WO2008010928A2

WO2008010928A2 - Method for discovery of anti-senescence genes

Info

Publication number: WO2008010928A2
Application number: PCT/US2007/015660
Authority: WO
Inventors: Abdelhadi Rebbaa
Original assignee: Childrens Memorial Hospital
Current assignee: Childrens Memorial Hospital
Priority date: 2006-07-19
Filing date: 2007-07-09
Publication date: 2008-01-24
Anticipated expiration: 2009-01-19
Also published as: WO2008010928A3

Abstract

Methods useful for identification of senescence-related genes are provided. The identified senescence-related genes may then be used to slow, halt, or reverse senescence. The methods of this invention may also be used to identify genes that are associated with senescence-related diseases, e.g. cancer.

Description

METHOD FOR DISCOVERY OF ANTI-SENESCENCE GENES

CROSS-REFERENCE TO RELATED APPLICATIONS

[00013 This invention claims priority to U.S. Provisional Patent Application

Serial No. 60/832,099, filed July 19, 2006, which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates generally to compounds and methods for the diagnosis and treatment of senescence and senescence-associated diseases.

BACKGROUND

[0003] Normal somatic cells invariably enter a state of permanent growth arrest and altered function after a finite number of divisions. This phenomenon, termed cellular senescence, is believed to be a contributing factor in aging. As we age, our organisms' ability to withstand the wear and tear of internal and environmental stress diminishes, rendering our bodies vulnerable to a variety of chronic illnesses including cancer, arthritis, diabetes, heart and neurodegenerative diseases. Conventional wisdom holds that the body's decreased resistance to stress during senescence may result from fading of normal maintenance and repair mechanisms. In light of this, better understanding of the signaling pathways leading to stress resistance would facilitate the discovery of ways to inhibit senescence and delay the onset of age-associated diseases.

[0004] Genetics has provided valuable insights into the mechanisms that limit the life-span of organisms (Hekimi and Guarente, 2003, Science 299: 1351-1354; Sinclair and Guarente, March 2006, ScL Amer. 48-57). Studies on genetic and comparative biology of senescence have also demonstrated that the life duration of both invertebrates and mammals can be extended by manipulating certain genes (Tatar et al., 2003, Science 299: 1346-1351 ; Weindruch et al., 1997, New Engl. J. Med. 337: 986-994). For instance, with the recent discovery that sir2 (silent information regulator 2) gene expression was up-regulated in yeast cells resistant to caloric restriction, and that this gene was responsible for increased longevity in these cells (Kaeberlein et al., 1999, Genes Dev. 13: 2570-2580; lmai et al., 2000, Nature 403: 795-800), as well as worms (Hekimi and Guarente, 2003, Science 299: 1351-1354), flies (Rogina and Helfand, 2004, Proc. Natl. Acad. ScL U.S.A. 101 : 15998-16003), and rodents (Cohen et al., 2004, Science 305: 390-392), it became evident that manipulation of a single gene could be sufficient to alter the aging process.

[0005] Recently, cancer cells were introduced as models for the discovery of senescence genes (Chang et ai, 2002, Proc. Natl. Acad. Sci. U.S.A. 99: 389-394). However, from a technical standpoint, the approaches used were mainly based on comparison of gene expression profiles between senescent and non-senescent cells or "subtraction differential" screening in which a pool of cDNA molecules is created from senescent cells and then hybridized to cDNA or RNA of growing cells in order to "subtract out" those cDNA molecules that are complementary to nucleic acid molecules present in growing cells (Kleinsek, 1989, Age 12: 55-60; Giordano et al., 1989, Exp. Cell Res. 185: 399-406). The difficulty with these methods is that many of the genes that appear to be associated with senescence do not have a causative relationship with it. This makes it difficult to identify authentic anti-senescence genes among the hundreds of differentially expressed genes between senescent and proliferating cells.

[0006] The inventor recently demonstrated that the stress level required for induction of senescence in cancer cells was much lower that the one that induce apoptotic cell death (Rebbaa et al., 2003, Oncogene 22: 2805-2811) suggesting that cancer cells must escape senescence first in order to become drug-resistant. Thus, manipulating cellular ability to undergo senescence could represent a novel approach to prevent and/or to reverse drug resistance in cancer. In support of this idea, it has been shown that: 1 ) forcing cancer cells to undergo senescence was sufficient to prevent and to reverse development of drug resistance (Zheng et al., 2004, Cancer Res. 64: 1773- 1780), and 2) over-expression of the longevity gene Sirti, known to inhibit cellular senescence, rendered cancer cells resistant to drugs (Chu et al., 2005, Cancer Res. 65: 10183-10187). Since the ability of somatic cells to live longer requires inhibition of senescence, drug resistance in cancer and longevity may share common mechanisms of stress resistance. Accordingly, the relationship between senescence and cancer can be viewed as follows: considering that somatic cells are continuously subjected to environmental insults (including but not limited to oxidation, UV radiation, chemical and microbial pollution, etc.), over time, as the cells age, they enter a state of irreversible growth arrest (senescence) leading to organ failure and ultimately to the organism's death.

