EP1723424A2 - Gene expression and polymorphisms that influence lung cancer susceptibility - Google Patents

Abstract

Methods are provided relating to the identification and treatment of cancers that involve altered expression of the polymerase (DNA directed) iota (POLI) (Pol ι) gene.

Description

GENE EXPRESSION AND POLYMORPHISMS THAT INFLUENCE LUNG CANCER SUSCEPTIBILITY

GOVERNMENT RIGHTS [001] This invention was supported, at least in part, by NLH grants R.01 CA099147 and CA58554. The United States Federal Government has certain rights in this invention.

PRIORITY CLAIM [002] This application claims priority to United States Provisional Patent Application 60/552,909, filed March 12, 2004, which is incorporated herein by reference, in its entirety.

BACKGROUND OF THE INVENTION [003] Lung cancer is the leading cause of mortality from cancer in both men and women in developed countries. There is evidence that although incidence is almost always associated with environmental factors such as smoking or occupational exposure to carcinogens, susceptibility has a genetic component, with early onset lung cancer following Mendelian inheritance. Moreover, susceptibility is largely intrinsic to the lung itself, as shown by classical experiments involving lung explants from sensitive and resistant mice. [004] Accordingly, it is desirable to have methods to provide additional information about the genetic profile of an individual. This information is useful in the context of cancer, including lung cancer, to identify individuals who are at risk for developing the disease so as to provide preventive care or prophylaxis. It is also useful for determining methods of treatment that are optimized for an individual's particular cancer profile. SUMMARY OF THE INVENTION [005] This invention relates to diagnosis and treatment of cancer. In particular, it relates to cancers that involve the expression of the polymerase (DNA directed) iota (POLI) (Pol i) gene and/or its product, the Pol . protein. Pol i is believed to be a modifier of lung tumorigenesis which alters DNA polymerase activity.

[006] Human groups exist that have an above average probability of being diagnosed with and/or of developing particular cancers (i.e., high-risk groups) and are appropriate candidates for evaluation of Pol i expression, and/or therapeutic or prophylactic use of Pol i. Such human groups may be at high risk because of exposure to particular environmental materials or circumstances (e.g., smoking or exposure to tobacco smoke, occupational exposure to carcinogens such as urethane and other agents), because of familial susceptibility to certain cancers (e.g., genetic inheritance of genes causing increased susceptibility), as a result of the presence of mutant forms of Pol t and/or reduced levels of non-mutant, wild type Pol i protein.

[007] In some embodiments, the invention provides methods for characterizing the etiology of a cancer in an individual by testing at least one cancer cell from the individual for one or more mutations in the at least one cancer cell's genomic Pol i gene. According to one embodiment, the at least one cancer cell is tested for the presence of a mutation at codon 706 of the human Pol i gene. More particularly, the at least one cancer cell is tested for the presence of a mutation at codon 706 wherein the mutation encodes for a change in the encoded amino acid from Threonine in the wild-type form of Pol i to Alanine in the mutant form of Pol i.

[008] According to one embodiment, the presence of a mutation at codon 706 ofthe human

Pol i gene is tested by analyzing the coding sequence ofthe Pol t gene.

[009] According to another embodiment, the presence of a mutation at codon 706 of the human Pol i gene is tested by using an antibody that detects the mutant Pol t protein.

[010] In some embodiments, the invention provides methods for identifying an individual who is at risk of developing cancer by testing at least one cell from the individual for one or more mutations in the at least one cell's genomic Pol t gene. According to one embodiment, the at least one cell is tested for the presence of a mutation at codon 706 of the human Pol i gene. More particularly, the at least one cell is tested for the presence of a mutation at codon 706 wherein the mutation encodes for a change in the encoded amino acid from Threonine in the wild-type form of Pol i to Alanine in the mutant form of Pol i.

[011] According to one embodiment, the presence of a mutation at codon 706 of the human Pol i gene is tested by analyzing the coding sequence ofthe Pol i gene. [012] According to another embodiment, the presence of a mutation at codon 706 of the human Pol i gene is tested by using an antibody that detects the mutant Pol i protein. [013] hi some embodiments the invention also provides methods for treating an individual identified as having a mutant Pol i gene or reduced expression of Pol i protein by administering to the individual an agent that restores or supplements wild-type, non-mutant Pol i protein function. According to some applications, the individual may have an adenocarcinoma, such as an adenocarcinoma of the lung. According to some embodiments, the agent is a Pol i protein. The Pol t protein may be administered in a fashion such that it is specifically targeted to cancer tissue in the individual. According to other embodiments, the agent is a polynucleotide encoding a Pol t protein, wherein the polynucleotide is in operable connection with a promoter that directs its expression. In some embodiments, the treatment is prophylactic.

[014] The compositions that are used according to the methods of this invention may be administered prior to, concurrent with, or after administration of other cancer therapeutic or prophylactic treatments, hi some embodiments, the agent or agents are administered to an individual identified as having a cancer associated with mutant Pol i genes. In other embodiments, the agent or agents are administered to an individual having an adenocarcinoma, in particular, an adenocarcinoma ofthe lung. [015] Additional features and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The features and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[016] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[017] The accompanying figures, which are incorporated in and constitute a part of this specification, and together with the description, serve to explain the principles of the invention. BRIEF DESCRIPTION OF THE DRAWINGS [018] Fig. 1 shows the amino acid sequence for the wild type Pol i protein from human; [019] Fig. 2 shows the nucleotide sequence that encodes a wild-type human Pol i protein; [020] Fig. 3. shows the amino acid sequence for the mouse Pol . protein; [021] Fig. 4 shows the nucleotide sequence that encodes a wild-type mouse Pol . protein; [022] Fig. 5 shows the annotated genes in the Par 2 candidate region. D18Mitl03 and D18Mitl88 were separately anchored to Celera and National Center for Biotechnology Information (NCBI) Build32 mouse genome maps by performing BLASTn search. The unit of gene position is "Mb."

[023] Fig. 6 shows amino acid alterations and transcript isoforms of the mouse Pol i gene. A, schematic illustration of amino acid-changing nαcleotide polymorphisms and alternative splicing. The full length of Pol imRNA consists of 10 exons ( ■ , E1-E10) with a total of 2497 bp (GenBank accession no. NM_011972). Exon 2d isoforai is produced by alternatively splicing out exon 2 (D) without changing the entire open reading frame. The asterisk sign (*) indicates that the exon 4a isoform has extra 32 bp (D) on regular exon 4, which produces a truncated protein caused by an early termination codon (tga) in exon 5. Ten amino acid changing codons are located in exon 1 (2), exon 8 (1), exon 9 (1), and exon 10 (6). The start codon (atg) is at 95 bp position, and the stop codon in the regular isoform is at 2246 bp position. B, amino acid-changing nucleotide polymorphism on codon 606 ofthe Pol i regular transcript. Direct sequencing results revealed that the codon 606 of the Pol i full-length transcript encodes a positive-charged arginine (CGA) in BALB/cJ but a neutral glutamine (CAA) in A/J mice. C, alternative mRNA splicing on exon 2. a, reverse transcription-PCR result using primer set 1 (see Materials and Methods in EXAMPLES). Two transcript isoforms were detected in A/J and BALB/cJ mouse lung. The longer one is regular Pol i transcript with exon 2. The shorter one is a new isoform without exon 2. b, sequencing result revealed that the new Pol i isoform is caused by in-frame exon 2 skip.

[024] Fig. 7 shows primer extension assays. A, differential activities in incorporation of dATP opposite template T at high enzyme concentration. Incorporations of dATP extend matched (a) and G/T mismatched (b) primer-template T. Pol t enzymatic activity is reflected by percentage of primers being converted. B, preferential incorporation of dGTP versus dATP opposite template T. Two ng of A/J and BALB/cJ full-length Pol i proteins were used in each reaction. At 0.1 and 1 mM deoxynucleoside triphosphate (dNTP) concentrations, both proteins exhibit classical Pol i enzymatic property, preferring incorporation of dGTP opposite template T. At 1 mM dNTP concentration, the BALB/cJ Pol i protein is less efficient (~2- fold) at incorporating dGTP opposite template T than the A/J protein.

[025] Fig. 8 shows alteration of POL I in human cancer cell lines. A, codon 706 polymorphism in human POL I gene. Sequencing has been done with reverse primer 2R. ACA to GCA (THR to ALA) was identified in A427, A549, CaLu-3, CaLu-6, NCI-H460, and NCIH596 cells. The polymorphism appeared to be heterozygous with the published sequence as indicated by arrow in Calu-3 sequence. B, Western Blot of human POL I protein in lung normal and cancer cell lines. A polyclonal antibody was used to examine protein expression. Ponceau Red staining has been performed to make sure that a similar amount of protein was loaded to each lane.

[026] Fig. 9 shows clustalw alignment of human, mouse, and fruit fly Pol i proteins. Mouse Pol i protein is represented by the A/J sequence. "*" indicates the identical residues in that column in the alignment.":" indicates the conserved substitutions. "." indicates semiconserved substitutions. The codon 706 is highlighted. DETAILED DESCRIPTION OF THE INVENTION [027] The present invention will now be described with occasional reference to the specific embodiments ofthe invention. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope ofthe invention to those skilled in the art.

