of cancer; however, dysplasia does not
 necessarily progress to cancer. Epithelial dysplasia results in the loss
 of normal orientation of one epithelial cell to another, accompanied by
 alterations in cellular and nuclear size and shape. Cancer is a
 proliferation of malignant cells that are no longer under normal
 physiologic control.
 The subgenomic polynucleotides of the invention contain less than a whole
 chromosome and are preferably intron-free. The subgenomic polynucleotides
 of the invention can be isolated and purified free from other nucleotide
 sequences by standard nucleic acid purification techniques, for example,
 using PCR, cloning, and/or restriction enzymes and probes to isolate
 fragments comprising the encoding sequences. Subgenomic polynucleotides of
 the invention can include all or a contiguous portion of a gene coding
 region. In one embodiment, an isolated and purified subgenomic
 polynucleotide ofthe invention comprises at least 10, 11, 12, 15, 18, 20,
 25, 30, 35, 40, 45, 50, 60, 70, 74, 80, 90, 100, 125, 150, 154, 175, 200,
 250, 300, or 350 contiguous nucleotides selected from the polynucleotide
 sequences as shown in SEQ ID NOS:1-15. In a preferred embodiment, the
 polynucleotide molecules comprise a contiguous sequence of at least twelve
 nucleotides selected from the group consisting of the polynucleotides
 shown in SEQ ID NOS:1-15.
 An open reading frame is a region of DNA that consists exclusively of
 triplets that represent amino acids. The open reading frame of the
 polynucleotide sequences of the invention can be determined by examining
 all three possible reading frames in both directions. If a reading frame
 contains termination codons it cannot be read into protein and is not
 considered an open reading frame. Usually, no more than one of the six
 possible frames is open in any single stretch of DNA. An extensive open
 reading frame is unlikely to exist by chance because of the lack of
 selective pressure to prevent the accumulation of nonsense codons.
 Therefore, the identification of a lengthy open reading frame is taken to
 be prima facie evidence that the sequence is translated into protein in
 that frame. Lewin, ed,. 1990, Genes IV, Cell Press, Cambridge, Mass.
 Subgenomic polynucleotides of the invention can be used, inlter alia, to
 produce proteins or polypeptides, as probes for the detection of mRNA of
 the invention in samples or extracts of human cells, to generate
 additional copies of the polynucleotides, and to generate ribozymes or
 antisense oligonucleotides. The subgenomic polynucleotides can also be
 used as single stranded DNA probes or as triple-strand forming
 oligonucleotides. The probes can be used to determine the presence or
 absence of the polynucleotide sequences as shown in SEQ ID NOS:1-15 or
 variants thereof in a sample.
 The sequence of a nucleic acid comprising at least 15 contiguous
 nucleotides of at least any one of SEQ ID NO:1-15, preferably the entire
 sequence of at least any one of SEQ ID NO:1-15, is not limited and can be
 any sequence of A, T, G, and/or C (for DNA) and A, U, G, and/or C (for
 RNA) or modified bases thereof, including inosine and pseudouridine. The
 choice of sequence will depend on the desired function and can be dictated
 by coding regions desired, the intron-like regions desired, and the
 regulatory regions desired.
 Where the entire sequence of any one of SEQ ID NO:1-15 is within the
 nucleic acid, the nucleic acid obtained is referred to herein as a
 polynucleotide comprising the sequence of any one of SEQ ID NO:1-15.
 Both secreted and membrane-bound polypeptides of the present invention are
 of interest. For example, levels of secreted polypeptides can be assayed
 conveniently in body fluids, such as blood and urine. Membrane-bound
 polypeptides are useful for constructing vaccine antigens or inducing an
 immune response. Such antigens would comprise all or part of the
 extracellular region of the membrane-bound polypeptides.
 Because both secreted and membrane-bound polypeptides comprise a fragment
 of contiguous hydrophobic amino acids, hydrophobicity predicting
 algorithms can be used to identify such polypeptides.
 A signal sequence is usually encoded by both secreted and membrane-bound
 polypeptide genes to direct a polypeptide to the surface of the cell. The
 signal sequence usually comprises a stretch of hydrophobic residues. Such
 signal sequences can fold into helical structures.
 Membrane-bound polypeptides typically comprise at least one transmembrane
 region that possesses a stretch of hydrophobic amino acids that can
 transverse the membrane. Some transmembrane regions also exhibit a helical
 structure.
 Hydrophobic fragments within a polypeptide can be identified by using
 computer algorithms. Such algorithms include Hopp & Woods, Proc. Natl.
 Acad. Sci. USA 78:3824-3828 (1981); Kyte & Doolittle, J. Mol. Biol.
 157:105-132 (1982); and RAOAR algorithm, Degli Esposti el al., Eur. J.
 Biochem. 190:207-219 (1990).
 Another method of identifying secreted and membrane-bound polypeptides is
 to translate the present polynucleotides, SEQ ID NO:1-15, in all six
 frames and determine if at least 8 contiguous hydrophobic amino acids are
 present. Those translated polypeptides with at least 8; more typically,
 10; even more typically, 12 contiguous hydrophobic amino acids are
 considered to be either a putative secreted or membrane bound polypeptide.
 Hydrophobic amino acids include alanine, glycine, histidine, isoleucine,
 leucine, lysine, methionine, phenylalanine, proline, threonine,
 tryptophan, tyrosine, and valine.