[0007] A subset of somatic cells with an active anti-senescence defense may escape the toxic effect of environmental stress and even become cancerous, suggesting that cancer may result at least in some cases from resistance to senescence. Regardless of the causes leading to carcinogenesis, most tumor cells can still be induced into senescence when subjected to chemotherapy or radiotherapy. However, some cells may withstand the toxicity of these treatments and become so-called resistant cancer cells. In light of this, stress-resistant cancer cells may represent a suitable model for the discovery of anti-senescence genes that can be used as targets to treat not only cancer, but senescence-associated diseases in general.

[0008] Due to the relationship between aging and the increased risk of developing cancer and other chronic diseases, a need exists for developing approaches and technologies that will allow identification of anti-senescence genes so that appropriate treatment strategies can be designed to delay onset of senescence-associated diseases and ameliorate the life style at older age. The identification and cloning of anti-senescence genes is a strongly felt need in the fields that investigate the control and mitigation of senescence-related processes and diseases. The present invention provides methods useful for the identification of genes associated with senescence and senescence- related diseases.

SUMMARY OF THE INVENTION

[0009] The present invention provides methods that are useful for identification of senescence-related genes. The senescence-related genes identified according to this invention may then be used to slow, halt, or reverse senescence. The methods are also useful for the identification of genes that are associated with senescence-related diseases, e.g. cancer. Thus the methods may be used to identify genes that may prevent or treat senescence-associated diseases.

[0010] The present invention provides methods for identification of anti- senescence genes, which include: generating a cDNA library from mRNA isolated from stress-resistant cells; transfecting a population of stress- sensitive cells with the library to obtain transfected stress-sensitive cells; conducting a functional screen for senescence-related genes on the transfected stress-sensitive cells to identify stress-sensitive cells that have escaped senescence; and identifying cDNA inserts from the stress-sensitive cells that have escaped senescence.

[0011] The present invention provides methods for identification of anti- senescence genes, which include: obtaining mRNA from stress-resistant cells; generating cDNA from the mRNA; generating a cDNA library that includes the cDNA operably linked to an inducible expression control system and a selectable gene; introducing the library into a population of stress-sensitive cells; culturing the stress-sensitive cells in the presence of an appropriate selection agent and under conditions that induce senescence; isolating stress- sensitive cells that have escaped senescence; and analyzing the DNA sequences in the isolated cells, thereby identifying anti-senescence genes. [0012] The stress-resistant cells can be stem cells, fibroblasts, hepatocytes, blood cells, intestinal cells, or cancer cells. Resistance in these cells can be either intrinsic or acquired. Selection for resistance to stress can be made by continuous exposure to increasing stress levels. The stress can be chemical, biological, physical or a combination of two or more stresses of different nature.

[0013] The stress-sensitive cells can be somatic cells, stem cells, intestinal cells, fibroblasts, blood cells, fibroblasts, or cancer cells. [0014] The cells that have escaped senescence can be cancerous or noncancerous.

[0015] The screen for cells transfected with cDNA library fragments may include one or more selectable genes. Examples of selectable genes include neomycin phosphotransferase, hygromycin, puromycin, G418 resistance, histidinol dehydrogenase, Sh ble, or dihydrofolate reductase. The screen for cells transfected with anti-senescence genes is carried out by subjecting cells transfected with the cDNA library to treatment with senescence-inducing stressors at senescence-inducing doses. Cells that have escaped senescence are selected for sequencing of the cDNA fragment contained within.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 shows electrophpretic images depicting expression of

UQCRC1 and Tsen34 in SKN-SH clones.

[0017] Figure 2 shows graphs depicting response of cDNA transfected clones to stress.

[0018] Figure 3 shows electrophoretic images (A, B) and a graph (C) depicting how classical drug resistance mechanisms mediated by superoxide dismutase (SOD), topoisomerase-llα (Topo-llα) and P-glycoprotein (P-gp) are not affected by UQCRC1 and Tsen34p.

[0019] Figure 4 shows a graph (A) and electrophoretic images (B) depicting how expression of Tsen34p inhibits senescence mediated by cellular exposure to doxorubicin.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

[0020] The present invention provides methods for identification of senescence-related genes, and genes identified using such methods. [0021] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunology, protein kinetics, and mass spectroscopy, which are within the skill of art. Such techniques are explained fully in the literature, such as Sambrook et ai, 2000, Molecular Cloning: A Laboratory Manual, third edition, Cold Spring Harbor Laboratory Press; Sambrook etal., 1989, Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press; Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc.; Kriegler, 1990, Gene transfer and expression: A laboratory manual. Stockton Press, New York.; Dieffenbach CW. et al., 1995, PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, each of which is incorporated herein by reference in its entirety. Procedures employing commercially available assay kits and reagents typically are used according to manufacturer-defined protocols unless otherwise noted. [0022] Generally, the nomenclature and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA, RNA, and protein isolation, nucleic acid amplification, and nucleic acid and protein purification. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications.

[0023] "Senescence" or "aging" according to this invention is a combination of processes of deterioration that follow the period of development of an organism. "Senescence" is derived from the Latin word senex, meaning "old man" or "old age." Senescence is generally characterized by an organism's declining adaptability to stress, increased homeostatic imbalance, and increased risk of disease. Because of this, death is the ultimate consequence of senescence.

