WO2004048520A2 - Redd1, a transcriptional target of p63 and p53, and methods of use therefor - Google Patents

Abstract

Identification of a novel shared transcriptional target, termed REDD1, that implicates ROS in the p53-dependent DNA damage response and in p63-mediated regulation of epithelial differentiation is disclosed.

Description

REDDl, A TRANSCRIPTIONAL TARGET OF P63 AND P53, AND METHODS OF USE THEREFOR

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 60/428,331, filed on November 21, _.2002. The entire teachings of the above application are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant K08 CA82831- 03 from the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The tumor suppressor gene TP53 encodes a transcriptional activator that functions as a key nodal point for integrating cellular responses to genomic damage (reviewed in Nogelstein et al., 2000). While p53 is largely dispensable for normal development, its disruption is a common event in human cancers. The role of p53 as a guardian of genomic integrity is supported by its activation following ionizing radiation and inappropriate cellular proliferation signals, and by the development of tumors in TP53-null mice and in humans with Li-Fraumeni Syndrome (Giaccia and Kastan, 1998). Ectopic expression of p53 triggers both cell cycle arrest and apoptosis in cultured cells. Gi phase arrest has been linked primarily to transcriptional activation of the cyclin-dependent kinase inhibitor p21^Cipl (Deng et al., 1995), while a number of candidate target genes have been implicated inp53-dependent apoptosis, including Box, TRAIL-DR5, PERP, Noxa, ΑnάAPAF-1 (Miyashita and Reed, 1995; Attardi et al., 2000; Oda et al., 2000; Takimoto and El-Deiry, 2000; Moroni et al., 2001). Additional p53 transcriptional targets are thought to contribute to apoptosis by regulating cellular redox status following DNA damage (Polyak et al., 1997; Li et al., 1999). Still others contribute to diverse cellular processes such as DNA repair, genome stability, angiogenesis and regulation of p53 turnover (El-Deiry, 1998; Tanaka et al., 2000).

The discovery of two IP53-related genes in mammals, TP63 and TP73, has provided new insight into the evolutionary function of this gene family (Irwin and Kaelin, 2001). TP63 appears to be the most ancient family member, and it is most closely related to the single gene present in Drosophila (Yang et al., 1998). In contrast to TP53, neither TP63 nor TP73 appears to be targeted by mutations in human cancer, although some links to the DNA damage response pathway have been demonstrated (Gong et al., 1999; Yuan et al., 1999; Flores et al., 2002). However, both p73 and p63 play essential roles in development, the former being essential for brain development, and the latter for formation of epithelial structures, including skin, limbs, hair follicles and mammary glands (Mills et al., 1999; Yang et al., 1999; Yang et al., 2000). Notably, p63 is expressed in primitive epithelial precursors, and TP63- null mice display an apparent depletion of epithelial stem cell reserve leading to severe developmental defects. While p63 -mediated regulation of cell cycle progression and apoptosis has been demonstrated in cultured cells, the mechanism by which this transcription factor modulates cellular differentiation is unknown (Yang et al., 1998; Dohn et al., 2001).

A fundamental question regarding p53 family members is whether their diverse physiological functions reflect tissue-specific differences in expression, or regulation of distinct sets of transcriptional targets. The DNA binding domain of p63 is highly homologous to that of p53, and ectopic overexpression of p63 leads to transcriptional activation of a subset of known p53 targets (Dohn et al., 2001). However, it is unclear whether these genes are physiologically regulated by p63. A further level of complexity stems from the multiple splicing variants derived from the TP63 gene. The N-terminal splice variant that is most similar to p53, TAρ63γ, mediates transcriptional activation in promoter-reporter assays, but it represents a very small subset of the cellular p63 transcript (Yang et al., 1998). The most abundant isoform, ΔNp63α, lacks the transactivational domain (Parsa et al., 1999). This truncated form has been postulated to function as a dominant negative protein, capable of inhibiting expression of p63 target genes, as well as targets of p53 and p73, through its heterotypic interaction with these family members (Yang et al., 1998; Gaiddon et al., 2001). As such, it has been uncertain whether physiological expression of p63 during normal development leads to induction or repression of specific target genes. In either case, the functional properties of p53 and p63-responsive gene products provide important insight into the cellular pathways regulated by these transcription factors.

SUMMARY OF THE INVENTION

As described herein, REDDl has been identified as a novel transcriptional target of p53 induced following DNA damage. During embryogenesis REDDl expression mirrors the tissue-specific pattern of the p53 family member p63, and TP63-τaxll embryos show virtually no expression of REDDl, which is restored in mouse embryo fibroblasts following p63 expression. In differentiating primary keratinocytes, TP63 and REDDl expression are coordinately downregulated, and ectopic expression of either gene inhibits in vitro differentiation. REDDl appears to function in the regulation of reactive oxygen species (ROS); it is shown herein that TP63-mxll fibroblasts have decreased ROS levels and reduced sensitivity to oxidative stress, which are both increased following ectopic expression of either TP63 or REDDl. Thus, REDDl encodes a shared transcriptional target that implicates ROS in the p53-dependent DNA damage response and in p63-mediated regulation of epithelial differentiation.

Accordingly, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1; the complement of SEQ ID NO: 1; a nucleic acid molecule which is at least about 60% identical to the nucleotide sequence SEQ ID NO: 1; a nucleic acid molecule which is at least about 60% identical to the nucleotide sequence of the complement of SEQ ID NO: 1; a nucleic acid molecule which hybridizes under high stringency conditions to the nucleotide sequence selected of SEQ ID NO: 1; and a nucleic acid molecule which hybridizes under high stringency conditions to the nucleotide sequence of the complement of SEQ ID NO: 1. In particular, the nucleic acid molecule is expressed in human cells. In one embodiment, the nucleic acid molecule is DNA. In another embodiment, expression of said nucleic acid molecule in a cell increases reactive oxygen species in said cell.

The invention further relates to an isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1; the complement of SEQ ID NO: 1 ; a portion of the nucleotide sequence of SEQ ID NO: 1, wherein the portion is at least about 10 nucleotides in length; a portion of the nucleotide sequence of the complement of SEQ ID NO: 1, wherein the portion is at least about 10 nucleotides in length; a portion of the nucleotide sequence of SEQ ID NO: 1, wherein the portion is at least about 20 nucleotides in length; and a portion of the nucleotide sequence of the complement of SEQ ID NO: 1, wherein the portion is at least about 20 nucleotides in length. The invention further relates to nucleic acid constructs comprising the nucleic acid molecules of the invention, and more particularly to nucleic acid constructs in which the isolated nucleic acid molecule is operatively linked to a regulatory sequence. The invention also pertains to recombinant host cells comprising the nucleic acid molecules or constructs of the invention. The invention also relates to methods for preparing a polypeptide encoded by an isolated nucleic acid molecule of the invention, comprising culturing the recombinant host cells of the invention.

In another embodiment, the invention relates to isolated polypeptides encoded by a nucleic acid molecule according to the invention. In one embodiment, the isolated polypeptide comprises SEQ ID NO: 2 or a functional portion thereof. The invention further pertains to an antibody, or an antigen-binding fragment thereof, which selectively binds to a polypeptide of the invention or to a portion of said polypeptide.

The invention further encompasses a method of assaying for the presence of a nucleic acid molecule in a sample, comprising contacting said sample with a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 ; the complement of SEQ ID NO: 1; a portion of SEQ ID NO: 1 which is at least 10 nucleotides in length; and a portion of the complement of SEQ ID NO: 1, which is at least 10 nucleotides in length, under conditions appropriate for selective hybridization, wherein hybridization of said nucleotide sequence to said sample is indicative of the presence of a nucleic acid molecule in said sample.

The invention also relates to a method of assaying for the presence of a polypeptide of the mvention in a sample, comprising contacting said sample with a compound, e.g., an antibody, which specifically binds to the polypeptide.

The invention also pertains to a method of inducing expression of REDDl in a cell comprising inducing expression of p53, whereby expression of REDDl is induced, as well as to a method of inducing expression of REDDl in a cell comprising inducing expression of p63, whereby expression of REDDl is induced. In one embodiment, the cell is an undifferentiated cell. With regard to this method and other methods of the invention, the phrase "inducing expression of shall include actual initiation of or increase in expression of a gene transcript as well as initiation of or increase in translation of a nucleic acid molecule into a protein. In addition, enhancement of a particular component will include both quantitative and qualitative increase in the level or activity of the component. Furthermore, inhibition of a particular component will include both quantitative and qualitative reduction in the level or activity of the component.

The invention also relates to a method of inducing activation of the ROS pathway in a cell, comprising inducing expression of REDDl . In one embodiment, expression of REDDl is induced by p53, and in another embodiment expression of REDDl is induced by p63. In a preferred embodiment, activation of the ROS pathway results in increased level of ROS. In one embodiment, activation of the ROS pathway results in increased sensitivity to oxidative stress. Another aspect of the invention relates to a method of causing cell death comprising inhibiting expression of REDDl in said cell; preferably said cell is a cancer cell.