[028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to that this invention belongs. The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular fomis "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[029] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth as used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical properties set forth in the following specification and claims are approximations that may vary depending on the desired properties sought to be obtained in embodiments of the present invention. Notwithstanding that the numerical ranges and parameters setting forth the broad scope ofthe invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from error found in their respective measurements. [030] The disclosure of all patents, patent applications (and any patents that issue thereon, as well as any corresponding published foreign patent applications), GenBank and other accession numbers and associated data, and publications mentioned throughout this description are hereby incorporated by reference herein, or in the provisional patent application to which this application claims priority. It is expressly not admitted, however, that any of the documents incorporated by reference herein teach or disclose the present invention.

[031] The present invention may be understood more readily by reference to the following detailed description of the embodiments of the invention and the Examples included herein. However, before the present methods and compositions are disclosed and described, it is to be understood that this invention is not limited to specific methods, specific nticleic acids, specific polypeptides, specific cell types, specific host cells or specific conditions, etc., as such may, of course, vary, and the numerous modifications and variations therein will be apparent to those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[032] "cDNA" means a DNA prepared using messenger RNA (mRNA) as template. In contrast to genomic DNA and DNA polymerized from a genomic, non- or partially-processed RNA template, cDNA contains coding sequences ofthe corresponding protein in the absence of introns and other non-translated nucleic acids.

[033] "Gene" refers broadly to any region or segment of DNA associated with a biological molecule or function. Thus, genes include coding sequence, and may further include regulatory regions or segments required for their expression. Genes may also include non- expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest, or synthesizing from known or predicted sequence information, and may include sequences encoding desired parameters.

[034] "Isolated," when used herein in the context of a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either dry form or an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant molecular species present in a preparation is substantially purified. An isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest. [035] "Malignant" or "cancerous" or "cancer" refers to the properties of cells or tissue that distinguish them from benign or normal cells. Malignant, cancerous, and cancer cells invade, grow and destroy adjacent tissue, metastasize, and usually grow more rapidly than benign cells.

[036] "Wild-type" is used herein to describe something that can be found in nature as distinct from being artificially produced by man, that is, "naturally-occurring", and in the context of Pol ., is non-mutant in that it lacks mutation at codon 706 ofthe human Pol t gene or the corresponding encoded amino acid. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is naturally- occurring. In particular, "wild-type" is used herein to refer to the naturally-occurring or native forms of proteins and their encoding nucleic acid sequences that lack mutations or polymorphisms that alter their function. Therefore, in the context of this application, 'wild- type' includes naturally occurring variant forms of Pol i genes, either representing splice variants or genetic variants between individuals, which may require different probes for selective detection, provided that such variants do not inch de variants in the sequence at codon 706 ofthe human Pol t gene.

[037] "Normal cell" means a non-cancerous or non-malignant cell.

[038] "Nucleic acid" and "polynucleotide" refer to deoxyribonucleotides or ribonucleotides, nucleotides, oligonucleotides, polynuclectide polymers and fragments thereof in either single- or double-stranded form. A nucl eic acid may be of natural or synthetic origin, double-stranded or single-stranded, and separate from or combined with carbohydrate, lipids, protein, other nucleic acids, or other materials, and may perform a particular activity such as transformation or form a useful composition such as a peptide nucleic acid (PNA). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and may be metabolized in a mannei similar to naturally-occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence e-xplicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19: 5081; Ohtsuka et al. (1985) J. Biol. Chem. 260: 2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes 8: 91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[039] "Proliferation" means growth and reproduction, i.e., division of cells. An important aspect of this invention is that the Pol i gene expressed in cells is believed to inhibit or suppress cell proliferation associated with cancer or malignancy. "Inhibition" and "suppression," as used with reference to cell proliferation are terms well known to those skilled in the art, and refer to slowing or stopping of cell division such that cells do not increase in number. The magnitude of such slowing of cell growth can be variable. Herein, any alteration of the growth of cells that comprise cancerous or precancerous cells or tissue falls within the scope of this application.

[040] "Sample" refers to an isolated sample of material, such as material obtained from an organism, containing nucleic acid molecules. A sample may comprise a bodily fluid; a cell; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; or a biological tissue or biopsy thereof. A sample may be obtained from any bodily fluid (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. [041] "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. Nucleic acids having longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes part I chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, N.Y. Generally, highly stringent hybridization and wash conditions are selected to be 5 °C. lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. Typically, under "stringent conditions" a probe will hybridize to its target subsequence, but to no other sequences. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T_m for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids that have more than 100 complementary residues on a filter in a Southern or northern blot is SO' ) formamide with 1 mg of heparin at 42 °C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72 °C for 15 minmtes. An example of stringent wash conditions is a 0.2x SSC wash at 65 °C for 15 minutes sse, Sambrook, infra., for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example med m stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45 °C for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at 40 °C for 15 minutes. For short probes (e.g., 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than 1.0 M Na ion, typicality 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is "typically at least 30 °C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2x (or higher) than t-hat observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under strLngent conditions are still substantially similar if the polypeptides that they encode are substantially similar. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. [042] "Target polynucleotide," as used herein, refers to a nucleic acid to which a polynucleotide probe can hybridize by base pairing and that comprises all or a fragment of a gene that encodes Pol i. In some instances, the sequences of target and probes may b-»e 100% complementary (no mismatches) when aligned. In other instances, there may be up to a 10% mismatch. Target polynucleotides represent a subset of all ofthe polynucleotides in a sample that encode the expression products of all transcribed and expressed genes in the cell or tissue from which the polynucleotide sample is prepared. The gene products of target polynucleotides are Pol i gene products, or fragments thereof.

[043] "Target Region" means a stretch of consecutive nucleotides comprising .all or a portion of a target sequence such as a gene or an oligonucleotide encoding the Po> 1 t gene product. Target regions may be 15, 16, 17, 18 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 5,6, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 61, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200 or more polynucleotides in length. In some embodiments, target regions are 70 nucleotides ira length, and lack secondary structure. Target regions may be identified using computer software programs such as OLIGO 4.06 software (National Biosciences, Plymoutbx MN), LASERGENE software (DNASTAR, Madison Wis.), MACDNASIS (Hitachi Software Engineering Co., San Francisco, Calif.) and the like.

[044] Methods For Characterizing The Etiology Of A Cancer and For Identifying An Individual Who Is At Risk Of Developing Cancer

[045] Based on the observations of Applicants, it is believed that certain cancer cells in individuals with cancer, such as adenocarcinoma of the lung, have Pol i proteins witt one or more mutations, such as a single nucleotide polymorphism at codon 706 of the hum^ Pol i gene. [046] Polynucleotides encoding wild-type, non-mutant forms of human and mouse Pol i protein are shown in Fig. 2 and Fig. 4. Polynucleotides comprising all or a portion of these sequences, or having sequences which are the complement thereof, are useful tools for designing hybridization probes for screening tissue samples for Pol i gene mutations, particularly tissues from patients at risk for, known to have, or suspected of having lung cancer, and for preparing primers useful for isolating and identifying cDNA clones and genomic clones encoding the Pol i gene and allelic forms thereof. Such hybridization techniques are known to those of skill in the art.

[047] In some embodiments of the invention, tissue samples are obtained from cancerous tissue or tissue that is believed to be or may become cancerous. In some embodiments, normal tissue is also obtained. According to such embodiments, a comparison may be made between the genetic profiles ofthe actual or suspected cancer cells and normal cells. [048] Polynucleotide primers

[049] Primers can be used to obtain Pol i polynucleotides from cDNA libraries, for screening tissue samples, or for diagnostic purposes. The primers may be used according to polymerase chain reaction (PCR) technologies to amplify transcripts of the genes which encode the Pol i gene products, or portions of such transcripts. Primers may comprise 15, 16, 17, 18 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 5,6, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 61, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 or more nucleotides, and have a G+C content of 40% or greater. Such oligonucleotides can be at least 98%, 99% or more complementary with a portion of the DNA strand, i.e., the sense strand, which encodes the Pol i gene or a portion of its corresponding antisense strand. Primers that have 100% complementarity with the antisense strand of a double-stranded DNA molecule which encodes a Pol i gene product have a sequence which is identical to a sequence contained within the sense strand. [050] Isolated allele specific primers can be used for diagnosis of an individual having or at risk of developing cancer, particularly lung cancer, more particularly adenocarcinoma of the lung. Allele specific primers for Pol i genes are produced based upon identification of regions within the Pol i gene encoding one or more polymorphisms, or SNPS, such as a polymorphism identified at codon 706 of the human Pol i gene. More particularly, allele specific primers for Pol i genes are produced for identification of one or more mutations at codon 706 that encode a change in the amino acid from Threonine in the wild-type form of Pol i to Alanine in the mutant fonri of Pol t. In some embodiments, the primers of the invention are designed to hybridize to the upstream and downstream (e.g., flanking) sequences of target regions of the Pol i gene so as to bracket the locus of such one or more SNPs.

[051] The primers of the invention embrace oligonucleotides of sufficient length and appropriate sequence so as to provide specific initiation of polymerization on a significant number of nucleic acids flanking the polymorphic locus. Conditions conducive to synthesis include the presence of nucleoside triphosphates and an agent for polymerization, such as DNA polymerase, and a suitable temperature and pH. In some embodiments, primers are single stranded for maximum efficiency in amplification. Primer length is determined based on many factors, including temperature, buffer, and nucleotide composition. [052] Primers are typically sufficiently complementary to hybridize with their respective strands under conditions which allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with the 5' and 3' sequences flanking the target sequence, for example, the Pol I coding sequence, to hybridize therewith and permit amplification of one or more polymorphic locus, such as a SNP at codon 706 of the human Pol t gene, and more particularly one or more mutations at codon 706 that encode a change in the amino acid from Threonine in the wild-type form of Pol i to Alanine in the mutant form of Pol i.