[0024] A "senescence-related gene" according to this invention is a gene that is involved in processes that control senescence. In one aspect of the invention, the senescence-related gene may be related to a disease associated with senescence. For example, the disease associated with senescence may be cancer (Rebbaa, 2005, Cancer Letters 219: 1-13) or diabetes (Geesaman, 2006, Am. J. Clin. Nutr. 83 suppl. 466S-9S), arthritis (Aigner et al., 2004, Rejuvenation Res. 7: 134-145), neurodegenerative diseases (Keller, 2006, Ageing Res. Rev. 5: 1-13), or atherosclerosis (Orlandi et al. , 2006, Atherosclerosis 188: 221-230).

[0025] A "screen", "functional screen", or "functional genetic screen" according to this invention is a genetic screening procedure that is focused on the identification of specific parameters, e.g. particular genotype, particular phenotype, etc. For example, a functional screen for senescence-related genes is a genetic screen designed to identify genes that are involved in the regulation of senescence-related processes, senescence-related diseases, etc. Examples of functional genetic screens are found in Raveh et al., 2000, Proc. Natl. Acad. ScL U.S.A. 97: 1572-1577, and in Erez et al., 2002, Oncogene 21 : 6713-6721 , both of which are incorporated herein by reference. [0026] "Transfection" according to this invention refers to the introduction of DNA into eukaryotic cells. Various methods of transfection are known in the art. These include transfection by calcium phosphate, calcium chloride, electroporation, heat shock, transfection with a gene gun, transfection using liposomes, transfection using viruses as carriers, etc.

Identification of senescence-related genes

[0027] The present invention describes a screen using a cDNA library constructed from cancer cells that have developed resistance to stress- induced senescence. This screen allows identification of novel anti- senescence genes. The screen is based on identification of genes contained within cells that have been selected for resistance to stress. Those genes may be responsible for protection against stress-induced senescence. The DNA from cells either having intrinsic resistance to stress-induced senescence or cells made (engineered) resistant to stress by exposure to increasing levels of stress is used to generate a cDNA library. That cDNA library is used for transfection of stress-sensitive cells. The transfected cells are then subjected to senescence-inducing conditions. Those transfected clones that escape the stress-induced senescence contain cDNA inserts that code for senescence-related genes.

[0028] The identification and targeting of anti-senescence genes may have applications to inhibit senescence in cell culture and to delay aging-associated diseases in animals and humans. As opposed to dealing with one disease at a time, which may not affect significantly the population's overall life span, identification of molecular targets to delay the senescence process itself may prove to be more effective in reducing the hurdles of late life. [0029] The present invention describes a method for discovery of anti- senescence genes. This method includes selection of stress-resistant cells. "Stress-resistant cells" are cells that are chosen for their intrinsic resistance to stress-induced senescence. Alternatively, the stress-resistant cells can be engineered, i.e. they can be made stress-resistant by exposure to stressors such as chemotherapeutic drugs, radiation, caloric restriction, or oxidant. [0030] The method includes generation of a cDNA library from the mRNA isolated from the stress-resistant cells. A cDNA library can be generated by a variety of methods known in the art, such as those described in Generation of cDNA Libraries: Methods and Protocols, 2003, S. Ying, ed., Humana Press, 2003, 352 pp. Alternatively, transcriptionally active DNA fragments can be generated from the nucleic acids isolated from stress-resistant cells using other known methods, e.g. as described in U.S. Patent No. 6,280,977. Also, a nucleic acid library useful for practicing the invention can be generated using the methods of U.S. Patent No. 6,846,655, incorporated herein by reference. [0031] The method includes transfection of stress-sensitive cells with the cDNA library made from stress-resistant cells. "Stress-sensitive cells" are defined in the context of this invention as cells that are more susceptible to stress-induced senescence than the cells from which the cDNA library used for transfection is derived.

[0032] The method also includes selection of cDNA library-transfected stress-sensitive clones for their ability to escape stress-induced senescence. [0033] The method further includes extraction of DNA from cDNA library- transfected clones that have escaped stress-induced senescence. The DNA inserts and sequenced, analyzed, and identified.

[0034] A variety of stresses may be used to practice the invention, so long as the stresses induce senescence. These include chemical, physical, and biological stresses. For example, senescence can be induced with stresses caused by UV and IR irradiation, drugs and other chemicals, mitogenic stimuli, oncogenic stimuli, toxic compounds, hypoxia, oxidants, caloric restriction, etc. (Jansen-Dϋrr and Osiewacz, 2002, EMBO Rep. 3: 1127-1132; Blagosklonny, 2003, EMBO Rep. 4: 358-362). In some aspects of the invention, combinations of stresses can be used (e.g., two or more chemical and physical stresses; two or more chemical and biological stresses; two or more physical and biological stresses; chemical, physical, and biological stresses in combination, etc.).

[0035] The practice of this invention initially involves comparison of two cell lines to determine their relative stress resistance. Upon determination of the relative stress resistance, the cell line with greater stress resistance will be designated "stress-resistant", and the other cell line (with lesser stress resistance) will be designated "stress-sensitive". [0036] A skilled artisan will know how to compare two cell lines to determine their relative stress resistance. The absolute resistance to stress may be identical between two cell lines, or it may vary from one cell line to another. Also, new cell lines with increased or decreased stress resistance may be discovered or generated over time, and their stress resistance may be compared to the stress resistance of existing cell lines, or between themselves. Nonetheless, it is possible to practice this invention so long as the comparison between the two cell lines yields a measurable relative difference in stress resistance. As explained below, determination of stress resistance can be accomplished through cell counting, cell staining, and other methods known in the art.