The invention also relates to a method of inhibiting differentiation of a cell comprising inducing expression of REDDl in said cell; preferably said cell is an epithelial precursor cell. The invention further relates to a method of enhancing cell death in response to oxidative stress, comprising enhancing expression of p63 in said cell, thereby enhancing susceptibility of said cell to oxidative stress, as well as to a method of enhancing cell death in response to oxidative stress, comprising enhancing expression of REDDl in said cell, thereby enhancing susceptibility of said cell to oxidative stress. In a preferred embodiment, cell death is mediated via the reactive oxygen species pathway. In another embodiment, the invention relates to a method of inhibiting cell death in response to oxidative stress, comprising inhibiting expression of p63 and/or REDDl in said cell, thereby reducing susceptibility of said cell to oxidative stress.

The invention also relates to a method of enhancing cell death in hypoxic cells, comprising inhibiting expression of REDDl in said cells. In one embodiment the hypoxic cells are proliferating cancer cells.

In another embodiment, the invention is drawn to a method of enhancing p53- induced cell death comprising enhancing expression of REDDl in said cell. In one embodiment, the cell is a nascent cancer cell or an established cancer cell. In one embodiment, the cell has reduced p53 expression. The invention also relates to a method of inhibiting the progression of cancer in an individual, comprising enhancing expression of REDDl in cancer cells in said individual, whereby death of said cancer cells is enhanced.

The invention also pertains to a method of inhibiting differentiation of a cell comprising inducing expression of the reactive oxygen species pathway in said cell. Preferably, the cell is an epithelial precursor cell.

The mvention also relates to a method of enhancing differentiation of a cell, comprising inhibiting expression of REDDl in said cell. In one embodiment, the cell is an epithelial cell. In one embodiment, the cell is a nascent cancer cell.

The invention also pertains to a method of decreasing abnormalities in breast cells, comprising inhibiting expression of REDDl or p63. The invention also relates to a method of treating breast disorders in an individual, comprising inhibiting expression of REDDl or p63 in cells of the breast of the individual. In a preferred embodiment, the breast disorder is breast cancer.

The invention also relates to a transgenic animal comprising a single germline copy of a DNA construct comprising the REDDl promoter region operably linked to a reporter gene. The reporter gene can be, for example, a β-gal/neomycin-resistance fusion gene. In one embodiment, an IRES is linked to the reporter gene. Preferably the animal is a mouse. The invention also relates to an isolated cell from the transgenic animal, as well as to methods of assaying for p53 and/or p63 activity utilizing said animals or isolated cells. These methods, animals and cells can also be used to identify agents which alter the expression or activity of p53, p63 and/or REDDl in accordance with methods known in the art. For example, an agent to be tested can be administered to said animal, and the activity of p53, p63 and/or REDDl can be assessed in the presence and absence of the agent. Alternatively, an isolated cell from said animal can be contacted with an agent to be tested, and the activity of p53, p63 and/or REDDl in said cell can be assessed in the presence and absence of the agent.

The invention also relates to a nucleic acid construct comprising the REDDl promoter region operably linked to a reporter gene. In one embodiment, the reporter gene is the beta galactosidase gene. The invention also relates to a recombinant host cell comprising such a nucleic acid construct. The invention also relates to a method of diagnosing a disorder associated with aberrant expression of REDDl in an individual, comprising assessing REDDl expression in an individual, wherein aberrant expression of REDDl as compared with expression in a normal individual is indicative of a disorder associated with aberrant expression of REDDl . In a preferred embodiment, the disorder is a proliferative disorder, e.g., cancer. In one embodiment, assessment of REDDl expression is performed by assessing the level of REDDl transcript. In another embodiment, assessment of REDDl expression is performed by assessing the level of REDDl protein. In a further embodiment, assessment of REDDl expression is performed by assessing the level of REDDl function, e.g., activation of the ROS pathway. The mvention further pertains to a method of identifying an agent which alters

REDDl expression, comprising contacting a cell comprising REDDl with an agent to be tested, assessing expression of REDDl in the presence of the agent as compared with expression of REDDl in the absence of the agent, and identifying an agent which alters REDDl expression. In one embodiment, assessment of expression of REDDl is performed by assessing the level of REDDl transcript. In another embodiment, assessment of expression of REDDl is performed by assessing the level of REDDl protein. In a further embodiment, assessment of expression of REDDl is performed by assessing the level of REDDl function, e.g., activation of the ROS pathway.

The invention also encompasses a method of identifying an agent which alters REDDl expression, comprising contacting a cell comprising a nucleic acid construct comprising the promoter region of the REDDl gene operably linked to a reporter gene with an agent to be tested, assessing expression of the reporter gene in the presence of the agent as compared with expression of the reporter gene in the absence of the agent, and identifying an agent which alters expression of the reporter gene, wherein an agent which alters expression of the reporter gene is an agent which alters REDDl expression. In one embodiment of the method, the cell is isolated from a transgenic animal.

The invention also relates to a method of identifying an agent which alters REDDl expression, comprising administering an agent to be tested to an animal, assessing expression of REDDl in the presence of the agent as compared with expression of REDDl in the absence of the agent, and identifying an agent which alters expression of REDDl. Assessment of REDDl expression can be performed, for example, by assessing the level of REDDl transcript, assessing the level of REDDl protein, or assessing the level of REDDl function, e.g., activation of the ROS pathway. The invention also relates to a method of identifying an agent which alters

REDDl expression, comprising administering an agent to be tested to a transgenic animal comprising a nucleic acid construct comprising the promoter region of the REDDl gene operably linked to a reporter gene, assessing expression of the reporter gene in the presence of the agent as compared with expression of the reporter gene in the absence of the agent, and identifying an agent which alters expression of the reporter gene, wherein an agent which alters expression of the reporter gene is an agent which alters expression of REDDl . BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1B demonstrate induction of REDDl following multiple forms of DNA damage. Figure 1A shows induction of REDDl following expression of mutant hTERT (hTERT-DA). Western blot analysis of lysates from cells with tetracycline (tet)-repressible expression of wild-type hTERT or hTERT-DA, demonstrating inducible expression of the transfected construct (top panel). Induction of REDDl mRNA 24 hrs following tetracycline withdrawal is shown by Northern blot (middle panel), with loading control (EtBr). The table summarizes the results of microarray analysis and northern blot validation by densitometry for the three most highly- induced sequences following hTERT-DA expression. Figure IB shows Northern blot analysis of primary diploid fibroblasts, either untreated (-), or 12 hours following lOGy ionizing radiation (IR), lOOμg/ml methyl-methane sulphonate (MMS), or 20J/m² ultraviolet radiation (UV). The same blot was sequentially probed with REDDl, p21^Cipl and GADD45 (EtBr, loading control).

Figures 2A-2C demonstrate that REDDl is a transcriptional target of p53. Figure 2A shows Northern blot analysis of 7P53-null primary fibroblasts from two patients with Li-Fraumeni syndrome (L3819 and L4402) and of ATM-nail cells from two patients with ataxia telangiectasia (AT960 and AT960) following DNA damage as in Figure 1. Induction of REDDl by IR is abrogated, while its induction following treatment with MMS is unaltered. Figure 2B shows Northern blot demonstrating expression of endogenous REDDl mRNA in Saos-2 and U2OS cells, following transient transfection with a p53 expression construct (p53) or vector control (N) (top panel). Expression of endogenous REDDl in U2OS cells with constitutive expression of temperature-sensitive p53 (U2tsp53) is shown following growth at 37°C (primarily mutant conformation) or temperature shift to 32°C (wild-type p53). No effect is seen with temperature shift in parental U2OS cells (middle panel). The fold-induction of REDDl following temperature shift in U2tsp53 cells is unaltered by treatment of cells with lOμg/ml cycloheximide (Chx), indicating that induction is independent of new protein synthesis (lower panel). Treatment with Chx itself induces an increase in baseline REDDl expression. Equal loading was demonstrated for all lanes (not shown). Figure 2C shows activation of a REDDl promoter reporter following transient transfection into U2,-tsp53 cells and shift to the permissive temperature (wild-type p53). Schematic representation of the p53 responsive sequence (p53RE, black box), located within a 0.6kb HindlH/Sacl fragment (0.6H/S), upstream of the transcriptional start (arrowhead). The three exons encoding REDDl are denoted by white boxes. Activation of this reporter by p53 is abrogated following disruption of the p53RE by four point mutations (0.6-mut). As further control, an adjacent 3.0kb Hindlll promoter fragment (3. OH) demonstrates no induction by p53, nor does vector backbone. Relative luciferase reporter activity is plotted with standard error indicated.

