Induction of tumor cell senescence by retinoid receptor agonists and antagonists

Abstract

The invention relates to the induction of tumor cell growth arrest. More particularly, the invention relates to the use of retinoic acid receptor (RAR) agonists and antagonists to mediate such growth arrest. The invention provides methods for using RAR-modulating compounds to induce growth arrest, methods for identifying such RAR-modulating compounds, and RAR-modulating compounds identified by such latter methods.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the induction of tumor cell growth arrest. More particularly, the invention relates to the use of retinoid receptor agonists and antagonists to mediate such induction of growth arrest.

2. Summary of the Related Art

Retinoids, natural and synthetic derivatives of vitamin A, are used in leukemia treatment and chemoprevention of cancers. These physiological regulators of gene expression were shown to be efficacious in the treatment of promyelocytic leukemia and (to a lesser degree) in chemoprevention of several cancers, in particular breast carcinoma.

Warrell, In Cancer, Principles and Practice of Oncology, V. T. H. S. DeVita and S. A. Rosenbert, eds. (Philadelphia: Lippincot Williams and Wilkins), pp. 489-494 (2001) teaches that retinoid treatment, however, produces a certain amount of systemic toxic responses, such as intracranial hypertension or hyperleukocytosis.

The antitumor effect of retinoids is most often attributed to the induction of differentiation (Altucci and Gronemeyer, Nat. Rev. Cancer 1, 181-193 (2001), but Roninson and Dokmanovic, J. Cell Biochem. 88: 83-94 (2003) teach that these compounds also stop the growth of tumor cells by activating the programs of apoptosis or senescence. Roninson and Dokmanovic, supra also teaches that senescence is observed at the lowest and generally non-toxic concentrations of retinoids, and (ii) it involves upregulation of several growth-inhibitory proteins, including secreted factors that arrest the growth of neighboring non-senescent cells. Senescent tumor cells may therefore be regarded as a reservoir of secreted factors that provide for long-term inhibition of tumor growth.

Dokmanovic et al., PCT/US01/17161 teaches that retinoid-induced senescence of human MCF-7 breast carcinoma cells is associated with increased RNA expression of several intracellular and secreted proteins with known growth-inhibitory activities. These include actin-binding protein EPLIN (Epithelial Protein Lost in Neoplasm) and an ubiquitin-like protein UBD (formerly known as FAT10), as well as secreted proteins insulin-like growth factor-binding protein 3 (IGFBP3) and an extracellular matrix component TGFB1 (formerly known as βIG-h3). Induction of these genes can be used as the test for identifying other compounds that are likely to induce the same form of senescence as retinoids.

Induction of gene expression by retinoids is mediated at the level of transcription, through binding to dimeric transcription factors formed by retinoic acid receptors (RAR) and rexinoid receptors (RXR). The best-known mechanism of action of these retinoid receptors involves their binding to retinoic acid response elements (RARE) in the promoters of retinoid-responsive genes. Nevertheless, Altucci and Gronemeyer, supra teaches that retinoid receptors also affect transcription through RARE-independent mechanisms, such as repression of transcription factor AP-1 (Jun/Fos) and Husmann et al, Biochem. J. 352: 763-772 (2000) teaches that they can act or by modulating the interaction of Sp1 and GC-rich DNA via ternary complex formation.

Remarkably, Dokmanovic et al., Cancer Biology & Therapy 1:24-27 (2002) teaches that only one of 13 genes that were found to be strongly upregulated by retinoids in senescent MCF-7 cells, TRIM31, contains a putative RARE sequence in its promoter, whereas the other genes, including EPLIN, UBD, IGFBP3 and TGFB1, showed no identifiable RARE sequences This suggests that retinoids upregulate these genes via a RARE-independent mechanism, but it is unknown whether this mechanism was mediated by retinoid receptors.

Induction of terminal cell growth arrest is of special interest in anticancer drug development. There is, therefore, a need to develop compounds that can induce growth arrest in tumor cells.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides methods for using one or more retinoic acid receptor (RAR)-modulating compounds to induce growth arrest in proliferating cells and that are efficient in inducing RARE-independent gene expression and that are inefficient in inducing RARE-dependent gene expression.

In a second aspect, the invention provides methods for identifying one or more RAR-modulating compounds that induce growth arrest in proliferating cells and that are efficient in inducing RARE-independent gene expression and that are inefficient in inducing RARE-dependent gene expression with relatively few toxic side effects.

In a third aspect, the invention provides compounds identified by the second aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effects of RAR agonists (RA and LGD1550, 100 nM each) and RAR antagonist LG100815 (10 μM) on luciferase expression from DR5 RARE-containing promoter in MCF-7 cells. The assays were carried out in triplicate. RA is all-trans retinoic acid. LGD1550 is a pan-RAR agonist. LG100815 is a pan-RAR antagonist.