[0037] A cell line "A" is designated stress-resistant if the cell line either possesses intrinsic resistance to stress or is made resistant to stress by continuous exposure to stress. Such a cell line can be used to generate the cDNA library.

[0038] A cell line A is considered to have intrinsic stress resistance in the context of this invention if, without any prior exposure to stressors or selection for drug resistance, it performs as a stress-resistant cell line in the test described below. A different cell line "B" is designated stress-sensitive and thus chosen for transfection with this cDNA library if its relative response to stress compared to that of the cell line A is higher. The relative response to stress can be measured by subjecting both cell lines A and B to treatment with increasing stress levels, and subsequent determination of the stress level that induces senescence in at least one of them. That can be done, for example, using senescence beta-galactosidase staining (a senescence beta- galactosidase staining kit can be obtained from Cell Signaling Technology, Danvers, Massachusetts; see also Takaoka et al., 2004, Oncogene 23: 6760- 6768). The number of cells that become senescent in response to the applied stress will be counted and the two cell lines A and B will be compared. If the number of senescent cells in cell line A is higher than in cell line B, this means that cell line A requires less stress level to reach the senescence state in comparison to cell line B- Therefore, cell line A is considered more sensitive to stress-induced senescence than the cell line B. If cell line A happens to contain fewer senescent cells than cell line B, cell line A will be considered more resistant to stress than cell line B. In that case, cell line A will not be used for transfection with the cDNA library from cell line B. [0039] A variety of stress-resistant cells may be used. In general, cells that have the ability to adapt to stress and can continue to proliferate in a stressful or toxic environment are stress-resistant cells. For example, cancer cells can be used, as cancer cells are easily adaptable to stress conditions and can become stress-resistant. Examples of agents that can be used to generate stress-resistant cells include ionizing radiation, DNA-damaging drugs, the p53 tumor suppressor, microtubule-active drugs (such as Taxol), oxidative stress and hypoxia-mimicking iron chelators, inhibitors of histone acetylase, transforming growth factor-beta (TGF-beta), retinoids, and other agents able to trigger premature cell senescence (Blagosklonny, 2003, EMBO Rep. 4: 358-362).

[0040] The administration of stress conditions can be carried out gradually, by increasing the stress level (e.g. gradual increase in the concentration of a stress-inducing drug). Alternatively, the administration of stress conditions can be carried out in a single step (e.g. subjecting the cells to a single dose of a stress-inducing drug). Furthermore, the cells can be selected using different stresses (e.g. physical and chemical) that are administered in parallel or in series. For example, the cells may be first selected for resistance to radiation, and then they may be selected for resistance to a drug. Alternatively, radiation and chemical stress may be applied together in the selection process.

[0041] Various cancer cell lines can be used for practicing the invention. For example, these cancer cell lines include: Mm5MT murine mammary tumor; K1735 melanoma; and MatLyLu prostate cancer cells. Additional cell lines include: SW-480 - colonic adenocarcinoma; HT-29 - colonic adenocarcinoma; A-427 - lung adenocarcinoma carcinoma; MCF-7 - breast adenocarcinoma; UACC-375 - melanoma line; DU 145 -prostate carcinoma; human osteosarcoma SaOS2 cells, and a number of other cancer cell lines that can be acquired from the American Tissue Type Collection (Manassas, Virginia). Numerous cancer cell lines can also be acquired from the United States National Cancer Institute where they are used for screening programs for new anti-cancer drugs.

[0042] Other stress-resistant cells useful for practicing the invention include stem cells, fibroblasts, hepatocytes, intestinal cells, and generally cells that can proliferate in the presence of stress, and can become, or can be made, stress-resistant. The stress-resistant cells may originate from different organisms. For example, the stress-resistant cells may be human cells, animal cells, yeast cells, plant cells, or insect cells.

[0043] In one example, the stress-resistant cells are human cells. Various sources for these cells exist. These cells may originate from a variety of available cell lines. For example, these include osteosarcoma cells, breast cancer cells, neuroblastoma cells, stem cells, etc. Alternatively, these cells may originate from patients that have been treated for cancer, and in which patients the tumor has relapsed. Because such patients have cells that have become resistant to stress (e.g. cancer-treating drug, radiation, etc.), the patients' tumor cells can be used as a source of stress-resistant cells for practicing the invention.