[053] The oligonucleotide primers of the invention may be prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof. In one such automated embodiment, diethylphosphoramidites are used as starting materials and may be synthesized as described by Beaucage, et al. (Tetrahedron Letters, 22:1859-1862, 1981). One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,066.

[054] Pol t-designed primers may be used in RT-PCR to quantify the amount of Pol i mRNA in the test tissues and cells. Pol i primers may also be used to analyze tissue sections from individuals by an RT in situ-PCR hybridization protocol as described Nuovo et al (1994) in Am J. Pathol, 144, 659-666, which is specifically incorporated herein by reference. [055] Polynucleotide Probes

[056] Polynucleotide probes are useful for detecting transcripts of genes which encode the Pol i protein. More particularly, polynucleotide probes are useful according to the instant invention for detecting transcripts of mutant Pol t having one or more mutations at codon 706 that encode a change in the amino acid from Threonine in the wild-type form of Pol t to Alanine in the mutant form of Pol i. Such polynucleotide probes may comprise 15, 16, 17, 18 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 5,6, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, Or 200 or more nucleotides. Polynucleotide probes have a sequence which is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more complementary with a contiguous sequence contained within the sense strand or antisense strand of a double stranded DNA molecule which encodes the Pol i protein (i.e., the target region of the Pol t o gene). Polynucleotide probes bind to the sense strand or antisense under stringent conditions, and in some instances under highly stringent conditions. The polynucleotide probes may be used in Northern assays to detect transcripts of Pol i homologous genes and in Southern assays to detect Pol i homologous genes. At least some of said polynucleotide probes comprise a polynucleotide sequence that is complementary to a target region of a Pol i gene.

[057] The polynucleotide probes may be genomic DNA or cDNA or mRNA, or any RNA- like or DNA-like material, such as peptide nucleic acids, branched DNAs and the like. The polynucleotide probes may be sense or antisense polynucleotide probes. Where target polynucleotides are double stranded, the probes may be either sense or antisense strands. Where the target polynucleotides are single stranded, the nucleotide probes may be complementary single strands.

[058] The polynucleotide probes may be prepared by a variety of synthetic or enzymatic schemes that are well known in the art. The polynucleotide probes can be synthesized, in whole or in part, using chemical methods well known in the art Caruthers et al. (1980) Nucleic Acids Res. Symp. Ser. 215-233). Alternatively, the probes can be generated, in whole or in part, enzymatically.

[059] Nucleotide analogues can be incorporated into the polynucleotide probes by methods well known in the art. The incorporated nucleotide analogues should serve to base pair with target polynucleotides. For example, certain guanine nucleotides can be substituted with hypoxanthine, which base pairs with cytosine residues. However, these base pairs are less stable than those between guanine and cytosine. Alternatively, adenine nucleotides can be substituted with 2,6-diaminopurine that can form stronger base pairs than those between adenine and thymidine. Additionally, the polynucleotide probes can include nucleotides that have been derivatized chemically or enzymatically. Typical chemical modifications include derivatization with acyl, alkyl, aryl or amino groups.

[060] The polynucleotide probes may be labeled with one or more labeling moieties to allow for detection of hybridized probe/target polynucleotide complexes. The labeling moieties can include compositions that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means. The labeling moieties include radioisotopes, such as P , P or S , chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, such as fluorescent markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, and the like.

[061] The polynucleotide probes can be immobilized on a substrate. Preferred substrates are any suitable rigid or semi-rigid support, including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which the polynucleotide probes are bound. Preferably, the substrates are optically transparent. [062] Target Polynucleotides

[063] In order to conduct sample analysis, a sample containing polynucleotides that will be assessed for the presence of target polynucleotides, that is, Pol i genes, or Pol t genes containing one or more SNPS, are obtained. The samples can be any sample containing target polynucleotides and obtained from any bodily fluid (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. In some embodiments, samples comprise cancer cells, other cells, or cell extracts from an individual or is at risk of developing, has or may have cancer, such as adenocarcinomas ofthe lung. [064] Any nucleic acid specimen, in purified or nonpurified form, can be utilized as the starting nucleic acid or acids, provided it contains, or is suspected of containing, the specific nucleic acid sequence containing the polymorphic locus. Thus, the process may employ, for example, DNA or RNA, including messenger RNA, wherein DNA or RNA may be single stranded or double stranded. In the event that RNA is to be used as a template, enzymes, and/or conditions optimal for reverse transcribing the template to DNA would be utilized. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized. A mixture of nucleic acids may also be employed, or the nucleic acids produced in a previous amplification reaction herein, using the same or different primers may be so utilized. The specific nucleic acid sequence to be amplified, i.e., the polymorphic locus, may be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be amplified be present initially in a pure form; it may be a minor fraction of a complex mixture, such as contained in whole human DNA.

[065] DNA utilized herein may be extracted using one of a variety of techniques such as that described by Maniatis, et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp 280, 281, 1982). If the extracted sample is impure, it may be treated before amplification with an amount of a reagent effective to open the cells, or animal cell membranes of the sample, and to expose and/or separate the strand(s) of the nucleic acid(s). This lysing and nucleic acid denaturing step to expose and separate the strands will allow amplification to occur much more readily. Additional methods of purification of nucleic acids are described in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Elsevier, New York N.Y. In one case, total RNA is isolated using the TRJZOL reagent (Life Technologies, Gaithersburg Md.), and mRNA is isolated using oligo d(T) column chromatography or glass beads. Alternatively, when polynucleotide samples are derived from an mRNA, the polynucleotides can be a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from that cDNA, an RNA transcribed from the amplified DNA, and the like. When the polynucleotide is derived from DNA, the polynucleotide can be DNA amplified from DNA or RNA reverse transcribed from DNA.

[066] Suitable methods for measuring the relative amounts of the target polynucleotide transcripts in samples of polynucleotides are Northern blots, RT-PCR, or real-time PCR, or RNase protection assays. Fore ease in measuring the transcripts for target polynucleotides, it is preferred that arrays as described above be used.

[067] The target polynucleotides may be labeled with one or more labeling moieties to allow for detection of hybridized probe/target polynucleotide complexes. The labeling moieties can include compositions that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means. The labeling moieties include radioisotopes, such as P , P , or S chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, such as fluorescent markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, and the like. [068] Genetic Analysis

[069] A variety of genetic analytical techniques are known for evaluating samples to detect and identify mutations in genomic Pol i genes, and more particularly for identifying one or more mutations at codon 706 of a human Pol i gene that encode for a change in the encoded amino acid from Tlireonine in the wild-type form of Pol t to Alanine in the mutant form of Pol l. [070] In one type of analysis, DNA isolated from the target tissue sample is analyzed by polymerase chain reaction (PCR). Regions of the Pol i open reading frame (ORF), or surrounding areas, are chosen and PCR primers are made that hybridize with the genomic DNA in the region. Such primers can be made to any known sequence within the Pol i gene or to regions surrounding the Pol i gene where the genomic sequence is known. One such set of regions surrounding the Pol i gene that can be used are polymorphic microsatellite markers, whose sequences and locations throughout the human, and some animal genomes, are known in the art. The primers are used in a PCR reaction to amplify the region of the genome that contains the Pol i gene. A single PCR reaction may be used to amplify the entire genomic region containing the Pol t gene. Alternatively, multiple PCR reactions, each amplifying a different region of the Pol . gene may be used. Preferably, PCR reactions are used such that the entire coding region of the Pol t gene is amplified. In addition, genomic regions within introns and surrounding the Pol i gene may also be amplified. [071] The amplified product may be detected by analyzing via a Southern blotting technique or similarly, using dot blot analysis. Suitable solid supports useful in Southern blotting techniques are membranes, beads, microtiter plates, etc. The use of non-radioactive probes or labels is facilitated by the high level of the amplified signal. Alternatively, probes used to detect the amplified products can be directly or indirectly detectably labeled. A detectable label is one that can be detected by physiochemical means, such as with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, by color absorbance, a metal chelator or an enzyme. Those of ordinary skill in the art will know of other suitable labels for binding to the probe, or will be able to ascertain such, using routine experimentation.

[072] Analysis ofthe size of a particular PCR product from the tumor or cancer cell genome as compared to the size of the same PCR product using DNA from a control cell (i.e., one known to have Pol i genes), can detect insertions or deletions of DNA in that area of the genome. It is well known in the art, that if there is an insertion of DNA in the area of a genome between the regions where two PCR primers are used to amplify the genome, the resulting PCR product is larger in size compared to the size of the same PCR product obtained using DNA from a genome where no insertion has occurred. Likewise, a deletion of DNA in the genome between two PCR primers results in a PCR product that is smaller in size compared to a control PCR product obtained using DNA from a genome not containing a deletion. Such analyses detect relatively large changes (e.g., minimum of 10% change) in size of a PCR product as compared to the product from a Pol i genome. Nonnally, size determination of PCR products is performed by comparing the relative sizes of two or more PCR products. For example, the size of a PCR product from a genome where a Pol i mutation is suspected is compared to the size of the same PCR product from a genome where Pol i mutations are known not to be present. Relative sizes are easily compared using migration of PCR products in an electric field, as occurs in gel electrophoresis. Agarose gel electrophoresis is often used for this purpose.