Figures 3A-3C evidence characterization of the REDDl protein. Figure 3 A shows the amino acid sequence alignment of human REDDl with its orthologs in mouse (mREDOl), Xenopus (xREDDl), and Drosophila (dREDDl and dREDD2, also known as Scylla and Charybde, respectively). Black shading represents identical residues, and grey shading conservative changes. Figure 3B shows immunoprecipitation-Western blot analysis of endogenous REDDl in U20S cells, either untreated (-), or 12 hrs following 10 Gy IR or lOOμg/ml MMS, using affinity- purified REDDl polyclonal antiserum. Migration of immunoglobulin heavy-chain (HC) and light chain (LC) are noted. Figure 3C shows cytoplasmic localization of REDDl protein in U2OS cells with constitutive expression of a REDDl construct, as shown by immunofluorescence using affinity-purified REDDl antiserum.

Figures 4A-4D demonstrate that REDDl expression parallels that of p63 during development. Figure 4 A shows the construction of the REDDl -beta galactosidase (β-gal) promoter reporter construct, which was synthesized by replacing the coding and 3' untranslated regions (hatched boxes) with the β-gal/neomycin fusion cDNA. This reporter was inserted by homologous recombination downstream of the endogenous REDDl locus (see Methods). Figure 4B shows β-gal activity in whole-mount embryos carrying one copy of the reporter at embryonic day 11 (El 1) or day 13.5 (El 3.5). Staining of the apical ectodermal ridge (AER) of the limb bud (LB), the branchial arches (BA), mammary primordia (MP), and developing follicles of the whisker pad (WP) are observed. Figure 4C demonstrates colocalization of p63 protein and REDDl-β-gal within the AER in tissue sections of limb bud from E9.5 embryos (top panels). Like p63, REDDl promoter is expressed in cells of the primitive ectoderm (PE; E9.5), and the root sheath (RS) of the developing hair follicle (lower panels; newborn). Figure 4D shows RNA in-situ hybridization of whole- mount E14.5 wild-type (+/+) or TP63-wxll (-/-) embryos, using a REDDl anti-sense probe. Like p63, REDDl mRNA expression is normally present at this stage in supraorbital and suborbital follicles (SF), whisker pad, limbs, and patches of developing epidermis (EP) that cover the embryo. These structures, along with REDDl expression, are absent in TP63-rmU embryos. No staining of wild-type embryos was observed with the control sense REDDl probe (not shown).

Figures 5A-5C show transcriptional regulation of REDDl by p63. Figure 5A shows Northern blot analysis of TP63, REDDl, p21^Cipl and GADD45 expression, at sequential intervals following the induction of differentiation in human primary keratinocytes by a change in calcium concentration. Involucrin expression is a marker for keratinocyte differentiation. The same blot was hybridized sequentially with these probes, and GAPDH expression is shown as a loading control. Figure 5B shows Western blot analysis of lysates from TP 63 -null (-/-) mouse embryo fibroblasts (MEFs) infected with the indicated adenoviral constructs and probed with monoclonal anti-p63 antibody (top panel). ΔNp63 (upper arrow) contains an additional C- terminal domain, explaining its slower migration than TAp63γ (lower arrow). Northern blot analysis (lower panels) from either TP63-heterozygous (+/-) or TP63- null (-/-) MEFs, demonstrating that baseline expression of REDDl is reduced in EPό^'3-null MEFs, and restored following adenoviral expression of TAp63γ or ΔNp63α. Note that ~10-fold more ΔNp63α than TAp63γ is required to induce equivalent levels of REDDl. Figure 5C demonstrates activation of REDDl promoter by both TAp63γ and ΔNp63α isoforms of p63. Promoter reporter constructs encoding the wild-type p53RE (0.6H/S) or mutant sequence (0.6-Mut) were transfected into U20S cells, along with the indicated p63 isoform or vector. The 0.6-Mut fragments lacks the 20-bp p53 consensus sequence. Bars show standard error from three independent experiments. Figures 6A-6B show inhibiton of keratinocyte differentiation by REDDl and p63. Figure 6A shows adenoviral expression of REDDl, TAp63γ (lower arrow) and ΔNp63 (upper arrow) in primary human keratinocytes. Lysates from cells infected with the indicated constructs were probed with affinity-purified polyclonal anti-REDDl antibodies or with monoclonal anti-p63. Endogenous ΔNp63α is detectable in undifferentiated cells (asterisk), and migrates slightly faster than epitope-tagged ectopically expressed ΔNp63α. Figure 6B shows Northern blot analysis of primary human keratinocytes infected with the indicated adenoviral constructs. Following infection, cells were left uninduced (U) or were induced (I) to differentiate by calcium shift. Induction of the differentiation marker involucrin (lanes 1 and 3; 7 and 8) is inhibited by REDDl (lanes 2 and 4) and TAp63γ (lanes 9 and 10) but not by ΔNp63α (lanes 11 and 12), while addition of the anti-oxidant NAC abrogates REDDl -mediated inliibition of differentiation (lanes 5 and 6).

Figures 7A-7C demonstrate regulation of ROS and oxidative stress sensitivity by p63 and REDDl. Figure 7A shows levels of intracellular ROS in I O^'S -heterozygous (+/- ) and TP63-null (-/-) primary and transformed MEFs, as assessed by the fluorescent indicator dye CM-H₂DCFDA. The difference in baseline ROS between TP63- heterozygous and null MEFs is comparable in magnitude to the ROS induced following acute treatment with 0.8mM H₂O₂ (lower panels). Similar differences in ROS levels were observed between wild-type and TP-63 null MEFs. Cellular transformation alters baseline ROS levels but does not change the difference in ROS observed between TPo^'3-heterozygous and null cells. Figure 7B shows induction of ROS following peroxide challenge. TP63-mιll MEFs were infected with the indicated adenoviral constructs, followed by treatment with 0.4mM H₂O₂ for 12 hours in the presence or absence of lOmM NAC. ROS induction was quantitated by CM- H₂DCFDA fluorescence (increase in mean fluorescence intensity). Expression of either REDDl or TAp63γ markedly enhances ROS induction, which is blocked by NAC. Error bars denote standard error for three independent experiments. Figure 7C demonstrates cellular sensitivity to oxidative stress. IPtfS-heterozygous or null MEFs were infected with the adenoviral constructs indicated, then treated with 0.8mM H₂O₂ in the presence or absence of lOmM NAC for 24 hours. Percent viable cells is shown compared to cultures without H₂O₂ treatment. TP63-mϊll MEFs are resistant to oxidative stress, and sensitivity is restored following expression of either REDDl or TAp63γ; NAC abrogates increased oxidative stress sensitivity mediated by REDDl or TAp63γ. Error bars denote standard error for at least three independent experiments.

Figures 8A-8B show the nucleic acid sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of REDDl.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows. REDDl (GenBank Accession No. AY090097) was identified here by expression profile analysis as a transcript that is dramatically upregulated following telomere disruption. This finding is consistent with the emerging parallels between pathways involved in telomere maintenance and response to DNA damage (Blackburn, 2000). In this setting, REDDl is activated to a greater extent than other well established DNA damage response genes, p21^Cipl and GADD45, and it exhibits a distinct induction profile in response to multiple DNA damage stimuli. Induction of endogenous REDDl following IR is dependent upon the presence of ATM and p53, placing it downstream of p53 within this well characterized DNA damage response pathway. Like p21^cipl and GADD45, REDDl is induced both by p53-dependent and independent pathways following DNA damage (Loignon et al., 1997; Jin et al., 2001). Consistent with recent observations that p63 participates in p53 -mediated DNA damage responses (Flores et al., 2002), TP63-null cells show decreased REDDl induction after ionizing radiation, but not MMS treatment (not shown). Characterization of additional elements within the REDDl promoter that mediate its particular DNA damage-induced transcriptional profile will require further analysis. In addition to the consensus p53-binding sequence, the proximal promoter of REDDl contains a functional heat shock element (HSE) (data not shown), and REDDl was also recently identified as RTP801, a gene induced by the hypoxia-inducible factor HIF-1 (Shoshani et al., 2002). The observations suggest that REDDl may be induced in response to multiple cellular stresses.

Transcriptional targets of p53 identified to date are thought to mediate several DNA-damage regulated processes, including cell cycle arrest, apoptosis, and regulation of p53 turnover. p53 is known to induce a number of genes implicated in cellular redox control, which ultimately contribute to p53 -mediated apoptosis via the mitochondrial apoptotic cascade (Polyak et al., 1997; Li et al., 1999). The direct transcriptional induction of REDDl by p53 underscores the existence of p53- dependent pathways that regulate ROS. Expression of REDDl alone is insufficient to induce apoptosis in fibroblasts, but reduced REDDl levels are associated with resistance to oxidative stress, while REDDl expression increases cellular sensitivity to lethal oxidative stress. In addition, expression of RTP801 [REDDl] maybe sufficient to induce apoptosis in some cellular contexts (Shoshani et al., 2002). Determining the precise contribution of REDDl to p53-dependent apoptotic functions will require studies in cells with targeted inactivation, since the high levels of REDDl transcript induced by DNA damage have prevented efficient suppression using RNA interference strategies (not shown).