[0044] A cDNA library is then generated from stress-resistant cells according to methods known in the art, using a variety of vectors and procedures. Some of these are described in Gubler and Hoffman, 1983, Gene 25: 263-269; Sartoris et al., 1987, Gene 56: 301-307; Generation of cDNA Libraries: Methods and Protocols, 2003, S. Ying, ed., Humana Press, 2003, 352 pp; Molecular CeIi Biology 2000, Lodish et al., eds., W.H. Freeman and Co.; Perkel, 2003, The Scientist 17: 43. [0045] The generated cDNA library is used for transfection of stress- sensitive cells. Transfection is performed according to procedures known in the art. For example, transfection can be performed using methods based on calcium phosphate precipitation, lipid-mediated transfection, DEAE-dextran, electroporation, lipofectin, etc. Manufactured reagents and kits for transfection can also be used, e.g. Transfectam and Profection® (Promega, Madison, Wisconsin), Lipofectamine™ (Invitrogen, Carlsbad, California), Polyfect, SuperFect, and Effectene, which are optimized for COS-7, NIH/3T3, HeLa, Hel_a-S3, 293, and CHO cells (Qiagen, Valencia, California), etc. To accomplish stable transfection, another gene can be co-transfected, which gives the transfected cell selection advantage, such as resistance towards a certain selection agent (e.g. toxin, antibiotic, etc.). Such co-transfected selectable genes include, for example, neomycin phosphotransferase, hygromycin, puromycin, G418 resistance, histidinol dehydrogenase, dihydrofolate reductase, etc. When the selection agent, towards which the co- transfected gene offers resistance, is then added to the cell culture, only those cells with the foreign genes inserted into their genome will be able to proliferate, while other cells will die. After applying this selection pressure for some time, only the cells with a stable transfection remain and can be cultivated further. A variety of selection agents can be used, as long as the co-transfected selectable gene confers resistance toward the selection agent to the transfected cell. For example, when the selection agent used for transfection is Geneticin, also known as G418, it can be neutralized by the product of the neomycin resistance gene. Similarly, when the selection agent used for transfection is zeocin or phleomycin, it can be neutralized by the product of the Sh ble gene from Streptoalloteichus hindustanus. [0046] A variety of stress-sensitive cells may be used for practicing the invention. In general, these stress-sensitive cells have higher intrinsic susceptibility to stress-induced senescence than those from which the cDNA library is generated. Stress-sensitive cells include, but are not limited to: stem cells, cancer cells, blood cells, fibroblasts, intestinal cells, hepatic cells, and other somatic cells from various organs.

[0047] It should be understood that for the purposes of this invention, stress-sensitive cells are those cells that, when subjected to the same stress conditions as the stress-resistant cells from which the cDNA library is derived, undergo senescence, while the stress-resistant cells do not undergo senescence.

[0048] The cDNA library-transfected stress-sensitive cells are then exposed to a selection process, under senescence-inducing conditions. A variety of senescence-inducing conditions may be utilized. Examples of senescence- inducing stresses include ionizing radiation, DNA-damaging drugs, the p53 tumor suppressor, microtubule-active drugs (such as Taxol), oxidative stress and hypoxia-mimicking iron chelators, inhibitors of histone acetylase, transforming growth factor-beta (TGF-beta), retinoids, and other agents able to trigger premature cell senescence (Blagosklonny, 2003, EMBO Rep. 4: 358-362).

[0049] The administration of stress conditions to the cDNA library- transfected cells can be carried out either as a single dose or gradually, by increasing the stress level (e.g. gradual increase in the concentration of a stress-inducing drug). Furthermore, the transfected cells can be selected for resistance to different stresses (e.g. physical and chemical) that are administered in parallel or in series. For example, the cells may be first stressed using radiation, and then they may be stressed using a drug. Alternatively, radiation and chemical stress may be applied together. [0050] Clones that have escaped from senescence under the senescence- Inducing conditions are then selected. The cDNA inserts contained within these clones are amplified and sequenced. DNA sequencing is performed using methods known in the art, e.g. by the chain termination method (Sanger sequencing), dye terminator sequencing, etc. The corresponding genes are then analyzed and identified using available GenBank and other nucleotide sequence databases. The identified genes are senescence-related genes or anti-senescence genes according to this invention. Examples of such genes are shown below.

[0051] Since resistance to stress is a common mechanism used by somatic cells to live longer and by cancer cells to become drug-resistant, the discovery of anti-senescence genes may have applications not only to delay the aging process, but also to treat cancer and other aging-associated diseases. [0052] This method is unique because it utilizes a functional screen for anti- senescence genes contained within cells that have been selected for resistance to senescence (e.g., stress-resistant cancer cells). In contrast, other, previously described approaches are based on differential gene expression, i.e. comparing gene expression profiles between senescent and non-senescent cells.

[0053] The methods of the- present invention are illustrated by the following examples, which are illustrative, but not limiting, of the combined preparations and methods of the present invention. Other suitable modifications and adaptations of a variety of conditions and parameters normally encountered in molecular and cell biology, obvious to those skilled in the art, are within the scope of this invention. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

EXAMPLES

[0054] It is to be understood that this invention is not limited to the particular methodology, protocols, patients, or reagents described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the claims. The following examples are offered to illustrate, but not to limit the claimed invention. Construction of a cDNA library from drug-resistant osteosarcoma cells [0055] Human osteosarcoma SaOS2 cells selected for resistance to doxorubicin (Zheng et ai, 2004, Cancer Res. 64: 1773-1780) were directly harvested in the RNA isolation solution (RNAzol) after aspirating the medium. The cell lysate was mixed with 0.2 volumes of chloroform, vortexed, and incubated on ice for 15 minutes. The mixture was centrifuged at 10,000x g for 10 minutes and the aqueous layer was collected. The aqueous layer was mixed with equal volume of isopropanol and centrifuged for 30 minutes at 10,000x g. The RNA pellet was washed with 70% ethanol, dried, and dissolved in DEPC-treated autoclaved water. An aliquot of RNA was checked on agarose-formaldehyde denaturing gel.