[073] Another method for analyzing PCR products is through determination of the nucleotide sequence of all or part of the PCR product. This method of analysis detects changes in relative size of PCR products that are less than 10%. This method also detects changes in the DNA sequence that do not result in relative size changes. For example, determination of the sequence and comparison of the sequence of the same PCR product obtained from amplification of DNA from two different cells can detect single or multiple nucleotide base changes, substitutions of regions of DNA, and the like. Methods for DNA sequence determination and for DNA sequence determination of PCR products are well known in the art of molecular biology. The chain termination method of sequencing is often used. DNA sequencing is often perfonned by automated sequencing machines. [074] In another type of analysis, RNA, preferably mRNA isolated from the tumor or cancer cells is used as a template to make DNA in a reverse transcription reaction. The reverse transcribed DNA is then used as a template in PCR reactions using PCR primers with sequences known to be within the mRNA of the Pol t gene. Various mRNA primers can be chosen, as described above in order to amplify the entire length ofthe mRNA sequence ofthe Pol I gene. This can be done using a single PCR reaction, or multiple PCR reactions as described above. Analysis ofthe PCR products is then performed much as already described. In one type of analysis, the presence of absence of a PCR product, or a change in its size as compared to controls is indicative of large changes, such as large insertions or deletions within the Pol i genome regions. Again, such analysis is commonly performed using gel electrophoresis of the PCR products, hi another type of analysis, the DNA sequence of the PCR products is determined, using methods well known in the art.

[075] According to one embodiment, a Pol t mutating polymorphism, such as a mutation at codon 706 of a human Pol t gene, may be detected using the reverse dot blot hybridization technique (RDB) (see for example, Bray, et al, Blood, 84(12):4361, 1994, incorporated herein by reference). Briefly, allele-specific ohgonucleotides are fixed to a solid support (e.g., a filter). Typically, an amino group is added to the terminus of the allele-specific ohgonucleotides for covalent attachment to the support. Labeled (e.g., biotinylated) ohgonucleotides flanking the polymorphic sequence in genomic are used to amplify genomic DNA by PCR, for example, and these PCR products are denatured into single stranded DNA and hybridized to the filters containing the allele-specific ohgonucleotides. [076] Other methods, well known in the art, can also be used to assay for presence of Pol t genes, transcripts, or changes in either as compared to wild type Pol i. Some of these methods include Southern blotting, Northern blotting, RNase protection assays, SI nuclease assays and the like. [077] Methods for Treating Individuals who have or at Risk of Developing Cancer [078] Various embodiments of the invention provides methods for preventing the formation of cancer or treating cancer in individuals in need of such treatment. As described herein, individuals may be identified as having cancer, such as adenocarcinoma of the lung, wherein at least one causative factor in their disease is the presence of a mutation in the Pol t gene, and more particularly, a mutation at codon 706 wherein the mutation encodes for a change in the amino acid from Threonine in the wild-type form of Pol t to Alanine in the mutant form of Pol i. Other individuals may be identified as being at risk for developing a cancer, such as adenocarcinoma of the lung, wherein at least one indicator of such risk is the presence of a mutation in the Pol i gene, for example, the mutation at codon 706. Such individuals are in need of treatment to prevent or stop proliferation of cancer cells. The methods of treatment described herein involve, in some embodiments, supplementing the levels of wild-type, non- mutant Pol i protein in the individual, some embodiments the level Pol i protein is supplemented or elevated by administering to an individual in need of treatment a Pol t protein, or a pharmaceutical composition containing a Pol i protein. In other embodiments the level Pol i protein is elevated by administering to an individual in need of treatment a polynucleotide encoding a Pol i protein, or a pharmaceutical composition containing a polynucleotide encoding a Pol i protein. References made herein with respect to therapeutic and prophylactic treatment with Pol i protein or polynucleotides encoding the same will be understood to describe treatment with wild-type, non-mutant forms of such Pol i proteins and polynucleotides. [079] Pol t protein

[080] The sequences of wild-type, non-mutant human and mouse Pol i proteins are shown in Fig. 1 and Fig. 3. The Pol t protein (collectively, "Pol i protein(s)"), may be produced by conventional peptide synthesizers. The Pol i proteins may also be produced using cell-free translation systems and RNA molecules derived from DNA constructs that encode the Pol i proteins. Pol i proteins may also be made by transfecting host cells with expression vectors that comprise a DNA sequence that encodes the respective Pol i protein or and then inducing expression of the protein in the host cells. For recombinant production, recombinant constructs comprising a sequence which encodes the Pol i protein are introduced into host cells by conventional methods such as calcium phosphate transfection, DEAE-dextran mediated transfection, trans vection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape lading, ballistic introduction or infection. [081] The Pol . protein may be expressed in suitable host cells, such as for example, mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters using conventional techniques. Following transformation of the suitable host strain and growth of the host strain to an appropriate cell density, the cells are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification ofthe Pol t protein.

[082] Conventional procedures for isolating recombinant proteins from transformed host cells, such as isolation by initial extraction from cell pellets or from cell culture medium, followed by salting-out, and one or more clrromatography steps, including aqueous ion exchange chromatography, size exclusion chromatography steps, and high performance liquid chromatography (HPLC), and affinity chromatography may be used to isolate recombinant Pol I protein.

[083] Inhibiting Lung Cancer Cell Proliferation with Pol . protein

[084] The present invention provides methods for inlribiting or suppressing growth of cells by introduce Pol t proteins into cells of an individual who has developed or is at risk of developing cancer. Such individuals include those who have or may develop adenocarcinomas ofthe lung. There are a variety of methods known in the art for introducing proteins into cells. According to one method, proteins are coupled or fused to short peptides that direct entry of the Pol i protein into cells. One such group of peptides are called protein transduction domains. Another method for intiroduction proteins into cells uses lipid carriers. For example, proteins that are associated with liposomes are able to enter cells when the liposomes enter or fuse with the cell membranes.

[085] Such methods include, but are not limited to, "protein transduction" or "protein therapy" as described in publications by Nagahara et al. (Nagahara, et al., 1998, Nat Med, 4:1449-52.) and in publications from the laboxatory of Dowdy (Nagahara, et al., 1998, Nat Med, 4:1449-52.; Schwarze, et al, 1999, Science, 285:1569-72.; Vocero-Akbani, et al., 20O0, Methods Enzymol, 322:508-21; Ho, et al., 20O1, Cancer Res, 61:474-7.; Vocero-Akbani,, et al, 2001, Methods Enzymol, 332:36-49; Snyder and Dowdy, 2001, Curr Opin Mol Ther, 3:147-52.; Becker-Hapak, et al., 2001, Methods, 24:247-56.), publications which are incorporated herein by reference.

[086] In one embodiment, an eleven amino acid sequence, the "protein transduction domain" (PTD), from the human immunodeficiency virus TAT protein (Green and Loewenstein, 1988, Cell, 55:1179-88.; Frankel and Pabo, 1988, Cell, 55:1189-93.) is fused to the Pol i protein. The purified protein is then put in contact with the surface of cells and the cells take up the Pol ι protein which functions to inhibit or suppress growth of that cell. In the case where it is desired to introduce the Pol i protein containing the fused PTD into cells comprising a tumor in a human or animal, ttie protein is administered to the human by a variety of methods. The Pol i protein may be administered by injection (e.g., intravenously) or by inhalation in an aerosol.

[087] Pol t proteins that contain the fused PTD are preferably made by fusing the DNA sequence encoding the Pol i gene with the DNTA sequence encoding the PTD. The resulting Pol i -PTD fusion gene may be incorporated into a vector, for example a plasmid or viral vector, that facilitates introduction of the fusion gene into a organism and expression of the gene at high levels in the organism such that large amounts of the fusion protein are made therein. One such organism in which the vector containing the fusion gene can be expressed is a bacterium, preferably Escherichia coli. Other organisms are also commonly used by those skilled in the art. After the fusion protein is expressed at a high level in any of these organisms, the fusion protein is purified from the organism using protein purification techniques well known to those skilled in the art. [088] Pol i Polynucleotides

[089] The present invention provides isolated polynucleotides which encode a Pol i protein. The Pol t-encoding polynucleotides may be single-stranded or double stranded. Such polynucleotides may be DNA or RNA molecules The isolated polynucleotide comprises all or a portion of the Pol t sequence shown in Fig. 2 or Fig. 4. The Pol i polynucleotides are useful in one embodiment for preparing Pol t proteins.

[090] The present invention also encompasses isolated polynucleotides whose sequence is the complement of the Pol i gene sequence, shown in Figs. 2 and 3, and polynucleotides that hybridize under stringent conditions, in some embodiments under highly stringent conditions, to the open reading frame sequence ofthe Pol . gene sequence, or the complement thereof. [091] Polynucleotides comprising sequences encoding a Pol i protein may be synthesized in whole or in part using chemical methods. Polynucleotides which encode a Pol i protein, particularly alleles ofthe genes which encode a Pol i protein, may be obtained by screening a genomic library or cDNA library with a probe comprising sequences identical or complementary to the sequences shown in Figs. 2 or 3, or with antibodies immunospecific for a Pol i protein, to identify clones containing such polynucleotide. Alternatively, polynucleotides encoding Pol i proteins may be made using polymerase chain reaction (PCR) technology and primers that bind specifically to sequences which are known to encode a Pol t protein.