Unlike p53, the functional pathways regulated by p63 and its native transcriptional targets are poorly understood. The striking colocalization of REDDl expression with p63 in the apical ectodermal ridge and in developing epithelial structures first pointed to a potential regulatory interaction. The highly restricted expression pattern shared by these two genes is also evident at the single cell level, with both present in the primitive ectoderm during mid-gestation, and later in the maturing epidermis and hair follicles (Parsa et al., 1999). Underscoring this similarity, REDDl is virtually undetectable in EPό^'3-null mouse embryos. The reduced REDDl levels in these embryos may result directly from loss of p63-mediated transcription, or may be an indirect consequence of epithelial differentiation abnormalities. Evidence supporting direct transcriptional activation of REDDl by p63 includes its transactivation of the REDDl promoter, coregulation of the two endogenous transcripts during in vitro differentiation of primary keratinocytes, and most significantly, restoration of baseline REDDl expression in TP63- ύl MEFs following adenoviral reconstitution of p63 expression. Of note, neither the dramatically decreased expression of REDDl in EPo^'3-null MEFs, nor the biphasic expression pattern during in vitro keratinocyte differentiation are observed with other p53- induced genes such asp21^CipI and GADD45. Regulation of REDDl by p63 therefore appears unique in defining a physiological interaction during epithelial differentiation. The transcriptional program activated by p63 is likely to underlie its regulation of epithelial differentiation. The phenotype of TP63-nυll mice has been explained either as a failure of basal stem cells to undergo differentiation, or as an initial wave of differentiation coupled with failure of maintenance and renewal of basal stem cells, leading to depletion of the basal stem cell pool (Mills et al., 1999; Yang et al., 1999). The latter hypothesis is based on the presence of differentiated epithelial structures in early embryos from these mice, followed by disappearance of the basal cell layer. While these studies of REDDl do not distinguish between these possibilities, the coexpression of p63 and REDDl in the undifferentiated basal cell layer, and their ability to inhibit keratinocyte differentiation point to a role in preserving undifferentiated basal cells. The mechanism underlying this effect is likely to be complex, but the observations described herein suggest a role for regulation of reactive oxygen species.

Results of work described herein show that TP63-null cells have significantly decreased levels of ROS and reduced sensitivity to oxidative stress, while ectopic expression of either TAp63γ or REDDl enhances ROS induction and restores sensitivity. In contrast, ΔNp63α did not regulate ROS or oxidative stress sensitivity, which may in part reflect its relatively weak ability to induce REDDl. Taken together, these studies implicate REDDl as a mediator of p63-dependent redox regulation, although the mechanism by which REDDl modulates intracellular ROS is_\ unknown. Direct generation of ROS by REDDl seems unlikely, since the REDDl protein does not possess homology to known cellular oxidative enzymes, and it does not appear to localize to sites of ROS production within mitochondria. Further insights may be derived from characterization of the specific reactive species generated by REDDl, since treatment of keratinocytes by H₂O₂ alone does not replicate the effect of REDDl on cellular differentiation (not shown). The identification of REDDl -RTP801 as a downstream target of both ρ53 and HIF-1 suggests that it may in fact function in concert with other redox-regulatory genes known to be induced by these two transcription factors.

The finding that loss of p63 alters cellular ROS levels may provide one mechanism by which p63 modulates differentiation and interacts functionally with other factors involved in epithelial development. In addition to effects on cellular stress and viability, subtle shifts in intracellular ROS levels have recently been shown to modulate cellular signaling through multiple tyrosine kinase growth factor receptors, including the epidermal growth factor receptor (EGF-R) (Bae et al., 1997; Finkel, 2000; Meng et al., 2002). Regulation of EGF-dependent responses by ROS may therefore contribute to the effect of p63 on keratinocyte differentiation in vitro, as well as the failure of epidermal differentiation in TP63 -null mice. In this regard, another growth factor, fibroblast growth factor-8 (FGF-8), is of particular interest, since it is specifically co-expressed with p63 and REDDl in the AER and is critical for induction of limb development (Capdevila and Izpisua Belmonte, 2001). Taken together, these data raise the possibility that regulation of ROS levels by p63 plays a role in its modulation of epithelial differentiation.

As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof. An individual is not limited to a human being but may also be other organisms including but not limited to mammals, plants, bacteria, or cells derived from any of the above.

Throughout this disclosure, various aspects of this invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. 1-W), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3^rd Ed., W.H. Freeman Pub., New York, NY and Berg et al. (2002) Biochemistry, 5^th Ed., W.H. Freeman Pub., New York, NY, all of which are herein incorporated in their entirety by reference for all purposes.

The present invention can employ solid substrates, including arrays in some preferred embodiments. Methods and techniques applicable to polymer (including protein) array synthesis have been described in U.S.S.N 09/536,841, WO 00/58516, U.S. Patents Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730 (International Publication Number WO 99/36760) and PCT/USO 1/04285, which are all incorporated herein by reference in their entirety for all purposes.

Patents that describe synthesis techniques in specific embodiments include U.S. Patents Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189, 5,889,165, and 5,959,098. Nucleic acid arrays are described in many of the above patents, but the same techniques are applied to polypeptide arrays. Nucleic acid aπays that are useful in the present invention include those that are commercially available from Affymetrix (Santa Clara, CA) under the brand name GeneChip®. Example arrays are shown on the website at affymetrix.com.

The present invention also contemplates many uses for polymers attached to solid substrates. These uses include gene expression monitoring, profiling, library screening, genotyping and diagnostics. Gene expression monitoring, and profiling methods can be shown in U.S. Patents Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248 and 6,309,822. Genotyping and uses therefore are shown in USSN 60/319,253, 10/013,598, and U.S. Patents Nos. 5,856,092, 6,300,063, 5,858,659, 6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other uses are embodied in U.S. Patents Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and 6,197,506.

The present invention also contemplates sample preparation methods in certain preferred embodiments. Prior to or concurrent with genotyping, the genomic sample may be amplified by a variety of mechanisms, some of which may employ PCR. See, e.g., PCR Technology: Principles and Applications for DNA Amplification (Ed. HA. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, CA, 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (Eds. McPherson et al, IRL Press, Oxford); and U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188,and 5,333,675, and each of which is incorporated herein by reference in their entireties for all purposes. The sample may be amplified on the array. See, for example, U.S Patent No 6,300,070 and U.S. patent application 09/513,300, which are incorporated herein by reference.

Other suitable amplification methods include the ligase chain reaction (LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO88/10315), self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and WO90/06995), selective amplification of target polynucleotide sequences (U.S. Patent No 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR) (U.S. Patent No 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Patent No 5, 413,909, 5,861,245) and nucleic acid based sequence amplification (NABSA). (See, US patents nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporated herein by reference). Other amplification methods that may be used are described in, U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and in USSN 09/854,317, each of which is incorporated herein by reference. Additional methods of sample preparation and techniques for reducing the complexity of a nucleic sample are described in Dong et al., Genome Research 11, 1418 (2001), in U.S. Patent No 6,361,947, 6,391,592 and U.S. Patent application Nos. 09/916,135, 09/920,491, 09/910,292, and 10/013,598.

Methods for conducting polynucleotide hybridization assays have been well developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those refeπed to in: Maniatis et al. Molecular Clotting: A Laboratory Manual (2^nd Ed. Cold Spring Harbor, N.Y, 1989); Berger and Kimmel Methods in En∑ymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, CA, 1987); Young and Davism, P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying out repeated and controlled hybridization reactions have been described in US patent 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which are incorporated herein by reference.

The present invention also contemplates signal detection of hybridization between ligands in certain preferred embodiments. See U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,225,625, in U.S. Patent application 60/364,731 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes. Methods and apparatus for signal detection and processing of intensity data are disclosed in, for example, U.S. Patents Numbers 5,143,854, 5,547,839, 5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723, 5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030, 6,201,639; 6,218,803; and 6,225,625, in U.S. Patent application 60/364,731 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes. The practice of the present invention may also employ conventional biology methods, software and systems. Computer software products of the invention typically include computer readable medium having computer-executable instructions for performing the logic steps of the method of the invention. Suitable computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. The computer executable instructions may be written in a suitable computer language or combination of several languages. Basic computational biology methods are described in, e.g. Setubal and Meidanis et al., Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2^nd ed., 2001). The present invention may also make use of various computer program products and software for a variety of purposes, such as probe design, management of data, analysis, and instrument operation. See, U.S. Patent Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170. Additionally, the present invention may have preferred embodiments that include methods for providing genetic information over networks such as the Internet as shown in U.S. Patent applications 10/063,559, 60/349,546, 60/376,003, 60/394,574, 60/403,381.

The present invention is related to U.S. Patent application No. 60/391,870 which is herein incorporated by reference in its entirety.