 The polypeptides of the invention include those encoded by the disclosed
 polynucleotides. These polypeptides can also be encoded by nucleic acids
 that, by virtue of the degeneracy of the genetic code, are not identical
 in sequence to the disclosed polynucleotides. Thus, the invention includes
 within its scope nucleic acids comprising polynucleotides encoding a
 protein or polypeptide expressed by a polynucleotide having the sequence
 of any one of SEQ ID NO:1-15. Also within the scope of the invention are
 variants; variants of polypeptides include mutants, fragments, and
 fusions. Mutants can include amino acid substitutions, additions or
 deletions. The amino acid substitutions can be conservative amino acid
 substitutions or substitutions to eliminate non-essential amino acids,
 such as to alter a glycosylation site, a phosphorylation site or an
 acetylation site, or to minimize misfolding by substitution or deletion of
 one or more cysteine residues that are not necessary for function.
 Conservative amino acid substitutions are those that preserve the general
 charge, hydrophobicity/hydrophilicity, and/or steric bulk of the amino
 acid substituted. For example, substitutions between the following groups
 are conservative: Gly/Ala, Val/lle/Leu, Asp/Glu, Lys/Arg, Asn/Gln,
 Ser/Cys,Thr, and Phe/Trp/Tyr.
 Cysteine-depleted muteins are variants within the scope of the invention.
 These variants can be constructed according to methods disclosed in U.S.
 Pat. No. 4,959,314, "Cysteine-Depleted Muteins of Biologically Active
 Proteins." The patent discloses how to substitute other amino acids for
 cysteines, and how to determine biological activity and effect of the
 substitution. Such methods are suitable for proteins according to this
 invention that have cysteine residues suitable for such substitutions, for
 example to eliminate disulfide bond formation.
 The protein variants described herein are encoded by polynucleotides that
 are within the scope of the invention. The genetic code can be used to
 select the appropriate codons to construct the corresponding variants.
 The invention encompasses polynucleotide sequences having at least 65%
 sequence identity to any one of SEQ ID NOS:1-15 as determined by the
 Smith-Waterman homology search algorithm as implemented in MSPRCH program
 (Oxford Molecular) using an affine gap search with the following search
 parameters: gap open penalty of 12, and gap extension penalty of 1.
 Polynucleotide probes comprising at least 12 contiguous nucleotides
 selected from the nucleotide sequence of a polynucleotide of SEQ ID
 NO:1-15 are used for a variety of purposes, including identification of
 human chromosomes and determining transcription levels.
 The nucleotide probes are labeled, for example, with a radioactive,
 fluorescent, biotinylated, or chemiluminescent label, and detected by well
 known methods appropriate for the particular label selected. Protocols for
 hybridizing nucleotide probes to preparations of metaphase chromosomes are
 also well known in the art. A nucleotide probe will hybridize specifically
 to nucleotide sequences in the chromosome preparations which are
 complementary to the nucleotide sequence of the probe. A probe that
 hybridizes specifically to a polynucleotide should provide a detection
 signal at least 5-, 10-, or 20-fold higher than the background
 hybridization provided with other unrelated sequences.
 Polynucleotides of the present invention are used to identify a chromosome
 on which the corresponding(gene resides. Using fluorescence in situ
 hybridization (FISH) on normal metaphase spreads, comparative genomic
 hybridization allows total genome assessment of changes in relative copy
 number of DNA sequences. See Schwartz and Samad, Current Opinions in
 Biotechnology (1994) 8:70-74; Kallioniemi el al., Seminars in Cancer
 Biology (1993) 4:41-46; Valdes and Tagle, Methods in Molecular Biology
 (1997) 68:1, Boultwood, ed., Human Press, Totowa, N.J.
 Preparations of human metaphase chromosomes are prepared using standard
 cytogenetic techniques from human primary tissues or cell lines.
 Nucleotide probes comprising at least 12 contiguous nucleotides selected
 from the nucleotide sequence of SEQ ID NOS:1-15 are used to identify the
 corresponding chromosome. The nucleotide probes are labeled, for example,
 with a radioactive, fluorescent, biotinylated, or chemiluminescent label,
 and detected by well known methods appropriate for the particular label
 selected. Protocols for hybridizing nucleotide probes to preparations of
 metaphase chromosomes are also well known in the art. A nucleotide probe
 will hybridize specifically to nucleotide sequences in the chromosome
 preparations that are complementary to the nucleotide sequence of the
 probe. A probe that hybridizes specifically to a polynucleotide-related
 gene provides a detection signal at least 5-, 10-, or 20-fold higher than
 the background hybridization provided with non-polynucleotide coding
 sequences.
 Polynucleotides are mapped to particular chromosomes using, for example,
 radiation hybrids or chromosome-specific hybrid panels. See Leach et al.,
 Advances in Genetics, (1995) 33:63-99; Walter el al., Nature Genetics
 (1994) 7:22-28; Walter and Goodfellow, Trends in Genetics (1992) 9:352.
 Such mapping can be useful in identifying the function of the
 polynucleotide-related gene by its proximity to other genes with known
 function. Function can also be assigned to the related gene when
 particular syndromes or diseases map to the same chromosome.
 A polynucleotide will be useful in forensics, genetic analysis, mapping,
 and diagnostic applications if the corresponding region of a gene is
 polymorphic in the human population. A particular polymorphic form of the
 polynucleotide may be used to either identify a sample as deriving from a
 suspect or rule out the possibility that the sample derives from the
 suspect. Any means for detecting a polymorphism in a gene are used,
 including but not limited to electrophoresis of protein polymorphic
 variants, differential sensitivity to restriction enzyme cleavage, and
 hybridization to an allele-specific probe.
 Any naturally occurring variants of the nucleotide sequences which encode
 variants thereof are within the scope of this invention. Allelic variants
 of subgenomic polynucleotides of the invention can occur and can be
 identified by hybridization of putative allelic variants with nucleotide
 sequences disclosed herein under stringent conditions. For example, by
 using the following wash conditions--2.times.SCC, 0.1% SDS, room
 temperature twice, 30 minutes each; then 2.times.SCC, 0.1% SDS, 50.degree.