[092] Inhibiting Lung Cancer Cell Proliferation with Pol i porvnucleotides [093] In one aspect, the present method comprises introduction of Pol i encoding polynucleotides, preferably contained within a vector, into cancer cells so that the cells achieve increased levels of Pol t expression. Herein, such introduction or transfer of a DNA molecule or molecules, specifically a DNA molecule encoding one or more Pol t encoding polynucleotide, into a cell refers to any of a variety of methods known in the art to achieve transfer of DNA molecules into cells. Whatever methodology is used to administer the Pol i genes to humans or animal, such methodologies comprise variations that result in the Pol i genes being introduced exclusively into normal and not being introduced into tumor cells. For example, techniques are known in the art that result in recombinant viruses specifically infecting certain cell types within a human or animal. For viruses, such "targeting" can be accomplished through manipulation of cellular receptors for the recombinant viruses and/or manipulation of viral ligands that recognize and bind to cellular receptors for the viruses. Such methodologies, as used to introduce Pol . genes into cane er cells in animals or humans, are within the purview of the present application. Targets on cancer cells include, but are not limited to, proteins such as carcino embryonic antigen, and other markers that are differentially expressed on cancer cells but not in corresponding normal cells. Specific ligands for such targets include, but are not limited to, known ligands, antibodies. [094] In one embodiment, polynucleotides encoding the Pol t protein or a functional equivalent thereof are introduced into such cells to permit expression or overexpression ofthe Pol i protein. Viral or plasmid vectors may be used to deliver the polynucleotide to the cells. Levels of Pol i may be increased in cancer cells by introducing a DNA fragment comprising an Pol i polynucleotide and a promoter into the cell and expressing the Pol i protein. Preferably, the promoter, which is operably linked to the Pol i polynucleotide is a tissue specific promoter. The DNA fragment may be incorporated into a viral vector or into a liposome which, preferably, further comprises a molecule which targets the liposome to the cancer cell.

[095] In one embodiment, polynucleotides encoding the Pol i protein or a functional equivalent or fragment thereof is introduced into cancer cells to permit expression or overexpression of the Pol i protein, hi one embodiment, Pol i delivery is specifically selective for cancer cells and is achieved using a targeting carrier that is selective for cancer cells and does not direct delivery to normal cells.

[096] In order to introduce the polynucleotide sequences encoding Pol i activity into cells, the protein coding region of the polynucleotide sequences is normally attached to sequences that facilitate its transcription into mRNA as well as translation of the mRNA into Pol i. A strategy common in the art for doing this is to clone the polynucleotide sequence encoding the Pol t protein into a vector which contains sequences facilitating expression of a protein coding sequence cloned therein.

[097] Expression vectors normally contain sequences that facilitate gene expression. An expression vehicle can comprise a transcriptional unit comprising an assembly of a protein encoding sequence and elements that regulate transcription and translation. Transcriptional regulatory elements generally include those elements that initiate transcription. Types of such elements include promoters and enhancers. Promoters may be constituti~ve, inducible or tissue specific. Transcriptional regulatory elements also include those that terminate transcription or provide the signal for processing of the 3' end of an RJSTA (signals for polyadenylation). Translational regulatory sequences are normally part of the protein encoding sequences and include translational start codons and translational termination codons. There may be additional sequences that are part of the protein encoding region, such as those sequences that direct a protein to the cellular membrane, a signal sequence for example.

[098] The Pol .-encoding polynucleotides that are introduced into cells are, in some embodiments, expressed at a high level (i.e., the introduced polynucleotide sequence produces a high quantity of Pol t protein within the cells) after introduction into the cells. Techniques for causing a high-level of expression of polynucleotide sequences introduced into cells are well known in the art. Such techniques frequently involve, but are not limited to, increasing the transcription of the polynucleotide sequence, once it has been introduced into cells. Such techniques frequently involve the use of transcriptional promoters that cause transcription of the introduced polynucleotide sequences to be initiated at a high rate. A variety of such promoters exist and are well known in the art. Frequently, such promoters are derived from viruses. Such promoters can result in efficient transcription of polynucleotide sequences in a variety of cell types. Such promoters can be constitutive (e.g., CMV enhancer/promoter from human cytomegalovirus) or inducible (e.g., MMTV enhancer/promoter from mouse mammary tumor virus). A variety of constitutive and inducible promoters and enhancers are known in the art. Other promoters that result in transcription of polynucleotide sequences in specific cell types, so-called "tissue-specific promoters," can also be used. A variety of promoters that are expressed in specific tissues exist and are known in the art. For example, promoters whose expression is specific to neural, liver, epithelial and other cells exist and are well known in the art. Methods for making such DNA molecules (i.e., recombinant DNA methods) are well known to those skilled in the art.

[099] Vectors for introducing Pol . polynucleotides into target cells

[0100] In the art, vectors refer to nucleic acid molecules capable of mediating introduction of another nucleic acid or polynucleotide sequence to which it has been linked into a cell. One type of preferred vector is an episome, i.e., a nucleic acid capable of extrachromosomal replication. Other types of vectors become part ofthe genome ofthe cell into which they are introduced. Vectors capable of directing the expression of inserted DNA sequences are referred to as "expression vectors" and may include plasmids, viruses, or other types of molecules known in the art.

[0101] Typically, vectors contain one or more restriction endonuclease recognition sites which permit insertion of the Pol i polynucleotide sequence. The vector may f rther comprise a marker gene, such as for example, a dominant antibiotic resistance gene, which encode compounds that serve to identify and separate transformed cells from non- transformed cells.

[0102] One type of vector used in the present invention is selected from viral vectors. ^"Viral vectors are recombinant viruses which are generally based on various viral families comprising poxviruses, herpesviruses, adenoviruses, parvoviruses and retroviruses. Such recombinant viruses generally comprise an exogenous polynucleotide sequence (herein, a Pol t gene) under control of a promoter which is able to cause expression of the exogenous polynucleotide sequence in vector-infected host cells.

[0103] One type of viral vector is a defective adenovirus which has the exogenous polynucleotide sequence inserted into its genome. The term "defective adenovirus" refers to an adenovirus incapable of autonomously replicating in the target cell. Generally, the genome of the defective adenovirus lacks the sequences necessary for the replication of the virus in the infected cell. Such sequences are partially or, preferably, completely removed from the genome. To be able to infect target cells, the defective virus contains sufficient sequences from the original genome to permit encapsulation of the viral particles duri-rig in vitro preparation of the construct. Other sequences that the virus contains are any such sequences that are said to be genetically required "in cis." [0104] It is desirable that the adenovirus is of a serotype which is not pathogenic for man. Such serotypes include type 2 and 5 adenoviruses (Ad 2 or Ad 5). In the case of the Ad 5 adenoviruses, the sequences necessary for the replication are the E1A and E1B regions. Methods for preparing adenovirus vectors are described in U.S. Patent No. 5,932,210, which issued in August, 1999 to Gregory et al., U.S Patent No. 5,985,846 which issued in November, 1999 to Kochanek et al, and U.S. Patent No. 6,033,908 which issued in March, 2000, to Bout et al.

[0105] It is also desirable that the virus vector is an immunologically inert adenovirus. As used herein the term "ii nunologically inert" means the viral vector does not encode viral proteins that activate cellular and humoral host immune responses. Methods for preparing immunologically inert adenoviruses are described in Parks et al., Proc Natl Acad Sci USA 1996; 93(24) 13565-70; Leiber, A. et al, J Virol. 1996; 70(12) 8944-60; Hardy s., et al, J. Virol. 1997, 71(3): 1842-9; and Morsy et al, Proc. Natl. Acad. Sci. USA 1998. 95: 7866-71, all of which are specifically incorporated herein by reference. Such methods involve Cre- loxP recombination. In vitro, Cre-/øxP recombination is particularly adaptable to preparation of recombinant adenovirus and offers a method for removing unwanted viral nucleotide sequences. Replication deficient recombinant adenovirus lacks the El coding sequences necessary for viral replication. This function is provided by 293 cells, a human embryonic kidney cell line transformed by adenovirus type. First generation adenoviruses are generated by co-transfecting 293 cells with a helper virus and a shuttle plasmid containing the foreign gene of interest. This results in the packaging of virus that replicates both the foreign gene and numerous viral proteins. More recently, 293 cells expressing Cre recombinase, and helper virus containing essential viral sequences and with a packaging signal flanked by loxP sites, have been developed (See Parks et al.) In this system, the helper virus supplies all of the necessary signals for replication and packaging in trans, but is not packaged due to excision of essential sequences flanked by loxP. When 293-Cre cells are co-transfected with this helper virus, and a shuttle plasmid (pRPlOOl) containing the packaging signal, nonsense "filler DNA", and the foreign gene, only an adenovirus containing filler DNA and the foreign gene is packaged (LoxAv). This results in a viral recombinant that retains the ability to infect target cells and synthesize the foreign gene, but does not produce viral proteins. [0106] Another type of viral vector is a defective retro virus which has the exogenous polynucleotide sequence inserted into its genome. Such recombinant retrovimses are well known in the art. Recombinant retrovimses for use in the present invention are preferably free of contaminating helper virus. Helper viruses are vi ses that are not replication defective and sometimes arise during the packaging ofthe recombinant retro vims. [0107] Non-defective or replication competent viral vectors can also be used. Such vectors retain sequences necessary for replication of the vims. Other types of vectors are plasmid vectors.

[0108] After Pol (.-encoding polynucleotides are introduced into cells, techniques may be used to determine the cells into which the polynucleotide sequences have been introduced and/or the specific cells that are expressing the introduced polynucleotide sequences. A variety of techniques to examine the presence of polynucleotide sequences and/or expression of polynucleotide sequences exist and are well known in the art. Some such techniques include Southern blotting, Northern blotting, polymerase chain reaction (PCR), Western blotting, RNase protection, radioiodide uptake assays, and others.