FIG. 2 shows the effects of retinoid agonists and antagonists on MCF-7 cell growth. The bars represent cell number after 7 days of culture with the addition of DMSO (control), 100 nM RA, 100 nM RAR agonist LGD1550, 100 nM RXR agonist LGD1268, 10 μM RAR antagonist LG100815, and 10 μM RXR antagonist LG101208. Experiments were done in triplicate, and the results are expressed relative to the average of the control.

FIG. 3 shows the effects of retinoid agonists and antagonists on growth and the senescent phenotype of MCF-7 cells. The bars represent percentages of SA-β-gal+ cells after 8 days of treatment with the indicated compounds (in triplicate). The compounds were used at the same concentrations as in FIG. 2.

FIG. 4 depicts microarray analysis of changes in gene expression in MCF-7 cells treated with RAR agonist LGD1550 or RAR antagonist LG100815, plotted using GeneSpring software. The X axis represents different time points of treatment with RAR ligands (0 point correspond to cells cultured for 3 days with DMSO carrier). The Y axis shows changes in gene expression on log scale.

FIG. 5 shows the comparison of changes in gene expression produced by RAR agonist and antagonist. The maximal changes in gene expression for 11,729 probe sets representing genes that show >1.3 fold effect by either the agonist or the antagonist (dots) are plotted on a log scale. Encircled dots correspond to the genes that are affected >5-fold by either ligand. Trend lines (power regression) correspond to the genes with >1.3-fold or >5-fold changes in gene expression The R² values for the regression lines are 0.6955 with n=11729 (for >1.3-fold) and 0.7876 with n=316 (for >5-fold).

FIG. 6 shows the comparison of changes in gene expression produced by RAR agonist and antagonist for the 62 genes listed at the top of Table 3 (similar induction by the agonist and the antagonist; circles) and in Table 4 (preferential induction by the agonist; triangles). The maximal changes in gene expression are plotted on a log scale.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to the induction of tumor cell growth arrest. More particularly, the invention relates to the use of retinoic acid receptor modulators to mediate such induction of growth arrest. The patents and publications cited herein reflect the level of knowledge in the art and are hereby incorporated by reference in their entirety. Any conflict between the teachings of these patents and publications and this specification shall be resolved in favor of the latter.

For purposes of determining the metes and bounds of claims containing this term, the term retinoic acid receptor (RAR) agonist is intended to mean those compounds recognized in the art as those capable of acting through retinoic acid receptors and are efficient at inducing RARE-dependent gene expression. Such compounds include, but are not limited to, all-trans-retinoic acid (RA), 13-cis retinoic acid and LGD1550.

As defined herein, “retinoid-responsive” gene is a gene that is induced by treatment with a known retinoic acid receptor ligand. As defined herein, a retinoic acid receptor ligand is intended to mean a retinoic acid receptor agonist and/or a retinoic acid receptor-modulating compound.

For purposes of determining the metes and bounds of claims containing this term, the term retinoic acid receptor (RAR)-modulating compound is intended to mean those compounds capable of acting through retinoic acid receptors and inducing expression of RARE-independent retinoid-responsive genes but inefficient at inducing RARE-dependent gene expression. Such compounds include, but are not limited to, LG100815.

As defined herein, “RARE-dependent” gene expression refers to the expression of retinoid-responsive genes that are preferentially induced by the RAR agonist relative to RAR-modulating compound (e.g. 3-fold or greater difference in their maximal induction), whether or not such genes contain RARE elements in their promoters.

As defined herein, “RARE-independent retinoid-responsive” gene expression refers to the expression of retinoid-responsive genes that (i) do not contain verified RARE elements in their promoters (see Balmer and Blomhoff, 2005 for a listing of RARE-containing genes) and (ii) are induced by the RAR agonist and the RAR-modulating compound to a similar degree (e.g. no more than 2.5-fold difference in their maximal induction).

In a first aspect, the invention provides methods for using one or more RAR-modulating compounds that are efficient in inducing RARE-independent gene expression and that are inefficient in inducing RARE-dependent gene expression to induce growth arrest in proliferating cells. In preferred embodiments, the proliferating cells are neoplastic cells. In preferred embodiments the proliferating cells are in a mammal, preferable the mammal is a human. In the methods according to this aspect of the invention, preferred compounds include ligands of retinoic acid receptors (RAR). Surprisingly, both agonists and antagonists of RAR induce growth arrest and senescence in proliferating cells, whereas neither agonists nor antagonists of rexinoid receptors (RXR) have this effect.

All-trans retinoic acid (RA) and other RAR agonists induce transcription both through a RARE-dependent mechanism and through RARE-independent mechanisms. The toxicity associated with these compounds may result from the stimulation of RARE-dependent transcription. RAR-modulating antagonists are inefficient in inducing RARE-dependent transcription and therefore should be less toxic. In fact, an RAR antagonist was reported to decrease the toxicity of RAR agonists. (See Standeven et al., Toxicol. Appl. Pharmacol. 138:169-175 (1996)).