The invention will be further illustrated by the following non-limiting examples. The present invention has many preferred embodiments and relies on many patents, applications and other references for details known to those of the art. Therefore, when a patent, application, or other reference is cited or repeated herein, it should be understood that it is incorporated by reference in its entirety for all purposes as well as for the proposition that is recited. EXAMPLES

MATERIALS AND METHODS

Cell Lines and Cell Culture

Saos-2 and U2OS are human osteosarcoma-derived cell lines. U2tsp53 is a U2OS cell line expressing the temperature-sensitive p53vall35 mutant. Human primary diploid fibroblasts and MEFs were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine calf serum (FBS). Primary human epidermal foreskin keratinocytes (HFK) were cultured in keratinocyte serum- free medium (Gibco/BRL) containing epidermal growth factor (EGF), bovine pituitary extract (BPE) and 0.4mM calcium as described (Dickson et al., 2000) and references therein). HFK differentiation was initiated by changing the calcium concentration of the growth media from 0.4 to 0.05 mM. U2OS-derived cell lines expressing inducible hTERT or hTERT-DA were created using the "tet-off system (Clontech).

Oligonucleotide array-base expression profiling

Gene expression profiles in inducible hTERT-DA cells were analyzed using Affymetrix Gene Chip microarrays as previously described (Ellisen et al., 2001b). Total RNA was isolated 12 and 24 hours after tetracycline washout, and was subjected to reverse transcription, labeling and hybridization as previously described (Lockhart et al., 1996). A five-fold cutoff was used to define significant message induction by microarray, and only genes whose induction was verified by Northern blot were analyzed further.

Cloning and Analysis of the REDDl Promoter

The human REDDl genomic locus was isolated from a BAG library (Research Genetics). Restriction enzyme fragments flanking and including the REDDl transcribed sequence were subcloned into the pGL-3 basic vector and screened for responsiveness to p53 or p63, as indicated using the Dual-Luciferase assay system (Promega). Secreted HGH or renilla expression (pRL-TK, Promega) was used as a transfection control. The mutant 0.6-mut and 0.6-Mut fragments were created by standard PCR-based site-directed mutagenesis procedures.

Targeted incorporation of the REDDl in vivo reporter A 14-kb mouse genomic fragment containing the REDDl transcription unit was isolated from a Lamda Zap II bacteriophage library (Stratagene). To generate the targeting vector, the entire REDDl coding region was excised and replaced with an IRES linked to the β-gal/neomycin-resistance fusion gene such that transcription is driven by the endogenous REDDl promoter. ES cell clones were screened for the presence of the targeting construct at the endogenous REDDl locus, and were found to have incorporated the construct immediately downstream of the intact REDDl transcription unit. Animals carrying a single germline copy of the inserted reporter were created using standard procedures (Hogan et al., 1994).

β-gal Staining and In Situ Hybridization of Whole Mount and Sectioned Embryos Whole-mount and frozen section staining for β-gal was carried out as described (Hogan et al., 1994). Immunohistochemical staining for p63 was performed using the 4A4 anti-p63 antibody as described (Yang et al., 1999). Whole-mount in situ hybridization was performed essentially as described (Wilkinson, 1992) using digoxigenin-labeled antisense and sense (control) mouse REDDl RNA probes. All embryos and sections were viewed by standard light microscopy.

Quantitation of Cellular ROS and Oxidative Stress Sensitivity

Intracellular levels of ROS were assayed using the fluorescent indicator dichlorofluorescein diacetate (CM-H₂DCFDA, Molecular Probes, Inc.). Cells were stained with lμg/ml of the indicator in serum-free medium for 30 minutes, then washed, trypsinized and analyzed immediately. Analyses were carried out using the FACScaliber flow cytometer and Cell Quest software (Becton Dickinson). For viability assays, following adenoviral infection sub-confluent cultures were left untreated or were treated with H₂0₂ for 24 hours, then cells were stained with 0.2% trypan blue and viable cells were counted in triplicate for each data point. Where indicated, cells were pretreated with NAC for 2 hours prior to addition of H₂O₂. Adenoviral-Based Gene Expression

REDDl, TAp63 and ΔNp63 cDNAs were subcloned into the pAdTrack-CMV vector and were co-transformed with the pAdEasy-1 plasmid into recombination- proficient BJ5183 cells (all from Stratagene). The γ splice variant of the TAp63 isoform and the α variant of ΔNp63 were used in these constructs, since they are the best characterized species. Resulting recombinants were transfected into 293 cells for viral production. Adenoviral stocks were titered using standard viral plaque assays and were used at an MOI of 50 for all infections, except as noted (Figure 5B). All cellular assays were initiated 24 hours following adenoviral infection.

RESULTS

REDDl mRNA is upregulated following multiple DNA damage stimuli

The initial objective of this work was to identify genes whose expression is upregulated following telomere destabilization, then to compare these with genes induced following other DNA damage stimuli. Tetracycline-regulated expression of the human telomerase catalytic subunit (hTERT) and a mutant with an aspartic acid to alanine substitution in the TERT enzymatic active site (hTERT-DA) were produced (Figure 1 A). The analogous mutant in yeast is catalytically inactive and provokes telomere-shortening when overexpressed (Lingner et al., 1997). As predicted, inducible expression of the mutant construct led to an increased frequency of telomere, fusions, accompanied by growth aπest and cell death, whereas expression of wild- type hTERT had no effect (data not shown). RNA samples were collected from cells at several time points following induction of hTERT-DA, labeled, and hybridized to oligonucleotide microaπays representing 5500 human genes and ESTs.

Hybridizations were performed in duplicate and results were verified using northern blot analysis (Figure 1 A). Only three genes were found to be reproducibly induced following expression of TERT-DA: the cyclin-dependent kinase inhibitor p21^c , the DNA damage responsive gene GADD45, and an EST that we have named REDDl. Induction of p21^cm and GADD45 following different DNA damaging agents is known to be mediated by distinct pathways (Lakin and Jackson, 1999). Therefore, the relative induction of these transcripts were compared with that of REDDl in primary human fibroblasts, using standard doses of ionizing radiation (IR), ultraviolet radiation (UN), or treatment with the DΝA alkylating agent Methyl Methane Sulphonate (MMS). Under these conditions, each gene showed a distinct pattern of induction: GADD45 was induced primarily by MMS and UV; p21^Cipl by IR, followed by MMS; and REDDl by MMS, followed by IR (Figure IB). Therefore REDDl, like these well-characterized DΝA damage responsive genes, is also a target of multiple pathways involved in the signaling of genomic injury.

REDDl is a direct transcriptional target of p53 The mechanism of p21^Cipl induction by MMS is not well understood, but its induction by IR has been used to define the classical DΝA damage response pathway, which involves ATM-dependent phosphorylation and stabilization of p53 (Morgan and Kastan, 1997). To determine whether the IR-mediated induction of REDDl is also dependent upon this pathway, REDDl expression was examined in human primary fibroblasts lacking either p53 or ATM. In L3819 and L4402, two independent EP53-null fibroblast lines derived from patients with Li-Fraumeni syndrome, induction of REDDl by IR was entirely absent, while induction in response to MMS was unchanged (Figure 2A). Identical results were observed with AT960 and AT582, two primary fibroblast lines derived from patients with ataxia telangiectasia (ATM- null). The induction of REDDl following IR is therefore dependent on an intact p53 pathway.

To determine whether ectopic overexpression of p53 alone leads to induction of REDDl, a CMV-driven p53 expression construct was transfected into Saos-2 cells, which lack endogenous p53, and into U2OS cells, in which endogenous p53 is destabilized by overexpression of MDM2. In both cell lines, expression of the endogenous REDDl transcript was induced following ectopic expression of p53 (Figure 2B). Induction of REDDl was also observed in cells with constitutive expression of a temperature-sensitive p53 mutant (U2-tsp53), following growth at the permissive temperature. Since the temperature switch allows activation of wild-type p53 without requiring protein synthesis, the question of whether induction of REDDl by p53 was independent of new protein synthesis, as expected for a direct transcriptional target, was tested. Indeed, while treatment with cycloheximide (Chx) increased baseline expression of REDDl, it did not alter the ~5-fold induction of endogenous REDDl mRNA by p53 (Figure 2B).

To establish that REDDl is a direct p53 transcriptional target, the putative promoter region within the compact (2kb) REDDl genomic locus was identified (Figure 2C). Luciferase reporter assays, using fragments of the REDDl promoter as well as intron sequences, mapped a single p53 -responsive element to a 0.6kb Hindlll/Sacl fragment, immediately upstream of the transcriptional start site. This element was induced ~7-fold following ectopic expression of p53 in Saos-2 cells or temperature switch in U2-tsp53 cells (Figure 2C). Within this element, a single consensus p53 binding site as defined by (El-Deiry et al., 1992) was identified.