[0109] Also encompassed by the present invention, are single stranded polynucleotides, hereinafter referced to as antisense polynucleotides, having sequences which are complementary to the DNA and RNA sequences which encode the Pol i protein. The term complementary as used herein refers to the natural binding of the polynucleotides under permissive salt and temperature conditions by base pairing. [0110] Administration of therapeutic compositions

[0111] Doses may be selected, depending on their dosage form, patient's age, sex and severity of disease, and other conditions, as appropriate, but the amount of the active ingredient may be generally about 0.0001 to 100 mg/kg a day. A unit dosage form may contain about 0.001 to 1000 mg of the active ingredient. The compositions may be administered using any mode that is medically acceptable, meaning any mode that produces effective levels of the active protein without causing clinically unacceptable adverse effects. Such modes of administration include parenteral routes (e.g., intravenous, intra-arterial, subcutaneous, intramuscular, mucosal or infusion), but may also include oral, rectal, topical, nasal or intradermal routes. Another route of introduction, of special use for treatment of patients with pulmonary fibrosis, is the respiratory route by inhalation into the lungs. Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations, increasing convenience to the patient and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. The pharmaceutical compositions of the present invention may also be administered by the respiratory route. The formulations administered by the respiratory route are generally oral aerosol formulations. Such formulations can be administered via the respiratory route in a variety of ways.

[0112] h the event that a response in an individual is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of peptides. The duration of therapy with the pharmaceutical compositions used in the methods of the present invention will vary, depending on the unique characteristics of the pharmaceutical composition and the particular therapeutic effect to be achieved, the severity of the disease being treated and the condition and potential idiosyncratic response of each patient. Ultimately the attending physician will decide on the appropriate duration of therapy with the pharmaceutical composition used in the method of the present invention. [0113] Pharmaceutical compositions

[0114] Therapeutic proteins and polynucleotides may be administered to an individual in need of the same in a pharmaceutical composition. Suitable formulations for delivery are found in Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Co., Philadelphia, Pa., 1985). These phannaceutical compositions are suitable for use in a variety of drug delivery systems (Langer, Science 249:1527-1533, 1990).

[0115] Pol i proteins and polynucleotides in pharmaceutical compositions are suitable for single administration or in a series of inoculations. The pharmaceutical compositions are intended for parenteral, topical or oral administration. Parenteral administration may be by intravenous, subcutaneous, intradermal, intraperitoneal or intramuscular administration. Parenteral administration may be preferentially directed to the patient's liver such as by catheterization to hepatic arteries or into a bile duct. For parenteral administration, the compositions can include Pol i proteins and a suitable sterile carrier such as water, aqueous buffer, 0.4% saline solution, 0.3%ι glycine, hyaluronic acid or emulsions of nontoxic nonionic surfactants as is well known in the art. The compositions may further include substances to approximate physiological conditions such a buffering agents and wetting agents such as NaCl, KC1, CaCl₂ sodium acetate and sodium lactate.

[0116] Solid compositions in conventional nontoxic solid carriers such as, for example, glucose, sucrose mannitol, sorbitol, lactose, starch, magnesium stearate, cellulose or cellulose derivatives, sodium carbonate and magnesium carbonate. For oral administration of solid compositions, the HCV-like particles preferably comprise 10% to 95%, and more preferably 25%ι to 75% ofthe composition. [0117] Therapeutic compositions may be administered as a single dose, but more likely as a series of dosages over a period of days, weeks or even months. Herein, an effective therapeutic dose is a dose that inhibits growth of a tumor, or causes tumor regression. EXAMPLES [0118] Pol i as a Tumor Suppressor Gene and Indicator of Cancer Risk [0119] Applicants have demonstrated the candidacy of Pol i for the Par2 locus. Linkage analysis and fine mapping in various mouse crosses and congenic mice have significantly narrowed the Par2 region. Among all of the annotated genes in the Par2 region, Pol twas identified as a candidate mouse lung susceptibility gene based on two lines of evidence: (a) genetic variants were identified between susceptible A/J and resistance BALB/cJ mice, and changes including genetic polymorphisms and altered expression of POL I were also observed in human lung cancer cells; (b) primer extension assays with purified BALB/cJ and A/J proteins in vitro found that both forms of Pol i are active but that they may differ in substrate discrimination. This result strongly supports the hypothesis that the amino acid differences between the two mouse isoforms may alter their substrate discrimination properties, which may affect the fonnation of Kras2 mutations in mouse lung tumors. A major characteristic of the highly error-prone human POL I is its preference for inserting wobble base G rather than A opposite template T. Primer extension assays demonstrated that the full-length BALB/cJ Pol i protein is less efficient than the A/J protein in incorporating G compared with A opposite a template T. At higher concentrations, the full-length BALB/cJ Pol _ protein incorporates Watson-Crick base A opposite template T more efficiently than the A/J protein. These results are consistent with a genetic modifier role for the Pol t variant in mouse and, possibly, human lung cancer susceptibility.

[0120] Multiple nucleotide polymorphisms and functional testing results (i.e., different enzymatic activities) provide evidence that the Pol i gene is a strong candidate for the mouse Par2 gene. As with tumor suppressors DCC, SMAD2, and SMAD4, the human POL I was also mapped to the chromosome 18q21.1. The gene belongs to the RadSO branch of the recently described UmuC/DinBXRevl/Rad30 family of DNA polymerases and encodes a 715- amino acid DNAdependent polymerase or POL I in human and a 717-amino acid Pol t protein in mouse. On the basis of in vitro studies, human POL I has the lowest fidelity of any eukaryotic polymerase studied to date, which suggests that its activities must be highly specialized. The fact that POL I orthologues are evolutionarily conserved in higher eukaryotes from Drosophila to humans also suggests that it provides some selective advantage. Several studies show that the POL I gene may play a role in somatic hypermutation by which specific mutations occur as part of antibody diversity. Human POL I protein has also been shown carrying an intrinsic 5'-deoxyribose phosphate lysase activity and can substitute for POL I during BER reactions in vitro.

[0121] Mouse model for Lung Cancer: Inbred strains of mice vary markedly in their susceptibility to spontaneous and chemically induced lung tumorigenesis, thus representing a valuable model to study genetic susceptibility to lung cancer. On the basis of their mean tumor multiplicities induced by a lung carcinogen, the inbred mouse strains can be categorized into sensitive, intermediate, and resistant groups. The A strain is the most susceptible strain, whereas the C57BL/6J strain is the most resistant. Other strains such as the BALB/cJ strain belong to the intermediate group and are less susceptible to lung tumorigenesis than the A strain. Experimental crosses of inbred mouse strains have revealed Pulmonary adenoma susceptibility (Pas) loci, Pulmonary adenoma resistance (Par) loci, and Susceptibility to lung cancer (Slues) loci.

[0122] A/J and BALB/cJ inbred strains of mice carry the same Pasl allele but show different susceptibility to urethane-induction of lung tumors. In (A/J X BALB/c) FI mice, the relatively resistant BALB/cJ phenotype was dominant over the high susceptibility of A/J mice. Additional analysis on F2 hybrids and backcross mice supported the hypothesis that a major locus named Par2 accounts for the difference in adenoma susceptibility between A/J mice and BALB/cJ mice. Par2 was mapped to the mouse chromosome 18 and accounts for 60% phenotype variance. The resistance of BALB/cJ mice appears due to the interaction between the Pasl QTL and Par2 QTL in the BALB/cJ mouse genome.

[0123] Here, Applicants provide evidence to support the candidacy of Pol i for the Par2 locus. It is the most error-prone DNA polymerase and preferentially incorporates G rather than A across from template T. Human POL i has at least two distinct catalytic activities including translesion DNA synthesis and 5'-deoxyribose phosphate lyase activity. Its 5'- deoxyribose phosphate lyase activity and capability for filling short gaps implicate that this polymerase may play a role in certain base excision repair (BER) reactions. It may also play a role in lung tumorigenesis by affecting DNA adduct repair and thus Kras2 mutations that are found in a high proportion of mouse and human lung tumors (13, 20). Applicant's disclosure herein reports identification of multiple nucleotide polymorphisms and alternatively spliced transcript isoforms in the Pol i gene between A/J and BALB/cJ mice. They also provide initial biochemical support for the hypothesis that on a mechanistic basis, the amino acid differences between the two mouse isoforms may affect their substrate discrimination properties. An amino acid-changing nucleotide polymorphism and altered expression of POL . protein in human lung cancer cell lines is also reported. These data support the Pol i gene as a modifier of lung tumorigenesis by altering DNA polymerase and, possibly, repair activity. [0124] Materials and Methods: Expression and Nucleotide Polymorphism Analyses of Genes in the Par2 Region. Inbred mouse strains were purchased from The Jackson Laboratory (Bar Harbor, ME). DNA was isolated from tail snips. For RNA isolation, 100 mg of lung tissue were pulverized and total RNA extracted using TRIzol reagent according to the manufacturer's protocol (Life Technologies, Inc., Gaithersburg, MD). The quality of the isolated RNA was assessed by absorbance at 260 nm, the A260/A280 ratio (1.7-1.9), and electrophoresis on 1% agarose/formaldehyde gels that indicated the intensity and integrity of

the 28S and 18S bands. Two μg of total RNA were used in a reverse transcription reaction to

synthesize the first-strand cDNA using oligo-dT primer. Primers were designed for each gene based on their published sequences. The following PCR condition was used for each primer set: 95°C for 3 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min, and finally 72°C for 6 min. Annealing temperature was optimized for each primer set. Electrophoreses on 1.5% agarose gels were performed to resolved PCR products. Genomic DNA- and DNase I-treated cDNA samples were used for those containing only one exon. Amplified PCR products were purified with QIAquick gel extraction kit (Qiagen, Valencia, CA) and subjected to direct sequencing.