 C. once, 30 minutes; then 2.times.SCC, room temperature twice, 10 minutes
 each--allelic variants of the polynucleotides of the invention can be
 identified which contain at most about 25-30% base pair mismatches. More
 preferably, allelic variants contain 15-25% base pair mismatches, even
 more preferably 5-15%, or 2-5%, or 1-2% base pair mismatches.
 Amplification by the polymerase chain reaction (PCR) can be used to obtain
 the polynucleotides of the invention, using either genomic DNA or cDNA as
 a template. The polynucleotides of the invention may also be obtained
 using reverse transcriptase and mRNA molecules that are complementary to
 the minus strand of a double-stranded sequence wherein said
 double-stranded sequence is selected from the group of polynucleotides
 comprising a sequence as shown in SEQ ID NOS:1-15. Using the
 polynucleotide sequences disclosed herein, subgenomic polynucleotide
 molecules of the invention can also be made usinc the techniques of
 synthetic chemistry.
 Probes specific to the polynucleotides of the invention may be generated
 using the polynucleotide sequences disclosed in SEQ ID NOS:1-15. The
 probes are preferably at least 12, 14, 16, 18, 20, 22, 24, or 25
 nucleotides in length and can be less than 2, 1, 0.5, 0.1, or 0.05 kb in
 length. The probes can be synthesized chemically or can be generated from
 longer polynucleotides using restriction enzymes. The probes can be
 labeled, for example, with a radioactive, biotinylated, or fluorescent
 tag.
 Subgenomic polynucleotides of the invention can be propagated in vectors
 and cell lines using techniques well known in the art. Expression systems
 in bacteria include those described in Chang el al., Nature (1978) 275:
 615; Goeddel et al., Nature (1979) 281: 544; Goeddel et al., Nucleic Acids
 Res. (1980) 8: 4057; EP 36,776; U.S. 4,551,433; deBoer et al., Proc. Natl.
 Acad. Sci. USA (1983) 80: 21-25; and Siebenlist et al, Cell (1980) 20:
 269.
 Expression systems in yeast include those described in Hinnen et al., Proc.
 Natl Acad. Sci USA (1978) 75: 1929; Ito et al., J. Bacteriol. (1983) 153:
 163; Kurtz et al., Mol. Cell. Biol. (1986) 6: 142; Kunze et al., J. Basic
 Microbiol. (1985) 25: 141; Gleeson es al, J. Gen. Microbiol. (1986) 132:
 3459; Roggenkamp et al., Mol. Gen. Genet. (1986) 202 :302, Das et al., J.
 Bacteriol. (1984) 158: 1165; De Louvencourt et al., J. Bacteriol. (1983)
 154: 737; Van den Berg et al., Bio/Technology (1990) 8: 135; Kunze et al.,
 J. Basic Microbiol. (1985) 25:141; Cregg et al., Mol. Cell. Biol. (1985)
 5: 3376; U.S. Pat. No. 4,837,148; U.S. Pat. No. 4,929,555; Beach and
 Nurse, Nature (1981) 300: 706; Davidow et al, Curr. Genet. (1985) 10: 380;
 Gaillardin et al., Curr. Genet. (1985) 10: 49; Ballance et al., Biochem.
 Biophys. Res. Commun. (1983) 112: 284-289; Tilburn et al., Gene (1983) 26:
 205-221; Yelton et al, Proc. Natl. Acad. Sci. USA (1984) 81: 1470-1474;
 Kelly and Hynes, EMBO J. (1985) 4: 475479; EP 244,234; and WO 91/00357.
 Expression of the subgenomic polynucleotides of the invention in insects
 can be accomplished as described in U.S. Pat. No. 4,745,051, Friesen el al
 (1986) "The Regulation of Baculovirus Gene Expression" in: THE MOLECULAR
 BIOLOGY OF BACULOVIRUSES (W. Doerfier, ed.); EP 127,839; EP 155,476; VIak
 et al., J. Gen. Virol. (1988) 69: 765-776; Miller et al., Ann. Rev.
 Microbiol. (1988) 42: 177; Carbonell et al., Gene (1988) 73: 409; Maeda et
 al., Nature (1985) 315: 592-594; Lebacq-Verheyden et al., Mol. Cell. Biol.
 (1988) 8: 3129; Smith et al., Proc. Natl. Acad. Sci. USA (1985) 82: 8404;
 Miyajima et al., Gene (1987) 58: 273; and Martin et al., DNA (1988) 7:99.
 Numerous baculoviral strains and variants and corresponding permissive
 insect host cells from hosts are described in Luckow et al.,
 Bio/Technology (1988) 6: 47-55; Miller et al., in GENETIC ENGINEERING
 (Setlow, J. K. et al. eds.), Vol. 8 (Plenum Publishing, 1986), pp.
 277-279; and Maeda et al., Nature, (1985) 315: 592-594.
 Mammalian expression of the subgenomic polynucleotides of the invention can
 be accomplished as described in Dijkema et al., EMBO J. (1985) 4: 76;
 Gorman et al., Proc. Natl. Acad. Sci. USA (1982) 79: 6777; Boshart et al.,
 Cell (1985) 41: 521; and U.S. Pat. No. 4,399,216. Other features of
 mammalian expression can be facilitated as described in Ham and Wallace,
 Meth. Enz. (1979) 58: 44; Barnes and Sato, Anal. Biochem. (1980) 102: 255;
 U.S. Pat. No. 4,767,704; US 4,657,866; U.S. Pat. No. 4,927,762; U.S. Pat.
 No. 4,560,655; WO 90/103430; WO 87/00195; and U.S. RE 30,985.