[0056] mRNA was isolated from total RNA using oligo dT cellulose resin. Briefly, total RNA was mixed with binding buffer (10 mM Tris pH 7.5, 500 mM NaCI) and heated at 70⁰C for 5 minutes. The samples were immediately chilled on ice for 5 minutes and mixed with oligo dT cellulose powder. The mixture was incubated at room temperature for 2 hours, and then the mixture was centrifuged at 5,00Ox g. The pellet was washed twice with binding buffer. The pellet was washed with excess of low salt buffer (10 mM Tris pH 7.5, 250 mM NaCI). This step was repeated 5-6 times to remove unbound RNA molecules. The mRNA was finally eluted in 10 mM Tris pH 8.0. [0057] The first strand was synthesized by using mRNA, first strand buffer, nucleotide mixture containing methylated dCTP, oligo dT primers containing Xho I and Not I sites at 3' end and reverse transcriptase. The mRNA was mixed with the primer oligos and heated at 70⁰C for 2 minutes. The mixture was chilled on ice and then nucleotides and reverse transcriptase were mixed. The reaction was performed at 42°C for 1 hour. An aliquot of the first strand was saved to check using gel electrophoresis.

[0058] The second strand was synthesized using DNA polymerase, RNase H, E. coli ligase, and dNTP mix. The cDNA ends were filled with cloned Pfu DNA polymerase. The second strand was extracted with equal volume of phenol: chloroform: isoamyl alcohol, and was then precipitated with ethanol. [0059] Adapter ligation was then performed. cDNA was re-suspended into ligation buffer, mixed with Eco Rl adapters and incubated with T4 DNA ligase. The complementary nucleic acid sequences of the Eco Rl adapter are given below. The nucleotide sequences of the 5' to 3' strand and the 3' to 5' strand of the adapter are shown as SEQ ID NO:1 and SEQ ID NO:2, respectively: 5'- OH-AATTCGGCACGAGG-3' (SEQ ID NO:1 ); and 3'-GCCGTGCTCCP-S¹ (SEQ ID NO:2).

[0060] After two days of ligation at 4°C, the second strand was again precipitated. The cDNA was phosphorylated by using YATP and T4 polynucleotide kinase. The cDNA was digested with Xho I (for large fraction library) and Not I (for small fraction library). The cDNA was fractionated on sephacryl S-400 column and pooled in two size ranges: 0.5 kb to 2.0 kb (small) and 2.0 kb to 6.0 kb (large). Fractions having less than 400 bp cDNA were discarded. Pooled cDNA was extracted with phenol-chloroform-isoamyl alcohol and precipitated with ethanol. The cDNA was dissolved in sterile water and stored at -20⁰C until further use.

[0061] The vector pFB-Neo was digested with Eco Rl / Not I (to ligate small fraction library) and Eco Rl / Xho I (to ligate large fraction library), and treated with calf intestinal alkaline phosphatase. The digested vector was purified on low melting agarose gel. An aliquot of the digested vector was ligated and electroporated into E. coli DH10B/XL-1 Blue cells to test the efficiency of the digestion. The cDNAs were separately ligated into the vector at various concentrations in the presence of T4 DNA ligase. The samples were incubated for 2 days at 4°C.

[0062] The ligated DNA was electroporated into DH10B/XL-1 Blue cells. Immediately after the electroporation, the cells were mixed with SOC medium and incubated for 1 hour at 37⁰C. The entire library (containing >1.0 x 10⁷ independent clones) was plated on LB-amp plates. After overnight incubation, the cells were harvested in LB medium containing ampicillin, centrifuged at 5,000 rpm for 15 minutes and re-suspended in LB medium containing ampicillin and 15% glycerol. The cells were frozen at -80⁰C. Quality Control of the cPNA Library

[0063] Restriction digestion was performed to check the quality of the library. Clones from small and large fraction libraries were randomly picked, grown overnight in liquid medium, and the plasmid DNA was isolated. The DNA was digested with Eco Rl / Not I and electrophoresed on agarose gels. More than 85% of the clones had inserts ranging from 0.5 kb to 2.0 kb and 2.0 to 6.0 kb, in the small and the large fraction libraries, respectively.

Specifications of the cDNA libraries

[0064] The library specifications are given in Table 1. DNA from the libraries was isolated using the CsCI density gradient method, and DNA was finally suspended in sterile water. During isolation the modified procedure was adapted to remove endotoxins from the DNA. Therefore, the libraries can be used directly for transfection.

Table 1. Library specifications

Transfection. selection, and cloning

[0065] The virus construct containing the library was amplified by infection of 293 cells using the calcium precipitation method and after 48 hours the culture medium containing the virus was harvested, aliquoted, and stored. [0066] Human neuroblastoma SKN-SH cells were seeded in 25 cm³ flasks and incubated in DMEM, 10% fetal bovine serum, until they reached 60% confluency. One ml of culture medium from 293 cells infected with small library was added to the flask containing SKN-SH cells and incubated for 48 hours. Cells were then selected with G418 1 mg/ml, a concentration that kills all non-transfected cells but not the transfected ones. Cells that survived were then subjected to treatment with a doxorubicin at a concentration of 0.1 μM that induces senescence in parental cells. Clones that survived were grown separately and genomic DNA was extracted. The cDNA inserts were amplified by PCR using primers sequences adjacent to the cloning site on the pFBNeo vector. The PCR fragments were then sequenced.