[0125] To detect alternative transcripts and nucleotide polymorphisms in the Pol t gene, five primer sets were used to cover the entire Pol t coding region: primer set 1: forward, 5'- GAGGAAGAAGACGCTCCTC-3', and reverse, 5'-TTCCTCCAACAATTCTGTGAC- 3'; primer set 2: forward, 5'-GAGCCGCTACAGAGAGATG- 3', and reverse, 5'- GTAGTAAGACCGTCTGCTG-3'; primer set 3: forward, 5'-

GTGGCTCCTAATAAACTCTTG-3', and reverse, 5'-AGGCCCTTTCTTAGCACTGC-3'; primer set 4: forward, 5'-ATGGTGAACGTGAAGATGCC- 3', and reverse, 5'- GCCTTGACTCGTTTGCTCAT- 3'; and primer set 5: forward, 5'- TCGTGCGGAAAGGACTGTTC-3' and reverse, 5'-ACTAGAATTTCCTTCCTGTGC-3'. Alternative mRNA splicing was detected by using primer sets 1 and 2. [0126] A/J and BALB/cJ GST-Pol i Plasmid Constructs. A full length of Pol i coding sequence was separately amplified using two sets of ohgonucleotides: PI set: forward, 5'- GAACGCGGATCCGCGGCCATGGAGCCCTTGCACGC- 3 '(A/J), forward, 5'- GAACGCGGATCCGCGGCCATGGAGCCCTCGCACGC- 3' (BALB/cJ), and reverse, 5'- ATAAGGATATCAATCAGGGGAGGC- 3'; and P2 set: forward, 5'- CCTCCCCTGATTGATATCCTTATG-3', and reverse, 5'-

CTCCCTCCATCGATGGACTTATCTGTGCGCCGAGG-3'. PI and P2 PCR products were digested with BamBI/EcoRV and EcoRV/Clal restriction enzymes (restriction sites are underlined in primer sequences). Digested PI and P2 PCR products were separately cloned into the pBluesript II SK vector, and their sequences were confirmed by direct sequencing in both directions. The Pol t -pBluesript II SK plasmid containing a full length of Pol t open reading frame was produced by subcloning the P2 fragment into PI plasmid. [0127] The GST-Pol . plasmid was generated by subcloning the ~2.2-kb Ba ϊΑl to Clal fragment from Pol i -pBluesript II SK plasmid into Bat Αll Clal doubledigested glutathione S-transferase (GST)-tagged expression vector pEBG-3XHV. The in-frame open reading frame of GST-Pol . fusion was confirmed by direct sequencing.

[0128] Purification of GST-Tagged Pol i Proteins. The GST-Pol i expression plasmids were transfected into HEK293 cells using Lipofectamine (Invitrogen, Carlsbad, CA), and 48 h after transfection, cells were lysed in NP40 buffer [20 mM Tris (pH 7.5), 100 mM NaCl,

1% NP40, 1 mM EDTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 5 μg/ml

aprotinin, and 5 μg/ml leupeptin]. Lysates were cleared by centrifugation, and NaCl was added to 1 M before the addition of glutathione-agarose beads and incubation for 2 h. Beads were washed by NP40 buffer and eluted in glutathione elution buffer [50 mM Tris (pH 8), 100 mM NaCl, 10 mM glutathione, and 1 mM DTT]. Eluted proteins were resolved by SDS- PAGE to determine purity and concentration. In addition, protein concentrations were estimated by Western blot analysis using human GST-i as a standard. The antibody to full- length human POL I was kindly provided by R. Prasad and S.H. Wilson. [0129] Primer Extension Assays. A set of experiments were designed to compare the incorporation of dATP by the A/J and PALB/c Pol i isofonns. The DNA template used was 5'-CTCGTCAGCATCTTCATCATACAGTCAGTG-3'. The matched primer was 5'- CACTGACTGTATGATGA-3', and the mismatched primer was 5'- CACTGACTGTATGATGG-3'. For matched primertemplate extension, 10, 25, 50, 110, and 225 ng of A/J and BALB/cJ Pol i proteins were used. Reactions were performed at 37°C degree for 15 min. For G/T mismatched primer extension, when dATP was added, 100, 200, 300, and 400 ng of A/J and BALB/cJ Pol ι proteins were used.

[0130] To examine the abilities of A/J and BALB/cJ Pol t proteins to incorporate dGTP compared with dATP opposite template T, the primer oligonucleotide (5'- AATTTCTGCAGGTCGACTCCAAAGGCT-3') was 5'-end labeled with 32P using T4 polynucleotide kinase and annealed at a 1.5:1 ratio with the template oligonucleotide (5'- CCAGCTCGGTACCGGGTTAGCCTTTGGAGTCGACCTGCAGAAAT- 3'). Reactions contained 40 mM Tris-Cl (pH 8.0), 30 mM NaCl, 5 mM MgC12, deoxynucleoside

triphosphate (as indicated in Fig. 7), 10 mM DTT, 1.25% glycerol, 250 μg/ml BSA, 2 pmols

of primer: template DNA, and 2 ng of GST-Pol i. Samples (5 μl) were removed at the

indicated times and added to an equal volume of formamide loading buffer (95 % deionized formamide, 25 mM EDTA, 0.01% bromphenol blue, and 0.01% xylene cyanol), heated to 94°C for 3 min, and separated by 12% denaturing PAGE. Gels were quantified by phosphorimage analysis.

[0131] Human POL I Mutation and Expression Analyses. Eleven human lung cancer cell lines, including A427 (carcinoma), A549 (carcinoma), CaLu-1 (metastatic epidermoid carcinoma), CaLu-3 (adenocarcinoma), CaLu-6 (adenocarcinoma), SK-LU1 (adenocarcinoma), SK-MES1 (squamous cell carcinoma), NCI-H460 (large cell carcinoma), NCI-H596 (adenosquamous carcinoma), NCI-H520 (squamous cell carcinoma), and NCI- H661 (large cell carcinoma), and the normal fetal lung cells MRC5 [all available from the American Type Culture Collection, Manassas, VA] were used in this study. The cells were grown in Eagle's MEM +10%FBS (MRC-5, A549, A427, CaLu-1, CaLu-3, CaLu-6, SK-LU- 1, and SK-MES-1) or RPMI 1640 + 10FBS (NCI-H460, NCI-H596, NCI-H520, and NCI- H661) and were harvested at 80-90% confluence. DNA, RNA, and protein were isolated from these cells by standard techniques. Primers used to amplify the human POL /gene (GenBank accession no. NM_007195) are the following: IF, 5'-GCGACG ACGAGGAAGACG- 3' (33-50); 2R, 5'-GTATTCCCTTGCTTTTCAGAC-3' (2230-2250); 3F, 5'-GATCTCAGATTGCAGCAGAG-3' (591-610); 4R, 5'-

AGAAGCCACTCCAGCACAG- 3' (649-667); 5F, 5'-GGATTTCCTACCAAGTGGAAG- 3' (1447- 1467); 6R, 5'-GAAGCTGCTTGAAGACTTCTTG-3' (1574-1595); 7F, 5'- CAAGAAGTCTTCAAGCAGCTTC- 3' (1574-1595); and 8R, 5'-

GTCCAATGTGGAAATCTGATC- 3' (1284-2204).

[0132] To examine the 2147 base coding region ofthe human POL L gene for polymorphisms or mutations, cDNA was made from the RNA and amplified overlapping regions with primers IF X 4R, 3F X 6R, and 5F X 2R. After Qiagen PCR purification (Qiagen), the PCR products were both sequenced directly and cloned using a TOPO-TA PCR cloning kit (Invitrogen). DYE-ET (Amersham-Phamiacia, Sunnyvale, CA) and BIG DYE (Applied Biosystems, Foster City, CA) fluorescent terminator cycle sequencing kits were used. Two to four clones were sequenced in each direction, and direct sequencing of products without cloning in at least two reactions was used to confirm the polymorphisms detected. Western blot analysis was performed to examine the protein expression of POL I in the human lung cancer cell lines and normal MRC5 line. A polyclonal antibody to the full-length POL I protein made in rabbits was used. Ponceau Red staining was used to confirm the even loading of protein.

[0133] Expression and Nucleotide Polymorphism Analyses of Genes in the Par2 Region. The minimum Par2 physical region is encompassed by the D18Mitl03 and D18Mitl88 markers. Applicants obtained the marker sequence information from the Whitehead Institute/ MIT Center for Genome Research.5 Using the marker sequences to blast against the Celera and National Center for Biotechnology Information (NCBI) mouse genome databases, and localized these two markers on both maps. The physical distance between the D18Mitl03 and D18Mitl88 markers was ~2.4 Mb in the Celera map with both markers sitting on the scaffold GA_x6K02T2NR3 J. In the NCBI mouse map, these two markers were localized to the contig NT_082383 and encompassed a ~2.6-Mb region (D18Mitl03 at the nucleotide position 2932731-2932845 and D18Mitl88 at the nucleotide position 5505960-5506058). [0134] The chromosome position for each gene in the candidate region is illustrated in Fig. 5. Generally, the gene annotations on both maps are consistent to each other. For example, four known genes, namely StarDό, Pol i, Mbd2, and Dec, are located in both Par2 regions. Sequence comparison revealed that the mCG9249 in the Celera map represents the same gene as the 2310002L13Rik does in the NCBI map. Both the mCG9258 and Loc225699 genes encode a protein similar to 60S ribosomal protein L5 and should represent a same gene. However, there are also some discrepancies between the Celera and NCBI mouse Par2 region. The Celera Par2 region has 7 annotated genes, whereas the NCBI Par 2 region has 11, which may reflect different gene prediction tools and prediction stringency used by amiotators. Another apparent discrepancy is the position of Mbd2 gene (Fig. 5). For the four known genes, their order in the NCBI mouse genome map is Mbd2-Stard6-Pol t-Dcc, which is different from their order (Stard6-Poli-Mbd2-Dcc) in the other three maps, i.e., the NCBI human, Celera mouse, and Celera human genome maps. Because of the high homology between human and mouse Par2 region, the Mbd2 gene apparently has been somehow mistakenly placed during the NCBI sequence assembling. Applicants used reverse

^ff ND, nul dolL'un' 'il h-i iii^ ul^'r ceM dtilabtisc updalii. transcription-PCR (RT-PCR) and direct sequencing to examine differential expression in lung tissues and coding-region nucleotide polymorphisms for the amiotated genes. The result is presented in Table 1.