 The subgenomic polynucleotides of the invention can be on linear or
 circular molecules. They can be on autonomously replicating molecules
 (vectors) or on molecules without replication sequences. They can be
 regulated by their own or by other regulatory sequences, as is known in
 the art. The subgenomic polynucleotides of the invention can be introduced
 into suitable host cells using a variety of techniques which are available
 in the art, such as transferrin-polycation-mediated DNA transfer,
 transfection with naked or encapsulated nucleic acids, liposome-mediated
 DNA transfer, intracellular transportation of DNA-coated latex beads,
 protoplast fusion, viral infection, electroporation, gene gun, and calcium
 phosphate-mediated transfection.
 The invention provides a method of detecting expression of a polynucleotide
 in, for example, a biological sample, which can be useful, inter alia, for
 diagnosing cancer or dysplasia. The basis for this method is the discovery
 that the polynucleotide sequence(s) as shown in:
 SEQ ID NO:12 is down-regulated in cancer,
 SEQ ID NOS:2, 5, and 15 are up-regulated in cancer and dysplasia; and
 SEQ ID NOS:1, 3-4, 6-11, and 13-14 are up-regulated in dysplasia only.
 In patients who have been diagnosed with pancreatic dysplasia or cancer,
 the detection of levels of the expression products of the polynucleotide
 sequences of the invention, either mRNA or protein, can be used to
 diagnose or prognose a disorder, to monitor treatment of the disorder, or
 to screen agents which affect the disorder.
 The expression products of the polynucleotide sequences of the invention,
 either mRNA or proteins, can be detected in a body sample for diagnosis or
 prognosis. The body sample can be, for example, a solid tissue or a fluid
 sample. The patient from whom the body sample is obtained can be healthy
 or can already be identified as having a condition in which altered
 expression of a protein of the invention is implicated.
 In one embodiment, the body sample is assayed for the levet of a protein
 expressed from a polynucleotide sequence of the invention. The protein
 could be detected by, for example, antibodies to the proteins. The
 antibodies can be labeled, for example, with a radioactive, fluorescent,
 biotinylated, or enzymatic tag and detected directly, or can be detected
 using indirect immunochemical methods, using a labeled secondary antibody.
 The presence of the protein can be assayed, for example, in tissue
 sections by immunocytochemistry, or in lysates, using Western blotting, as
 is known in the art.
 The levet of the protein in a tissue sample suspected of being cancerous or
 dysplastic is compared with the levet of the protein in a normal tissue. A
 higher level of the polypeptides expressed from polynucleotide sequences
 as shown in SEQ ID NOS:1, 3-4, 6-11, and 13-14 in the suspect tissue, as
 compared to the normal tissue, indicates the presence of dysplastic cells
 in the suspect tissue. A higher levet of the polypeptides expressed from
 polynucleotide sequences as shown in SEQ ID NOS:2, 5, and 15 in the
 suspect tissue, as compared to the normal tissue, indicates the presence
 dysplastic cells or cancerous cells or both in the suspect tissue. A lower
 levet of the polypeptide expressed from the polynucleotide sequence as
 shown in SEQ ID NO:12 in the suspect tissue, as compared to the normal
 tissue, indicates the presence of cancerous cells in the suspect tissue.
 Additionally, a differentiation between cancer or dysplasia in a patient's
 diagnosis can be made. The expression of a polynucleotide sequence of the
 invention that is up-regulated in dysplastic cells only (i.e., SEQ ID
 NOS:1, 3-4, 6-11, and 13-14) and the expression of a polynucleotide that
 is up-regulated in both dysplastic cells and cancerous cells (i.e., SEQ ID
 NOS:2, 5, and 15) can be used to screen a patient's tissues. If
 examination of a patient's tissues reveals that there is no up-regulation
 of a polynucleotide sequence that is up-regulated in dysplastic cells only
 (i.e., SEQ ID NOS: 1, 3-4, 6-11, and 13-14), and that there is
 up-regulation of a polynucleotide sequence that is up-regulated in both
 cancerous cells and dysplastic cells (i.e., SEQ ID NOS:2, 5, and 15), then
 the patient is diagnosed with cancer.
 Alternatively, the presence of mRNA expressed from the polynucleotide
 sequences of the invention in two tissues can be compared. mRNA can be
 detected, for example, by ini silil hybridization in tissue sections, by
 reverse transcriptase-PCR, or in Northern blots containing poly A+mRNA.
 One of skill in the art can readily determine differences in the size or
 amount of mRNA transcripts between two tissues, using Northern blots and
 nucleotide probes. For example, the levet of mRNA of the invention in a
 tissue sample suspected of being cancerous or dysplastic is compared with
 the expression of the mRNA in a normal tissue. Any methods known in the
 art for determining the amounts of specific mRNAs can be used.
 A higher levet of mRNA expressed from polynucleotide sequences as shown in
 SEQ ID NOS:1, 3-4, 6-11, and 13-14 in the suspect tissue, as compared to
 the normal tissue, indicates the presence dysplastic cells in the suspect
 tissue. A higher level of mRNA expressed from the polynucleotide sequences
 as shown in SEQ ID NOS:2, 5, and 15 in the suspect tissue, as compared to
 the normal tissue, indicates the presence dysplastic cells or cancerous
 cells or both in the suspect tissue. A lower levet of the mRNA expressed
 from the polynucleotide sequence as shown in SEQ ID NO:12 in the suspect
 tissue, as compared to the normal tissue, indicates the presence of
 cancerous cells in the suspect tissue. Any combinations of these sequences
 can be used to determine a diagnosis.