Sequences obtained using the method

[0067] Shown below are examples of some sequences obtained using the methods of the present invention.

[0068] A ubiquinol cytochrome c reductase core protein 1 (UQCRC1 ) was identified as an anti-senescence molecule. The nucleotide sequence of UQCRC1 is shown in SEQ ID NO:3. UQCRC1 is a component of complex III of the respiratory chain in mitochondria. This complex is part of a succession of membrane proteins that facilitate electron transfer from NADPH to oxygen, and participate in energy generation. It is also known to be the site of leakage for reactive oxygen species (ROS) out of the mitochondrial respiratory chains. ROS have been reported to be responsible for acceleration of the senescence process and their leakage rate out of the mitochondrial respiratory chain has been found to be inversely proportional with longevity and the onset of senescence associated illnesses (Barja, 1998, Ann. N. Y. Acad. Sci. 854: 224- 2238; Moghaddas et ai, 2003, Arch. Biochem. Biophys. 414: 59-66). [0069] Another gene that was identified using the methods of the present invention corresponds to a tRNA splicing endonuclease 34 (Tsen34), which is involved in cell division. The nucleotide sequence of tRNA splicing endonuclease 34 (Tsen34) is shown as SEQ ID NO:4. In yeast, deficiency of this enzyme was found to be lethal, and its decrease resulted in G1 cell cycle arrest (Volta et al., 2005, Biochem. Biophys. Res. Comm. 337: 89-94). [0070] Yet another gene that was identified using the methods of the present invention corresponds to the human cysteine protease legumain. This protease was identified as an inhibitor of osteoclast formation and bone resorption, both of which are hallmarks of osteoporosis, a disease of old age (Choi et al., 1999, J. Biol. Chem. 274: 27747-27753). The nucleotide sequence of legumain is shown as SEQ ID NO:5.

[0071] Figure 1 shows electrophoretic images depicting expression of UQCRC1 (SEQ ID NO:3) and Tsen34 (SEQ ID NO:4) in SKN-SH (human neuroblastoma SKN-SH cells) clones. RNA from stress-resistant clones was extracted and PCR-amplified using primers corresponding to UQCRC1 and Tsen34, respectively. The SK lane indicates SKN-SH cells; the pFB lane indicates SKN-SH cells transfected with the empty vector pFB neo; the UQ lane indicates SKN-SH clone expressing the sequence corresponding to UQCRC1 ; the Tsen lane indicates SKN-SH clone expressing the, sequence corresponding to the Tsen34p (Tsen34 protein) sequence. GADPH (glyceraldehyde-3-phosphate dehydrogenase) represents the loading control. [0072] Expression of UQCRC1 was greater in the corresponding clone than in parental cells or those transfected with the empty vector. Similarly, the expression of Tsen34p was higher in the corresponding clone than in parent cell line or the cells transfected with the empty vector. [0073] Figure 2 shows graphs depicting response of cDNA transfected clones to stress. Cells were treated with doxorubicin (Dox), cisplatin (Cisp), hydrogen peroxide (H₂O₂), or trichostatin A (TSA) at the indicated concentrations for 96 hours. Viable cells were then counted and their relative numbers represented in percent relative to control, non-treated cells. SK indicates SKN-SH cells; pFB indicates SKN-SH cells transfected with the empty vector pFB neo.

[0074] The clone expressing UQCRC1 was resistant to doxorubicin but not to cisplatin, H₂O₂ or TSA. However, the clone expressing Tsen34p was resistant not only to doxorubicin, but also to cisplatin and TSA. Since senescence can be caused by various stresses, the anti-senescence function of the Tsen34 gene may be broader than that of UQCRC1. [0075] Figure 3 shows electrophoretic images and a graph depicting how classical drug resistance mechanisms mediated by superoxide dismutase (SOD), topoisomerase-llα (Topo-llα), and P-glycoprotein (P-gp) are not affected by UQCRC1 and Tsen34p. Figure 3A: expression of SOD and Topo- llα (measured by PCR) in the parental cell line SKN-SH (SK), cells transfected with the empty vector pFB neo (pFB), the clone expressing the UQCRC1 sequence (UQ)₅ and the clone expressing the Tsen34p sequence (Tsen). Figure 3B: expression of the drug transporter P-gp (measured by Western blot) in the parental cell line (SK) and the derived clones expressing UQCRC1 (UQ) and Tsen34p (Tsen). Figure 3C: accumulation of radiolabeled doxorubicin in the clones versus the parental cell line. The cells were incubated with radiolabeled doxorubicin for 1 hour or 24 hours. After that, the cells were washed three times with PBS, solubilized and the associated radioactivity counted. The numbers of DPM/10⁶ cells are graphed. The data represent the average of three determinations ± SE.