[0135] The Stardό gene is expressed exclusively in the mouse testis, and its function has been suggested to be specific for fertility. RT-PCR results indicate that Stardό is expressed in mouse lung tissues but has no differential expression or nucleotide polymorphism between A/J and BALB/cJ inbred strains. Applicants' previous study has revealed neither expression difference nor nucleotide polymorphism between A/J and BALB/cJ mice for the Dec gene. Real-time RT-PCR analysis for the Mbd2 gene only revealed a slight expression difference (A/J: BALB/cJ, 0.84 +/- 0.03; data not shown), and no nucleotide polymorphism has been found for this gene either. Thus, Stardό, Mbd2, and Dec are less likely to be the Par2 gene. Two other genes, i.e., A430085C19 and Loc225699, only have one exon and were not detected by DNase I-treated RT-PCR in lung. Applicants could not detect 2310002L13Rik/mCG9249 in either A/J or BALB/cJ lung tissues.

[0136] Nucleotide Polymorphisms and Alternative mRNA Splicing in the Pol i Gene. The remaining known gene in the Par2 region, Pol i, has a 2154-bp open reading frame and encodes a 717-amino acid protein. By sequencing its entire coding region, Applicants found a total of 25 nucleotide polymorphisms in the Pol i coding region between A/J and BALB/cJ mice. As shown in Fig. 6A, Applicants have identified 10 amino acid-changing polymorphisms and two alternatively spliced exons in Pol i between A/J and BALB/cJ mice. Among them, 9 amino acid polymorphisms and 1 alternative splicing variant were consistent with those reported previously [0137] . The newly observed amino acid changing polymorphism at codon 606 encodes a positively charged arginine (CGA) in BALB/cJ but a neutral Glutamine (CAA) in A/J (Tig. 6B).

[0138] Pol i exon 4a transcript isoform was reported as a product of alternative splicing on exon 4. Applicants have found that in addition to this isoform (detected by our primer set 2; data not shown), another isoform also exists in mouse lung tissues. The new alternartive transcript was detected by primer set 1 and has a shorter nucleotide sequence compared with the regular one (Fig. 6C, a). Directly sequencing this shorter PCR product has revealed "that the exon 2 in the full-length Pol imRNA is spliced out without changing open reading frame (Fig. 6C, b). Applicants present this isoform as exon 2d ("d" means "deleted"). In C57BLV6J mice, the exon 2d isoform seems to be more abundant than the regular isoform that contains exon 2, whereas A/J, BALB/cJ, 129/SvJ, and C3H/HeJ lung tissues have more ofthe regular- length isoform transcript than the exon 2d isoform. Applicants did not observe differential expression ofthe new exon 2d transcript isoform between A/J and BALB/cJ mice. [0139] Initial Biochemical Characterization of Pol t Protein. To examine the properties of the Pol t isoforms encoded in A/J and BALB/cJ mice, Applicants expressed and purified t- ofh enzymes as full-length GST fusion proteins and measured their polymerization activity. Both enzymes were active in extending a correctly paired primer-template tlirough the addition of dAMP opposite template T (Fig. 7-4, a). Thus, the amino acid differences between the two polymerases do not result in loss of polymerization activity by either protein. At concentrations of Pol i estimated to be equivalent, more extension of the correctly paired primer was observed for the BALB/cJ enzyme than for the A/J enzyme. However, in parallel reactions using somewhat higher enzyme concentrations, a difference between the "two isoforms was not apparent for extension of a primer containing a terminal T-G mismatch (Fig. 7-4, b). These data suggested that the two isofonns of Pol i may differ in their ability to use aberrant substrates. To further test this hypothesis, Applicants examined a property that distinguishes Pol i from all other DNA polymerases studied to date — the preferential incorporation of incorrect dGMP over correct dAMP opposite template thymine. The results show that both mouse isoforms of Pol t do exhibit this noncanonical behavior (Fig. IB). Thus, when examined at two different concentrations of dGTP or dATP (0.1 and 1.0 mM), incorporation of dGTP is preferred by both enzymes (compare squares to circles). However, at the higher deoxynucleoside triphosphate concentration (black symbols), the BALB/cJ Pol i prefers dGTP by a factor of 3- -fold, whereas the A/J Pol t prefers dGTP by a factor of ~2- fold. This observation additionally supports the hypothesis that the amino acid differences between the two mouse isoforms may alter their substrate discrimination properties. [0140] Human POL I Mutation and Expression in Lung Cancer Cell Lines. To explore the possible role of POL I in human lung cancer development, Applicants examined 11 human lung cancer cell lines for mutations in the coding region of the human POL I gene. Two silent polymorphisms at codon 12 (TCG to TCT, Ser) and codon 293 (GTG to GTC, Val) were identified in the CaLu-1 cells, and a common polymorphism at position 2180, which changes the amino acid at codon 706 (ACA to GCA, Thr to Ala), was identified in A427, A549, CaLu-3, CaLu-6, NCI-H460, and NCI-H596 cells. These polymorphisms in the lung cancer cell lines all appeared to be heterozygous with the published sequence. Fig. 8.4 shows a sample without and one with the polymorphism at codon 706. [0141] In addition to the sequence analyses, an assessment of POL I protein expression in the human lung cancer cell lines was made. Western analysis demonstrated that POL I expression varied among the cell lines from low expression in the normal MRC-5 and the A427, SK-LU-1, and NCI-H460 cancer cells to very high in the CaLu-1 and NCI-H520 cell lines with the other cells in between (Fig. 8R). These expression differences did not correlate with the amino acid change at codon 706 observed in certain cell lines or with the tumor morphological subtypes (e.g., adenocarcinoma or squamous cell carcinoma) from which the cells were derived.

[0142] The protein similarities between the human POL I, mouse Pol , and other species such as Arabidopsis thaliana, Caenorhabditis elegans, Escherichia coli, as well as Saccharomyces cerevisiae Pol t-like proteins are 76, 29, 29, 32, and 20%, respectively (UniGene). Most of conserved amino acids are clustered within the NH2-terminal region, which contains five conserved motifs. The evolutionary conservation of the Pol i codons was examined using the human, mouse, and fruit fly's Pol tsequences. The fruit fly Pol tsequence was derived from the Drosophila melanogaster transcript CG7602-RA (GenBank accession no. AAF54198). Applicants aligned these three sequences using the Clustalw software. As shown in Fig. 9, the conserved amino acids are mainly clustered in NH2-terminal region consistent with the previous study (22). The codon 706 shows weak conservation among these three species. At this codon, the fruit fly Pol i has a proline, whereas mouse Pol i has an alanine. Both are hydrophobic and neutral amino acids.

Claims

What is claimed is:

1. A method of characterizing the etiology of a cancer in a human individual by testing at least one cancer cell from the individual for one or more mutations in the at least one cancer cell's Pol i gene, said one or more mutations comprising a mutation at codon 706.

2. The method according to claim 1, wherein the mutation at codon 706 encodes alanine.

3. The method according to claim 1, wherein at least one cancer cell is tested by analyzing the sequence ofthe Pol i gene.

4. The method according to claim 1, wherein t herein at least one cancer cell is tested by using an antibody that detects the mutant Pol i protein.

5. A method of identifying an individual who is at risk of developing cancer by testing at least one cell from the individual for one or more mutations in the at least one cell's Pol i gene, said one or more mutations comprising a mutation at codon 706.

6. The method according to claim 5, wherein the mutation at codon 706 encodes alanine.

7. The method according to claim 5, wherein at least one cancer cell is tested by analyzing the sequence ofthe Pol i gene.

8. The method according to claim 5, wherein t herein at least one cancer cell is tested by using an antibody that detects the mutant Pol t protein.

9. A method for treating an individual identified as having a mutant Pol i gene by administering to the individual an agent that supplements or restores Pol i protein function.

10. The method according to claim 9, wherein the individual has an adenocarcinoma.

11. The method according to claim 10, wherein the individual has adenocarcinoma of the lung.

12. The method according to claim 9, wherein the agent that supplements or restores Pol i protein function is a wild-type, non-mutant Pol i protein, a polynucleotide encoding a wild- type, non-mutant Pol I protein, said polynucleotide in operable connection with a promoter that directs its expression, phannaceutical compositions thereof, or combinations thereof.

13. The method according to claim 9, wherein the agent is administered in a fashion such that it is specifically targeted to cancer tissue in the individual.

14. The method according to claim 9, wherein the treatment is prophylactic.

15. The method according to claim 9, wherein the treatment is therapeutic.