 Optionally, the levet of a particular expression product of a
 polynucleotide sequence of the invention in a body sample can be
 quantitated. Quantitation can be accomplished, for example, by comparing
 the levet of expression product detected in the body sample with the
 amounts of product present in a standard curve. A comparison can be made
 visually or using a technique such as densitometry, with or without
 computerized assistance. Alternative methods can be used, for example
 ELISA, western blot, immunoprecipitation, radioimmunoassay, etc. Any
 method known in the art for detecting and quantitating a particular
 protein can be used.
 Reagents specific for the polynucleotides and polypeptides of the
 invention, such as antibodies and nucleotide probes, can be supplied in a
 kit for detecting the presence of an expression product in a biological
 sample. The kit can also contain buffers or labeling components, as well
 as instructions for using the reagents to detect and quantify expression
 products in the biological sample.
 Polynucleotide expression in a cell can be increased or decreased, as
 desired. Polynucleotide expression can be altered for therapeutic
 purposes, as described below, or can be used to identify and study the
 role of therapeutic agents in cancer and other diseases.
 Decreasing the expression of genes containing sequences selected from the
 group consisting of the sequences as shown in SEQ ID NOS:1, 3-4, 6-11, and
 13-14 is useful, for example, as a therapeutic for altering the abnormal
 characteristics of dysplastic cells. Decreasing the expression of
 polynucleotide sequences selected from the group consisting of the
 sequences as shown in SEQ ID NOS:2, 5, and 15 is useful, for example, as a
 therapeutic agent for decreasing the growth rate of dysplastic and cancer
 cells.
 Expression of the polynucleotide sequences of the invention can be altered
 using an antisense oligonucleotide sequence. Therapeutic compositions for
 decreasing gene expression comprise an expression construct containing
 polynucleotides encoding all or a portion of a polynucleotide sequence
 selected from the group consisting of SEQ ID NOS:1-11, and 13-15. Within
 the expression construct, the polynucleotide segment is orientated in the
 antisense direction and is located downstream from a promoter.
 Transcription of the polynucleotide segment initiates at the promoter.
 Preferably, the antisense oligonucleotide sequence is at least ten
 nucleotides in length, but longer sequences of at least 11, 12, 15, 20,
 25, 30, 35, 40, 45, 50, 60, 70, 74, 80, 90, 100, 125, 150, 162, 175, 200,
 250, 300, or 350 contiguous nucleic acids can also be used. Antisense
 oligonucleotide molecules can be provided in a DNA construct and
 introduced into cells whose division is to be decreased, as described
 above. A more complete description of gene transfer vectors, especially
 retroviral vectors is contained in U.S. Ser. No. 08/869,309, which is
 incorporated herein by reference.
 The antisense oligonucleotides can be composed of deoxyribonucleotides,
 ribonucleotides, or a combination of both. Oligonucleotides can be
 synthesized manually or by an automated synthesizer, by covalently linking
 the 5' end of one nucleotide with the 3' end of another nucleotide with
 phosphodiester or non-phosphodiester internucleotide linkages such as
 alkylphosphonates, phosphorothioates, phosphorodithioates,
 alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate
 esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and
 phosphate triesters. See Brown, 1994, Meth. Mol. Biol. 20:1-8; Sonveaux,
 1994, Meth. Mol. Biol. 26:1-72; Uhlmann et al., 1990, Chem. Rev.
 90:543-583.
 Although precise complementarity is not required for successful duplex
 formation between an antisense molecule and the complementary coding
 sequence of a gene, antisense molecules with no more than one mismatch are
 preferred. One skilled in the art can easily use the calculated melting
 point of an antisense-sense pair to determine the degree of mismatch which
 will be tolerated between a particular antisense oligonucleotide and a
 particular coding sequence of the selected gene.
 The antisense oligonucleotides of the invention can be modified without
 affecting their ability to hybridize to a polynucleotide coding sequence
 of the present invention. These modifications can be internal or at one or
 both ends of the antisense molecule. For example, internucleoside
 phosphate linkages can be modified by adding cholesteryl or diamine
 moieties with varying numbers of carbon residues between the amino groups
 and terminal ribose. Modified bases or sugars or both, such as arabinose
 instead of ribose, or a 3', 5'-substituted oligonucleotide in which the 3'
 hydroxyl group or the 5' phosphate group are substituted, can also be
 employed in a modified antisense oligonucleotide. These modified
 oligonucleotides can be prepared by methods well known in the art. Agrawal
 et al., 1992, Trends Biotechnol. 10: 152-158; Uhlmann et al., 1990, Chem.
 Rev. 90:543-584; Uhlmann et al., 1987, Tetrahedron. Lett. 215:3539-3542.
 Expression of the polynucleotides of the invention can also be decreased by
 delivering polyclonal, monoclonal, or single chain antibodies that
 specifically bind to polypeptides expressed from the polynucleotide
 sequences as shown in SEQ ID NOS:1-11 and 13-15. Antibodies specific to
 these proteins bind to the protein and prevent the protein from
 functioning in the cell. Blocking protein expression or function is
 usefuil for preventing, reducing the effects of, or curing, cancer and
 dysplasia.
 In one embodiment of the invention, expression of the polynucleotides
 selected from the group consisting of the polynucleotide sequences shown
 in SEQ ID NOS:1-11, and 13-15 are decreased using a ribozyme, an RNA
 molecule with catalytic activity. See, e.g., Cech, 1987, Science 236:
 1532-1539; Cech, 1990, Ann. Rev. Biochem. 59:543-568; Cech, 1992, Curr.
 Opin. Struct. Biol. 2: 605-609; Couture and Stinchcomb, 1996, Trends
 Genet. 12: 510-515. Ribozymes can be used to inhibit gene function by
 cleaving an RNA sequence, as is known in the art (e.g., Haseloffet al.,
 U.S. Pat. No. 5,641,673).