Generation of a mutant construct by altering the four critical residues within the core consensus sequence, "Ca/t a/tG", abolished p53-mediated transactivation of the REDDl promoter (Figure 2C). Taken together, these observations suggest that REDDl is a direct transcriptional target of p53.

REDDl is a member of an evolutionarily conserved gene family.

The REDDl open reading frame encodes a predicted protein of 232 amino acids, with orthologs in mouse, Xenopus and Drosophila (Figure 3A). The amino acid sequence predicts an acidic, serine-rich protein with strongest evolutionary sequence conservation at the carboxy terminus. While several short, conserved helical regions are predicted from secondary structure analysis, no known functional motifs are apparent. A related human transcript is predicted to encode a novel protein with overall 50% identity to REDDl, which we refer to as REDD2. Based on northern blot analysis of multiple tissue panels, REDDl is expressed in most adult tissues, while REDD2 expression is only detectable by RT-PCR analysis (data not shown). Of note, REDDl was also recently identified as RTP801, a gene induced in the cellular stress response to hypoxia (Shoshani et al., 2002).

While REDDl has a predicted molecular weight of 25 kDa, the protein migrates at 34 kDa. This was confirmed for endogenous protein recognized by specific polyclonal antisera (Figure 3B), for transiently transfected and in vitro translated protein products recognized by these antibodies, and for epitope-tagged constructs (data not shown). The aberrant migration is likely to result from multiple lysine residues at the carboxy terminus. REDDl protein is present in the cytoplasm, without evidence of localization to specific organelles (Figure 3C), and both endogenous and epitope-tagged REDDl remain cytoplasmic following DNA damage stimuli (data not shown). In most cells, REDDl protein is expressed at low levels despite readily detectable mRNA. This most likely results from the short half-life of this protein, estimated to be 15-20 minutes for transfected REDDl; treatment of cells with proteasome inhibitors stabilizes REDDl, suggesting that the protein is regulated via this degradative pathway (data not shown). Following IR or MMS treatment, endogenous REDDl protein is increased to detectable levels (Figure 3B). However, this increase in protein expression is correlated with the induction of the REDDl message, and does not result from any change in the REDDl protein half-life (not shown).

REDDl expression during development parallels that of p63. In striving to generate REDE'i-null mice, mice were developed in which the targeting construct, encoding β-galactosidase (β-gal) linked to the REDDl promoter, was integrated in tandem to the endogenous gene. While this did not disrupt endogenous gene expression, it allowed for measurement of REDDl transcription during normal development. This in vivo reporter construct included 4 kb of REDDl upstream regulatory sequences, the normal start site, first intron and first splice junction, but replaced the entire REDDl coding sequence with β-gal (Figure 4 A and Methods). Mice expressing a single integrated copy of this reporter were produced. Their embryonic fibroblasts induced β-gal following DNA damage in a pattern consistent with that demonstrated for endogenous REDDl in human fibroblasts, indicating that these regulatory sequences were maintained in the integrated construct (data not shown). β-gal staining of developing embryos containing the reporter showed a striking tissue-specific pattern, reminiscent of the p53-family member p63. Like p63, the most intense REDDl staining in early embryos (Εl 1) is noted in the apical ectodermal ridge (AΕR), a cluster of primitive ectoderm at the tip of the limb bud critical for induction of limb development (Capdevila and Izpisua Belmonte, 2001) (Figures 4B, 4C). At embryonic day 13.5 (Ε13.5), REDDl promoter activity is present predominantly in ectoderm-derived tissues known to express p63, such as the whisker pad, eyelid, breast primordia, and the developing limb (Figure 4B). Staining in developing cartilage of the limbs, tail and cranium is also detectable. p63 is also characteristically expressed in the single cell-layer of primitive ectoderm throughout the embryo, and at later stages within the developing epidermis and the root sheath of hair follicles (Mills et al., 1999; Parsa et al., 1999; Yang et al., 1999). Tissue sections from embryos and newborn skin stained for β-gal confirmed that the REDDl pattern is essentially identical to that of p63 (Figure 4C). p63 is required for normal differentiation of ectoderm-derived tissues, and as noted above rp<53 -null mice exhibit failure of skin, limb and mammary development. Therefore, KNA-in-situ hybridization was used to determine whether REDDl expression is altered in EP<53-null mice. Staining for REDDl RNA in wild-type littermates confirmed the pattern of REDDl expression detected in the β-gal reporter mice. Remarkably, REDDl expression is virtually absent in TP63-null embryos (Figure 4D). Together, these data demonstrate spatial and temporal colocahzation of these two genes during embryonic development, consistent with physiological regulation of REDDl expression by p63.

Transcriptional regulation of REDDl by p63 Unlike ρ53, which is regulated by post-translational modification, p63 appears to be regulated primarily at the transcriptional level. TP63 mRNA is expressed at high levels in undifferentiated primary keratinocytes and undergoes rapid downregulation following differentiation (Parsa et al., 1999; Pellegrini et al., 2001). To determine whether REDDl is co-regulated with TP63 during this process, expression of both genes during in vitro differentiation of human primary keratinocytes was examined. Within hours of differentiation, initiated either by addition of serum or a change in calcium concentration, expression of both TP63 and REDDl transcripts declined markedly, followed by a concordant increase in both transcripts late in differentiation (Figure 5A). In contrast, expression of the p53-regulated genes GADD45 anάp21^Cipl remained unaltered following differentiation stimuli.

The parallels between REDDl and p63 expression in vivo and during induced differentiation suggest positive regulation of REDDl by p63. To test this, REDDl expression was first examined in mouse embryo fibroblasts (MEFs) derived from TP63-χvλl mice. REDDl mRNA is readily detectable in wild-type and TP63- heterozygous MEFs, but is virtually absent in MEFs from ZP6^"5-null mice. Reintroduction of adenoviral TAp63γ restores REDDl expression (Figure 5B), directly confirming the ability of p63 to regulate endogenous REDDl expression. Of note, REDDl expression is also induced by the ΔNp63 isoform, which lacks the N- terminal transactivation domain, although much less strongly than by TAp63γ. In contrast to cells lacking p63, expression of REDDl is not altered in ZP53-null cells, which however fail to induce REDDl transcription following IR. These data are consistent with a model in which p63 predominates in regulating baseline REDDl expression, while p53 induces REDDl specifically in response to DNA damage stimuli.

Transcriptional regulation of REDDl by p63 is expected to depend on the p53 family response element in the REDDl promoter. Indeed, cotransfection of reporter constructs containing this promoter sequence and constructs encoding the TAp63γ isoform into U2OS cells demonstrated approximately 5-fold activation, which was largely dependent upon the presence of this consensus p53 response element (Figure 5C). Similar activation was seen following transfection into ZPo^'3-null MEFs (data not shown). The observed transactivation by TAp63γ cannot be attributed to an indirect effect through endogenous p53, since these MEFs were immortalized by expression of SV40 large T antigen (SV40TAg), which inactivates p53. The ΔNp63α isoform demonstrated weak but significant activation of the reporter, consistent with its ability to regulate endogenous REDDl (Figure 5C). This activity is in keeping with the direct or indirect transactivation potential of ΔNp63 identified in some studies (Dohn et al., 2001), and suggests that transcriptional activation, rather than repression, is the net result of p63 expression.

Regulation of keratinocyte differentiation, ROS, and oxidative stress sensitivity by REDDl and p63 Despite the critical function of p63 during epithelial development, assays to examine its functional properties have not been well defined. The observation that both p63 and REDDl are rapidly downregulated during the differentiation of human primary keratinocytes prompted us to ask whether suppression of these genes is required for differentiation to occur. Human adenoviral vectors were used to express REDDl, TAp63γ, or ΔNp63α in primary human keratinocytes (Figure 6A). Adenoviral expression of these proteins alone did not significantly alter the survival or proliferation of undifferentiated cultures (data not shown). However, constitutive expression of REDDl or TAp63γ nearly completely inhibited induction of keratinocyte differentiation following calcium shift, as measured by induction of the differentiation marker involucrin (Figure 6B). In contrast, expression of ΔNp63α or the adenoviral vector alone had little or no effect on baseline or differentiation- induced involucrin expression (Figure 6B). The inability of ΔNp63α to inhibit cellular differentiation is consistent with its weak transactivational properties, compared with TAp63γ (Figure 5C). These data suggest that p63 and REDDl expression contribute to preservation of the undifferentiated state, and that their downregulation is required for normal keratinocyte differentiation. REDDl has also been identified as RTP801, a hypoxia-inducible gene involved in the regulation of cellular ROS (Shoshani et al., 2002). Ectopic expression of RTP801 was shown to modulate cellular ROS levels and sensitivity to oxidative stress. ROS levels are known to be regulated by cellular stress and to contribute to p53-dependent apoptosis (Polyak et al., 1997; Li et al., 1999). However, recent evidence has also implicated ROS as intracellular messengers in receptor signaling pathways linked to cellular proliferation and differentiation (Adler et al., 1999; Finkel, 2000; Meng et al., 2002). To determine whether p63 is involved in regulation of cellular ROS, ROS levels were first compared in EP< 3-heterozygous and TP63-mήl MEFs, using the indicator dye CM-H₂DCFDA, whose fluorescence is proportional to the level of multiple forms of intracellular ROS (Ohba et al., 1994). Remarkably, TP63 -null MEFs had significantly lower levels of ROS compared with cells from wild-type or heterozygous littermates (Figure 7A). This difference was consistently observed between fibroblasts derived from multiple embryos, and was detectable in both early and late-passage MEFs. The difference in ROS was also detectable following transformation of multiple MEF lines by SV40TAg, implying that p63- dependent changes in cellular redox status are not mediated indirectly through modulation of p53 function (Figure 7A). The magnitude of the difference in cellular ROS attributable to p63 is potentially physiologically significant, as it is comparable to the change observed following acute treatment of cells with a lethal dose of hydrogen peroxide (0.8mM H₂O₂), a major cellular mediator of oxidative stress (Figure 7A). However, H₂O₂ itself is likely to constitute only a subset of the multiple ROS species regulated by p63. Adeno viral-mediated expression of TAp63γ in TP63- null cells did not significantly change baseline ROS, but it led to a marked enhancement of H₂O₂-induced ROS levels (Figure 7B). Expression of ΔNp63α had little or no effect as compared to the vector control. Adenoviral expression of RED l in TP63-mxll MEFs enhanced H₂O₂-induced ROS levels to a similar degree as TAp63γ expression (Figure 7B). These data suggest that reduced REDDl expression in TP63-wιll cells contributes to their decreased ROS levels, and that regulation of REDDl by TAρ63γ modulates cellular ROS.