[0076] This data suggests that drug resistance mechanisms mediated by SOD, Topo-llα or P-gp are not affected by UQCRC1 or Tsen34p expression. Doxorubicin accumulation in the cells did not change between the three cell lines (panel C), suggesting that drug transporter associated mechanisms were not affected by UQCRC1 or Tsen34p. These two genes appear to protect cells against stress through mechanisms that are not yet known. [0077] Figure 4 shows a graph and electrophoretic images depicting how expression of Tsen34p (Tsen 34 protein) inhibits senescence mediated by cellular exposure to doxorubicin. Figure 4A shows cell proliferation in the absence or in the presence of the senescence-inducing concentration of doxorubicin (5x10^"8 M). Parental SKNSH (SK) and the clone expressing Tsen34p (Tsen) were subjected to treatment with doxorubicin and subsequently, the cell number was counted every day for up to 5 days. Figure 4B shows how doxorubicin-induced expression of the cell cycle inhibitor p21/WAF1 was compared between parental cells (SK) and the clone expressing Tsen34p (Tsen). Cells were treated with 5x10⁸ M doxorubicin for 24 hrs. After that, proteins were extracted, separated by electrophoresis and probed by Western blot using antibody specific for p21 A/VAF1. An antibody to beta actin (β-actin) was used as a loading control.

[0078] The data in Figure 4 show that, while doxorubicin treatment of the parental cells (SK) results in complete arrest of proliferation (panel A), the Tsen-expressing clone continued to proliferate under these conditions, suggesting that this clone was able to escape from drug-induced senescence. In support of this, expression of p21/WAF1 (panel B), which represents a common molecular marker for senescence, was up-regulated in parental cells treated with doxorubicin but not in the clone expressing Tsen34p. [0079] The above examples demonstrate how the present invention can be used for identification of genes that may be involved in senescence and in senescence-associated diseases.

[0080] It is to be understood that this invention is not limited to the particular devices, methodology, protocols, subjects, or reagents described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the claims. Other suitable modifications and adaptations of a variety of conditions and parameters normally encountered in molecular biology, medical diagnostics, and clinical prevention and therapy, obvious to those skilled in the art, are within the scope of this invention. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

Claims

CLAIMSWhat is claimed is:

1. A method for identification of anti-senescence genes comprising: generating a cDNA library from mRNA isolated from stress-resistant cells; transfecting a population of stress-sensitive cells with the library to obtain transfected stress-sensitive cells; conducting a functional screen for senescence-related genes on the transfected stress-sensitive cells to identify stress-sensitive cells that have escaped senescence; and identifying cDNA inserts from the stress-sensitive cells that have escaped senescence.

2. The method of claim 1 , wherein the stress-resistant cells possess intrinsic stress resistance and are selected from the group consisting of stem cells, fibroblasts, hepatocytes, intestinal cells, and cancer cells.

3. The method of claim 2, wherein the stress-resistant cells are cancer cells.

4. The method of claim 1 , wherein the stress-resistant cells are engineered to be stress-resistant and are selected from the group consisting of stem cells, fibroblasts, hepatocytes, intestinal cells, and cancer cells.

5. The method of claim 1 , wherein the stress resistance is induced with chemical, physical, or biological agents that are applied in series or simultaneously.

6. The method of claim 1 , whereiη the stress-sensitive cells are stress-sensitive somatic cells, stem cells, or cancer cells.

7. The method of claim 1 , wherein the cDNA library comprises an expression system that is based on an inducible promoter.

8. The method of claim 1 , wherein the cDNA library comprises a selectable gene.

9. The method of claim 8, wherein the selectable gene is neomycin phosphotransferase, hygromycin, puromycin, G418 resistance, histidinol dehydrogenase, Sh ble, or dihydrofolate reductase.

10. The method of claim 1 , wherein the cDNA library comprises an expression system that is based on a repressible promoter.

11. The method of claim 1 wherein the functional screen comprises inducing of senescence with chemical, physical, or biological senescence- inducing agents that are applied in series or simultaneously.

12. The method of claim 1 , wherein the cells that have escaped senescence are cancerous.

13. The method of claim 1 , wherein the cells that have escaped senescence are non-cancerous.

14. A method for identification of anti-senescence genes comprising: obtaining mRNA from stress-resistant cells; generating cDNA from the mRNA; generating a library comprising the cDNA operably linked to an inducible expression control system and a selectable gene; introducing the library into a population of stress-sensitive cells; culturing the stress-sensitive cells in the presence of an appropriate selection agent and under conditions that induce senescence; isolating stress-sensitive cells that have escaped senescence; and analyzing the sequences of the cDNA in the isolated cells, thereby identifying anti-senescence genes.

15. The method of claim 14, wherein the stress-resistant cells are stem cells, fibroblasts, hepatocytes, intestinal cells, or cancer cells.

16. The method of claim 14, wherein the stress-resistant cells are cancer cells.

17. The method of claim 14, wherein the stress-sensitive cells are somatic cells or stem cells.

18. The method of claim 14, wherein the inducible expression control system comprises an inducible promoter.

19. The method of claim 14, wherein the inducible expression control system comprises a repressible promoter.

20. The method of claim 14 wherein the cells expressing the cDNA are cancerous.

21. The method of claim 14, wherein the cells expressing the cDNA are non-cancerous.

22. The method of claim 14, wherein the selectable gene is neomycin phosphotransferase, hygromycin, puromycin, G418 resistance, histidinol dehydrogenase, Sh ble, or dihydrofolate reductase.