 The coding sequence of a polynucleotide of the invention can be used to
 generate a ribozyme which will specifically bind to RNA transcribed from
 said polynucleotide. Methods of designing and constructing ribozymes which
 can cleave other RNA molecules in trans in a highly sequence specific
 manner have been developed and described in the art (see Haseloff, J. et
 al. (1988), Nature 334:585-591). For example, the cleavage activity of
 ribozymes can be targeted to specific RNAs by engineering a discrete
 "hybridization" region into the ribozyme. The hybridization region
 contains a sequence complementary to the target RNA and thus specifically
 hybridizes with the target (see, for example, Gerlach, W. L. et al., EP
 321,201). Longer complementary sequences can be used to increase the
 affinity of the hybridization sequence for the target. The hybridizing and
 cleavage regions of the ribozyme of the invention can be integrally
 related; thus, upon hybridizing to the target RNA through the
 complementary regions, the catalytic region of the ribozyme can cleave the
 target.
 Ribozymes of the invention can be introduced into cells as part of a DNA
 construct, as is known in the art. The DNA construct can also include
 transcriptional regulatory elements, such as a promoter element, an
 enhancer or UAS element, and a transcriptional terminator signal, for
 controlling the transcription of the ribozyme in the cells.
 Mechanical methods, such as microinjection, liposome-mediated transfection,
 electroporation, gene gun, or calcium phosphate precipitation, can be used
 to introduce the ribozyme-containing DNA construct into cells whose
 division it is desired to decrease, as described above. Alternatively, if
 it is desired that the DNA construct be stably retained by the cells, the
 DNA construct can be supplied on a plasmid and maintained as a separate
 element or integrated into the genome of the cells, as is known in the
 art.
 As taught in Haseloff et al., U.S. Pat. No. 5,641,673, the ribozymes of the
 invention can be engineered so that their expression will occur in
 response to factors which induce expression of a polynucleotides of the
 invention. The ribozyme can also be engineered to provide an additional
 levet of regulation, so that destruction of RNA occurs only when both the
 ribozyme and the corresponding gene are induced in the cells.
 Preferably, the mechanism used to decrease expression of the
 polynucleotides of the invention, whether antisense nucleotide sequence,
 antibody, or ribozyme decreases expression of the polynucleotide by 50%,
 60%, 70%, or 80%. Most preferably, expression of the polynucleotide is
 decreased by 90%, 95%, 99%, or 100%. The effectiveness of the mechanism
 chosen to alter expression of the polynucleotide can be assessed using
 methods well known in the art, such as hybridization of nucleotide probes
 to mRNA of the polynucleotide, quantitative RT-PCR, or detection of a
 protein using specific antibodies of the invention.
 Increased expression of a polynucleotide is useful to decrease the growth
 rate of cancer cells where the particular polynucleotide is down-regulated
 in cancer cells, such as the polynucleotide sequence as shown in SEQ ID
 NO:12. Therapeutic compositions for increasing polynucleotide expression
 comprise an expression construct containing all or a portion of the
 polynucleotide sequence as shown in SEQ ID NO:12. Within an expression
 construct, the polynucleotide segment is oriented in the sense direction
 and is located downstream from the promoter. Transcription of the
 polynucleotide segment initiates at the promoter. The expression construct
 can be introduced into cells along with a pharmaceutically acceptable
 carrier to decrease the growth rate of cancer cells or ameliorate other
 abnormal characteristics. Expression of the polynucleotide sequence can be
 monitored by detecting production of mRNA which hybridizes to the
 delivered polynucleotide or by detecting protein encoded by the delivered
 polynucleotide.
 Proteins that are expressed from the polynucleotide sequences of the
 invention can be produced recombinantly in prokaryotic or eukaryotic host
 cells, such as bacteria, yeast, insect, or mammalian cells, using
 expression vectors known in the art. Enzymes can be used to generate less
 than full length polypeptides by enzymatic proteolysis of full-length
 proteins of the invention. Alternatively, synthetic chemistry methods,
 such as solid-phase peptide synthesis, can be used to synthesize the
 proteins and polypeptides.
 Species homologs of human subgenomic polynucleotides or the encoded
 polypeptides can be identified by making suitable probes or primers and
 screening cDNA expression libraries from other species, such as mice,
 monkeys, yeast, or bacteria. Mammalian homologs are preferred, however.
 Proteins or polypeptides expressed from the polynucleotide sequences as
 shown in SEQ ID NO:1-15 can be isolated and purified from human cells that
 express the proteins. The proteins can be obtained substantially free from
 other human proteins by standard protein purification methods, such as
 size exclusion chromatography, ion exchange chromatography, ammonium
 sulfate fractionation, affinity chromatography, or preparative get
 electrophoresis.
 Proteins or polypeptides expressed from the polynucleotides of the
 invention can also be used in a fusion protein, for example, as an
 immunogen. The fusion protein comprises two protein segments. The first
 protein segment consists of at least six, eight, ten, twelve, fifteen,
 twenty or thirty contiguous amino acids of a polypeptide sequence
 expressed from a polynucleotide sequence as shown in SEQ ID NOS:1-15. The
 first protein segment is fused to a second protein segment by means of a
 peptide bond. The second protein segment can be a full-length protein or a
 fragment of a protein. Techniques for making fusion proteins, either
 recombinantly or by covalently linking two protein segments, are well
 known in the art.