To determine the functional significance of altered ROS levels in Pό^'3-null cells, the survival of TPό^'3-heterozygous or TPό^'3-null MEFs was compared following treatment with H₂O₂. TP63-nu\l cells displayed 2-fold enlianced resistance to peroxide challenge, as compared to rP6^"3-heterozygous cells (Figure 7C). This difference was observed in both primary and SV40TAg-transformed MEFs, indicating that the difference does not depend on intact p53 function. The resistance to oxidative stress was specific, since EPo^'3-null cells were not markedly more resistant to DNA damage induced either by the alkylating agent methyl methane sulphonate (MMS) or by ionizing radiation (not shown), consistent with previous observations (Flores et al., 2002). Adenoviral expression of either REDDl or TAp63γ in 7P6^"3-null MEFs did not alter baseline cell viability or proliferation (not shown), but it doubled the number of cells killed following H₂O₂ challenge (Figure 7C). This effect was specifically linked to the increase in ROS associated with expression of REDDl or TAp63γ, since treatment of cells with the anti-oxidant N-acetyl cysteine (NAC) blocked induction of ROS and effectively suppressed the increased cell killing observed in p63 and REDDl -reconstituted cells exposed to H₂O₂ (Figures 7B and 7C).NAC did not further suppress oxidative stress sensitivity of vector-infected TP63-mxll cells (Figure 7C). Remarkably, treatment of primary keratinocytes with NAC completely abolished the inhibition of differentiation mediated by REDDl (Figure 6B). NAC itself had no effect on calcium-induced keratinocyte differentiation (not shown). These observations suggest that the enhancement of cellular ROS by REDDl contributes to p63-mediated inhibition of keratinocyte differentiation.

Thus, increasing REDDl or p63 expression increases cell death in response to oxidative stress. Consistent with this direct experimental observation, by correlation it was also shown that cells with decreased expression of both genes have decreased sensitivity (i.e. decreased death) following oxidative stress. Therefore a role for REDDl in mediating cell death in response to oxidative stress has been defined. Data is further provided that REDDl does so by increasing reactive oxygen species (ROS). Additional data (not shown) suggests that REDDl may have the opposite effect in cells that are hypoxic (i.e. grown in a low oxygen state). By using cells with particular mutations in the hypoxia response pathway, it is shown herein that cells exhibiting the hypoxia response are exquisitely sensitive to inhibition of REDDl expression. In other words, inhibiting REDDl expression causes cell death preferentially in cells that are hypoxic. This observation is important in regard to potential anti-cancer therapy, since proliferating cancer cells are commonly hypoxic. Further, our unpublished data show that REDDl enhances cell death induced by p53. This observation is important therapeutically, since the ability of p53 to induce cell death has been directly linked to its tumor suppression function. Thus, increasing REDDl levels may help restore the normal cell death response in nascent cancer cells and therefore function as a cancer preventative. In addition, increasing REDDl levels specifically in established cancers that have lost p53 may be of benefit in combination with other treatments, by restoring the death response. Loss of p53 has been shown to cause decreased sensitivity of cancer cells to radiation and chemotherapy, and increasing REDDl levels may help restore sensitivity.

Work described herein also shows that activation of the ROS pathway (in this case via REDDl) can inhibit epithelial cell differentiation. Results of work described herein have also shown directly (data not shown) that inhibiting REDDl expression can induce features of normal differentiation in epithelial cells. This observation may also be relevant to potential therapeutic applications in cancer treatment, since one hallmark of most cancer cells is that they do not undergo normal differentiation. Elevation of REDDl Levels in Progression of Human Breast Carcinoma

Additional data have shown that increasing REDDl (or p63) expression causes abnormalities in early events of breast cell development. Specifically, REDDl expression causes morphologic abnormalities in breast cells as they begin to differentiate and form normal breast structures. This observation therefore defines a method to inhibit abnormalities in the breast by inhibiting REDDl levels. REDDl inhibition may therefore be useful in the prevention of breast cancer or other breast diseases. Work described herein has also demonstrated that REDDl levels are elevated during the progression of human breast carcinoma. Specifically, a series of human breast tissue specimens that had undergone microdissection were analyzed in order to separate cells from each specimen coπesponding to normal epithelium, pre- invasive ductal carcinoma in situ (DCIS) and invasive carcinoma. These normal and neoplastic epithelia were then analyzed for REDDl expression (by quantitative realtime PCR analysis). In 40% of cases there was a significant (>2-fold) increase in REDDl expression in DCIS and invasive carcinoma as compared to normal epithelium, with some cases showing more than 20-fold increased expression. Subsequent (RNA in situ hybridization) analysis on tissue sections from the same specimens used for PCR analysis confirmed that REDDl was markedly overexpressed in the neoplastic cells compared to normal epithelium. These findings support the utility of inhibiting the REDDl pathway as a means of inhibiting breast abnormalities, including cancer. Most notably, REDDl levels were highest in breast tumors that lacked both estrogen receptor staining and Her-2 amplification. These tumors represent a highly aggressive subset of breast cancers for which fewer therapeutic options are available. Therefore, inhibiting the REDDl pathway in these tumors may be particularly attractive.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

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Claims

CLAIMSWhat is claimed is:

1. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: a) SEQ ID NO: 1; b) the complement of SEQ ID NO: 1 ; c) a nucleic acid molecule which is at least about 60% identical to the nucleotide sequence SEQ ID NO: 1; d) a nucleic acid molecule which is at least about 60% identical to the nucleotide sequence of the complement of SEQ ID NO: 1; e) a nucleic acid molecule which hybridizes under high stringency conditions to the nucleotide sequence selected of SEQ ID NO: 1; and f) a nucleic acid molecule which hybridizes under high stringency conditions to the nucleotide sequence of the complement of SEQ ID NO: l.

2. An isolated nucleic acid molecule according to Claim 1 , wherein said nucleic acid molecule is expressed in human cells.

3. An isolated nucleic acid molecule according to Claim 1 , wherein said nucleic acid molecule is DNA.

4. An isolated nucleic acid molecule according to Claim 1 , wherein expression of said nucleic acid molecule in a cell increases reactive oxygen species in said cell.

5. An isolated nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of: a) SEQ ID NO: 1; b) the complement of SEQ ID NO : 1 ; c) a portion of the nucleotide sequence of SEQ LO NO: 1, wherein the portion is at least about 10 nucleotides in length; d) a portion of the nucleotide sequence of the complement of SEQ ID NO: 1, wherein the portion is at least about 10 nucleotides in length; e) a portion of the nucleotide sequence of SEQ ID NO: 1 , wherein the portion is at least about 20 nucleotides in length; and f) a portion of the nucleotide sequence of the complement of SEQ ID NO: 1, wherein the portion is at least about 20 nucleotides in length.

6. A nucleic acid construct comprising the isolated nucleic acid molecule of Claim 1 or Claim 5.

7. The nucleic acid construct of Claim 6 wherein the isolated nucleic acid molecule is operatively linked to a regulatory sequence.

8. A recombinant host cell comprising the isolated nucleic acid molecule of Claim 1 or Claim 5.

9. The recombinant host cell of Claim 8 wherein the isolated nucleic acid is operatively linked to a regulatory sequence.