 The second protein or protein fragment of a fusion protein can be derived
 from another type of protein or a similar protein. The second protein or
 protein fragment can be labeled with a detectable marker, such as a
 radioactive or fluorescent tag, or can be an enzyme that will generate a
 detectable product. Enzymes suitable for this purpose, such as
 .beta.-galactosidase, are well-known in the art. A fusion protein can be
 used, for example, to target the proteins of the invention or polypeptides
 to a particular location in a cell or tissue, in various assays, such as
 the yeast two-hybrid technique, or as an immunogen.
 The proteins or polypeptides expressed from the polynucleotides of the
 invention can be used for generating antibodies. The antibodies can be
 used, inter alia, to detect and quantitate expression of the cognate
 protein. Proteins or polypeptides expressed from the polynucleotides of
 the invention comprising at least six, eight, ten, twelve, fifteen, twenty
 or thirty consecutive amino acids can be used as immunogens. The proteins
 or polypeptides can be used to obtain a preparation of antibodies which
 specifically bind to a protein or polypeptide of the invention. The
 antibodies can be polyclonal or monoclonal. Techniques for raising both
 polyclonal and monoclonal antibodies are well known in the art.
 Single chain antibodies can also be constructed. Single chain antibodies
 which specifically bind to a protein or polypeptide expressed from the
 polynucleotides of the invention can be isolated, for example, from
 single-chain immunoglobulin display libraries, as are known in the art.
 The library is "panned" against a protein or polypeptide, and a number of
 single chain antibodies which bind different epitopes of the polypeptide
 with high-affinity can be isolated. Hayashi et al., 1995, Gene 160:129-30.
 Such libraries are known and available to those in the art. The antibodies
 can also be constructed using the polymerase chain reaction (PCR), using
 hybridoma cDNA as a template. Thirion et al., 1996, Eur. J. Cancer Prev.
 5:507-11.
 The single chain antibody can be mono- or bi-specific, and can be bivalent
 or tetravalent. Construction of tetravalent bispecific single chain
 antibodies is taught in Coloma and Morrison, 1997, Nat. Biotechnol.
 15:159-63. Construction of bivalent bispecific single chain antibodies is
 taught in Mallender and Voss, 1994, J. Biol. Chem. 269:199-206.
 A nucleotide sequence encoding the single chain antibody can then be
 constructed using manual or automated nucleotide synthesis, cloned into
 DNA expression vectors using standard recombinant DNA methodologies, and
 introduced into cells which express the selected gene, as described below.
 Alternatively, the antibodies can be produced directly using filamentous
 phage technology. Verhaar et al., 1995, Int. J. Cancer 61:497-501;
 Nicholls et al., 1993, J. Immunol. Meth. 165:81-91.
 The antibodies bind specifically to the epitopes of the proteins or
 polypeptides expressed from the polynucleotides of the invention. In a
 preferred embodiment, the epitopes are not present on other human
 proteins. Typically a minimum number of contiguous amino acids to encode
 an epitope is 6, 8, or 10. However, more can be used, for example, at
 least 15, 25, or 50, especially to form epitopes which involve
 non-contiguous residues or particular conformations.
 Antibodies that bind specifically to the proteins or polypeptides include
 those that bind to full-length proteins or polypeptides. Specific binding
 antibodies do not detect other proteins on Western blots of human cells,
 or provide a signal at least ten-fold lower than the signal provided by
 the target protein of the invention. Antibodies which have such
 specificity can be obtained by routine screening. In a preferred
 embodiment of the invention, the antibodies immunoprecipitate the proteins
 or polypeptides expressed from the polynucleotides of the invention from
 cell extracts or solution. Additionally, the antibodies can react with
 proteins or polypeptides expressed from the polynucleotides of the
 invention in tissue sections or on Western blots of polyacrylamide gels.
 Preferably the antibodies do not exhibit nonspecific cross-reactivity with
 other human proteins on Western blots or in immunocytochemical assays.
 Techniques for purifying antibodies to the proteins or polypeptides
 expressed from the polynucleotides of the invention are available in the
 art. In a preferred embodiment, the antibodies are passed over a column to
 which a particular protein or polypeptide expressed from the
 polynucleotides of the invention is bound. The bound antibodies are then
 eluted, for example, with a buffer having a high salt concentration.
 Therapeutic compositions of the invention also comprise a pharmaceutically
 acceptable carrier. Pharmaceutically acceptable carriers are well known to
 those in the art. Such carriers include, but are not limited to, large,
 slowly metabolized macromolecule, such as proteins, polysaccharides,
 polylactic acids, polyglycolic acids, polymeric amino acids, amino acid
 copolymers, and inactive virus particles. Pharmaceutically acceptable
 salts can also be used in the composition, for example, mineral salts such
 as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as the
 salts of organic acids such as acetates, proprionates, malonates, or
 benzoates.
 Therapeutic compositions can also contain liquids, such as water, saline,
 glycerol, and ethanol, as well as substances such as wetting agents,
 emulsifying agents, or pH buffering agents. Liposomes, such as those
 described in U.S. Pat. No. 5,422,120, WO 95/13796, WO 91/14445, or EP
 524,968 B1, can also be used as a carrier for the therapeutic composition.
 Typically, a therapeutic composition is prepared as an injectable, either
 as a liquid solution or suspension; however, solid forms suitable for
 solution in, or suspension in, liquid vehicles prior to injection can also
 be prepared. A composition can also be formulated into an enteric coated
 tablet or get capsule according to known methods in the art, such as those
 described in U.S. Pat. No. 4,853,230, EP 225,189, AU 9,224,296, and AU
 9,230,801.
 Administration of the therapeutic agents of the invention can include local
 or systemic administration, including injection, oral administration,
 particle gun, or catheterized administration, and topical administration.
 Various methods can be used to administer a therapeutic composition
 directly to a specific site in the body.