10. A method for preparing a polypeptide encoded by an isolated nucleic acid molecule, comprising culturing the recombinant host cell of Claim 9.

11. An isolated polypeptide encoded by an isolated nucleic acid molecule according to Claim 1.

12. An isolated polypeptide encoded by an isolated nucleic acid molecule according to Claim 5.

13. An isolated polypeptide comprising SEQ ID NO: 2 or a functional portion thereof.

14. An antibody, or an antigen-binding fragment thereof, which selectively binds to the polypeptide encoded by an isolated nucleic acid molecule according to Claim 1 or Claim 5, or to a portion of said polypeptide.

15. An antibody, or an antigen-binding fragment thereof, which selectively binds to the polypeptide of Claim 13.

16. A method of assaying for the presence of a nucleic acid molecule in a sample, comprising contacting said sample with a nucleotide sequence selected from the group consisting of: a) SEQ ID NO: 1; b) the complement of SEQ ID NO: 1 ; c) a portion of SEQ ID NO: 1 which is at least 10 nucleotides in length; and d) a portion of the complement of SEQ ID NO: 1 , which is at least 10 nucleotides in length, under conditions appropriate for selective hybridization, wherein hybridization of said nucleotide sequence to said sample is indicative of the presence of a nucleic acid molecule in said sample.

17. A method of assaying for the presence of a polypeptide encoded by an isolated nucleic acid molecule according to Claim 1 in a sample, comprising contacting said sample with an antibody which specifically binds to the polypeptide.

18. A method of assaying for the presence of a polypeptide according to Claim 13 in a sample, comprising contacting said sample with an antibody which specifically binds to the polypeptide.

19. A method of inducing expression of REDDl in a cell comprising inducing expression of p53, whereby expression of REDDl is induced.

20. A method of inducing expression of REDDl in a cell comprising inducing expression of p63, whereby expression of REDDl is induced.

21. A method according to Claim 20, wherein said cell is an undifferentiated cell.

22. A method of inducing activation of the ROS pathway in a cell, comprising inducing expression of REDDl.

23. A method according to Claim 22, wherein expression of REDD 1 is induced by p53.

24. A method according to Claim 22, wherein expression of REDDl is induced by p63.

25. A method according to Claim 22, wherein activation of the ROS pathway results in increased level of ROS.

26. A method according to Claim 22, wherein activation of the ROS pathway results in increased sensitivity to oxidative stress.

27. A method of causing cell death comprising inhibiting expression of REDDl in said cell.

28. A method according to Claim 27, wherein said cell is a cancer cell.

29. A method of inhibiting differentiation of a cell comprising inducing expression of REDDl in said cell.

30. A method according to Claim 29, wherein said cell is an epithelial precursor cell.

31. A method of enhancing cell death in response to oxidative stress, comprising enhancing expression of p63 in said cell, thereby enhancing susceptibility of said cell to oxidative stress.

32. A method of enhancing cell death in response to oxidative stress, comprising enhancing expression of REDDl in said cell, thereby enhancing susceptibility of said cell to oxidative stress.

33. A method according to Claim 31 or 32, wherein cell death is mediated via the reactive oxygen species pathway.

34. A method of inhibiting cell death in response to oxidative stress, comprising inhibiting expression of p63 in said cell, thereby reducing susceptibility of said cell to oxidative stress.

35. A method of inhibiting cell death in response to oxidative stress, comprising inhibiting expression of REDDl in said cell, thereby reducing susceptibility of said cell to oxidative stress.

36. A method of inhibiting cell death in response to oxidative stress, comprising inhibiting expression of p63 and REDDl in said cell, thereby reducing susceptibility of said cell to oxidative stress.

37. A method of enhancing cell death in hypoxic cells, comprising inhibiting expression of REDDl in said cells.

38. A method according to Claim 37, wherein said hypoxic cells are proliferating cancer cells.

39. A method of enhancing p53-induced cell death comprising enhancing expression of REDDl in said cell.

40. A method according to Claim 39, wherein said cell is a nascent cancer cell.

41. A method according to Claim 39, wherein said cell is an established cancer cell.

42. A method according to Claim 41 , wherein said cell has reduced p53 expression.

43. A method of inhibiting the progression of cancer in an individual, comprising enhancing expression of REDDl in cancer cells in said individual, whereby death of said cancer cells is enhanced.

44. A method of inhibiting differentiation of a cell comprising inducing expression of the reactive oxygen species pathway in said cell.

45. A method according to Claim 44, wherein said cell is an epithelial precursor cell.

46. A method of enhancing differentiation of a cell, comprising inhibiting expression of REDDl in said cell.

47. A method according to Claim 46, wherein said cell is an epithelial cell.

48. A method according to Claim 46, wherein said cell is a nascent cancer cell.

49. A method of decreasing abnormalities in breast cells, comprising inhibiting expression of REDD 1.

50. A method of decreasing abnormalities in breast cells, comprising inhibiting expression of p63.

51. A method of treating breast disorders in an individual, comprising inhibiting expression of REDDl in cells of the breast of the individual.

52. A method of treating breast disorders in an individual, comprising inhibiting expression of p63 in cell of the breast of the individual.

53. A method according to Claim 51 or 52, wherein the breast disorder is breast cancer.

54. A transgenic animal comprising a single germline copy of a DNA construct comprising the REDDl promoter region operably linked to a reporter gene.

55. A transgenic animal according to Claim 54, wherein the reporter gene is a β- gal/neomycin-resistance fusion gene.

56. A transgenic animal according to Claim 54, wherein an IRES is linked to the reporter gene.

57. A transgenic animal according to Claim 54, Claim 55 or Claim 56, wherein said animal is a mouse.

58. An isolated cell from a transgenic animal according to Claim 54, Claim 55 or Claim 56.

59. A nucleic acid construct comprising the REDDl promoter region operably linked to a reporter gene.

60. A nucleic acid construct according to Claim 59, wherein the reporter gene is the beta galactosidase gene.

61. A recombinant host cell comprising the nucleic acid construct of Claim 59 or Claim 60.

62. A method of diagnosing a disorder associated with aberrant expression of REDDl in an individual, comprising assessing REDDl expression in an individual, wherein aberrant expression of REDDl as compared with expression in a normal individual is indicative of a disorder associated with aberrant expression of REDDl.

63. A method according to Claim 62, wherein the disorder is cancer.

64. A method according to Claim 62, wherein assessment of REDDl expression is performed by assessing the level of REDDl transcript.

65. A method according to Claim 62, wherein assessment of REDDl expression is performed by assessing the level of REDDl protein.

66. A method according to Claim 62, wherein assessment of REDDl expression is performed by assessing the level of REDDl function.

67. A method according to Claim 66, wherein REDDl function is activation of the ROS pathway.

68. A method of identifying an agent which alters REDDl expression, comprising contacting a cell comprising REDDl with an agent to be tested, assessing expression of REDDl in the presence of the agent as compared with expression of REDDl in the absence of the agent, and identifying an agent which alters REDDl expression.

69. A method according to Claim 68, wherein assessment of expression of REDDl is performed by assessing the level of REDDl transcript.

70. A method according to Claim 68, wherein assessment of expression of REDDl is performed by assessing the level of REDDl protein.

71. A method according to Claim 68, wherein assessment of expression of REDDl is performed by assessing the level of REDDl function.

72. A method according to Claim 71, wherein REDDl function is activation of the ROS pathway.

73. A method of identifying an agent which alters REDDl expression, comprising contacting a cell comprising a nucleic acid construct comprising the promoter region of the REDDl gene operably linked to a reporter gene with an agent to be tested, assessing expression of the reporter gene in the presence of the agent as compared with expression of the reporter gene in the absence of the agent, and identifying an agent which alters expression of the reporter gene, wherein an agent which alters expression of the reporter gene is an agent which alters REDDl expression.

74. A method according to Claim 73, wherein the cell is isolated from a transgenic animal.

75. A method of identifying an agent which alters REDDl expression, comprising administering an agent to be tested to an animal, assessing expression of REDDl in the presence of the agent as compared with expression of REDDl in the absence of the agent, and identifying an agent which alters expression of REDDl.

76. A method according to Claim 75, wherein assessment of REDDl expression is performed by assessing the level of REDDl transcript.

77. A method according to Claim 75, wherein assessment of REDDl expression is performed by assessing the level of REDDl protein.

78. A method according to Claim 75, wherein assessment of REDDl expression is performed by assessing the level of REDDl function.

79. A method according to Claim 78, wherein REDDl function is activation of the ROS pathway.

80. A method of identifying an agent which alters REDDl expression, comprising administering an agent to be tested to a transgenic animal comprising a nucleic acid construct comprising the promoter region of the REDDl gene operably linked to a reporter gene, assessing expression of the reporter gene in the presence of the agent as compared with expression of the reporter gene in the absence of the agent, and identifying an agent which alters expression of the reporter gene, wherein an agent which alters expression of the reporter gene is an agent which alters expression of REDDl .