 For treatment of tumors, for example, a small tumor or metastatic lesion
 can be located and a therapeutic composition injected several times in
 several different locations within the body of the tumor. Alternatively,
 arteries which serve a tumor can be identified, and a therapeutic
 composition injected into such an artery, in order to deliver the
 composition directly into the tumor.
 A tumor which has a necrotic center can be aspirated and the composition
 injected directly into the now empty center of the tumor. A therapeutic
 composition can be directly administered to the surface of a tumor, for
 example, by topical application of the composition. X-ray imaging can be
 used to assist in certain of the above delivery methods. Combination
 therapeutic agents, including a protein or polypeptide or a subgenomic
 polynucleotide and other therapeutic agents, can be administered
 simultaneously or sequentially.
 Receptor-mediated targeted delivery can be used to deliver therapeutic
 compositions containing subgenomic polynucleotides, proteins, or reagents
 such as antibodies, ribozymes, or antisense oligonucleotides of the
 invention to specific tissues. Receptor-mediated delivery techniques are
 described in, for example, Findeis et al. (1993), Trends in Biotechnol.
 11, 202-05; Chiou et al. (1994), GENE THERAPEUTICS: METHODS AND
 APPLICATIONS OF DIRECT GENE TRANSFER (J. A. Wolff, ed.); Wu & Wu (1988), J
 Biol. Chem. 263, 621-24; Wu et al. (1994), J. Biol. Chem. 269, 542-46;
 Zetike et al. (1990), Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59; Wu et al.
 (1991), J. Biol. Chem. 266, 338-42.
 Alternatively, therapeutic compositions can be introduced into human cells
 ex vivo, and the cells then replaced into the human. Cells can be removed
 from a variety of locations including, for example, from a selected tumor
 or from an affected organ. In addition, a therapeutic composition can be
 inserted into non-affected, for example, dermal fibroblasts or peripheral
 blood leukocytes. If desired, particular fractions of cells such as a T
 cell subset or stem cells can also be specifically removed from the blood
 (see, for example, PCT WO 91/16116). The removed cells can then be
 contacted with a therapeutic composition utilizing any of the
 above-described techniques, followed by the return of the cells to the
 human, preferably to or within the vicinity of a tumor or other site to be
 treated. The methods described above can additionally comprise the steps
 of depleting fibroblasts or other non-contaminating tumor cells subsequent
 to removing tumor cells from a human, and/or the step of inactivating the
 cells, for example, by irradiation.
 Both the dose of a composition and the means of administration can be
 determined based on the specific qualities of the therapeutic composition,
 the condition, age, and weight of the patient, the progression of the
 disease, and other relevant factors. Preferably, a therapeutic composition
 of the invention increases or decreases expression of a polynucleotide by
 50%, 60%, 70%, or 80%. Most preferably, expression of the polynucleotide
 is increased or decreased by 90%, 95%, 99%, or 100%. The effectiveness of
 the mechanism chosen to alter expression of the polynucleotide can be
 assessed using methods well known in the art, such as hybridization of
 nucleotide probes to mRNA of the polynucleotide, quantitative RT-PCR, or
 detection of a protein or polypeptide using specific antibodies.
 If the composition contains protein, polypeptide, or antibody, effective
 dosages of the composition are in the range of about 5 .mu.g to about 50
 .mu.g/kg of patient body weight, about 50 .mu.g to about 5 mg/kg, about
 100 .mu.g to about 500 .mu.g/kg of patient body weight, and about 200 to
 about 250 .mu.g/kg.
 Therapeutic compositions containing subgenomic polynucleotides can be
 administered in a range of about 100 ng to about 200 mg of DNA for local
 administration in a gene therapy protocol. Concentration ranges of about
 500 ng to about 50 mg, about 1 .mu.g to about 2 mg, about 5 .mu.g to about
 500 .mu.g, and about 20 .mu.g to about 100 .mu.g of DNA can also be used
 during a gene therapy protocol. Factors such as method of action and
 efficacy of transformation and expression are considerations that will
 effect the dosage required for ultimate efficacy of the subgenomic
 polynucleotides. Where greater expression is desired over a larger area of
 tissue, larger amounts of subgenomic polynucleotides or the same amounts
 re-administered in a successive protocol of administrations, or several
 administrations to different adjacent or close tissue portions of, for
 example, a tumor site, may be required to effect a positive therapeutic
 outcome. In all cases, routine experimentation in clinical trials will
 determine specific ranges for optimal therapeutic effect.
 The therapeutic compositions are useful in treating pancreatic cancer and
 pancreatic dysplasia, as well as other types of cancers such as: bone
 cancer; brain tumors, breast cancer; endocrine system cancers, such as
 cancers of the thyroid, pituitary, and adrenal glands and the pancreatic
 islets; gastrointestinal cancers, such as cancer of the anus, colon,
 esophagus, gallbladder, stomach, liver, and rectum; genitourinary cancers
 such as cancer of the penis, prostate and testes; gynecological cancers,
 such as cancer of the ovaries, cervix, endometrium, uterus, fallopian
 tubes, vagina, and vulva; head and neck cancers, such as hypopharyngeal,
 laryngeal, oropharyngeal cancers, lip, mouth and oral cancers, cancer of
 the salivary gland, cancer of the aerodigestive tract and sinus cancer;
 leukemia; lymphomas including Hodgkin's and non-Hodgkin's lymphoma;
 metastatic cancer; myelomas; sarcomas; skin cancer; urinary tract cancers
 including bladder, kidney and urethral cancers; and pediatric cancers,
 such as pediatric brain tumors, leukemia, lymphomas, sarcomas, liver
 cancer and neuroblastoma and retinoblastoma.
 The following example provides data and experimental procedures. However,
 the invention is not limited to the example. The invention is defined in
 the specification as a whole which includes the claims.