In a second aspect, the invention provides methods for identifying one or more RAR-modulating compounds that induce growth arrest in proliferating cells with relatively few toxic side effects that are efficient in inducing RARE-independent gene expression and that are inefficient in inducing RARE-dependent gene expression. In a preferred embodiment, the method according to this aspect of the invention comprises providing proliferating cells, contacting the cells with a test compound, determining the level of RARE-independent retinoid-responsive gene expression (as defined above), determining the level of RARE-dependent gene expression (as defined above), and comparing the ratio of RARE-independent retinoid-responsive gene expression to RARE-dependent gene expression. Test compounds that induce RARE-independent retinoid-responsive gene expression and that have the highest ratio of RARE-independent retinoid-responsive gene expression to RARE-dependent gene expression are determined to be RAR-modulating compounds that induce growth arrest in proliferating cells and should have relatively non-toxic side effects. In preferred embodiments, the level of RARE-independent retinoid-responsive and RARE-dependent gene expression is normalized against cells not treated with the test compound. In certain preferred embodiments, the level of RARE-independent retinoid-responsive and RARE-dependent gene expression is compared to cells treated with a compound known to induce RARE-dependent gene expression, such as RA or another RAR agonist.

In certain embodiments, RARE-independent retinoid-responsive and/or RARE-dependent gene expression can be determined by quantitative reverse-transcription PCR. This embodiment provides a method for identifying one or more RAR-modulating compounds that induce growth arrest in proliferating cells and that are efficient in inducing RARE-independent gene expression and that are inefficient in inducing RARE-dependent gene expression, contacting the cells with a test compound, obtaining cells that have undergone growth arrest, determining the level of expression of RARE-independent retinoid-responsive genes and determining the level of expression of RARE-dependent genes wherein test compounds that increase the expression of RARE-independent retinoid-responsive genes relative to cells not treated with the test compound and that have the highest ratio of the expression of RARE-independent retinoid-responsive genes to the expression of RARE-dependent genes, are determined to be RAR-modulating compounds that are inefficient in inducing RARE-dependent gene expression and induce cell growth arrest.

For example, quantitative reverse-transcription PCR assays in Table 2 show that the ratio of the fold-induction of TGFB1 relative to TRIM31 is 0.19 for 100 nM RA and 0.12 for all three concentrations of RAR agonist LGD1550, but in the case of RAR antagonist LG100815, this ratio increases to 0.56 at 1 μM and 0.38 at 10 μM.

In certain embodiments, RARE-independent retinoid-responsive and/or RARE-dependent gene expression can be determined by hybridization with oligonucleotide or cDNA arrays. For example, FIG. 6 shows that plotting fold increase in gene expression upon treatment with RAR agonist or RAR-modulating antagonist can be used to identify groups of genes that show either similar response to both RAR ligands or preferential response to RAR agonist.

In certain embodiments RARE-independent retinoid-responsive and/or RARE-dependent gene expression can be determined by providing cells transfected with a first gene encoding a first detectable protein operatively linked to a promoter of a RARE-independent retinoid-responsive gene and a second gene encoding a second detectable protein, that is different from and separately detectable in the presence of the first detectable protein, operatively linked to a promoter of a RARE-dependent gene. This embodiment provides a method for identifying one or more RAR-modulating compounds that induce growth arrest in proliferating cells and that are efficient in inducing RARE-independent gene expression and that are inefficient in inducing RARE-dependent gene expression, by contacting the cells with a test compound, measuring the levels of first and second detectable proteins, and comparing the levels of detectable protein expression. Test compounds that induce RARE-independent retinoid-responsive gene expression and that have the highest ratio of RARE-independent retinoid-responsive gene expression to RARE-dependent gene expression are determined to be RAR-modulating compounds that induce growth arrest in proliferating. Preferred detectable proteins include, without limitation, firefly luciferase, Renilla luciferase, beta-galactosidase, chloramphenicol acetyltransferase, horseradish peroxidase, green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, fluorescent protein DsRed, alkaline phosphatase and immunologically detectable proteins or peptides.

The methods according to this aspect of the invention can be used for testing derivatives of existing RAR agonists or antagonists (see e.g. Hammond et al., Br J. Cancer 85: 453-462 (2001); Standeven et al., Toxicol. Appl. Pharmacol. 138: 169-175 (1996); Toma et al., Int. J. Cancer 78: 86-94 (1998); Yang et al., Breast Cancer Res. Treat. 56: 277-291 (1999)), which can be generated by standard methods of combinatorial chemistry or combinatorial biocatalysis. This method can also be used with other natural or synthetic retinoids with unknown receptor specificity.

In a third aspect, the invention provides compounds identified by the second aspect of the invention, as well as optimized derivatives of such compounds.

The examples below are intended to further illustrate certain preferred embodiments of the invention, and are not intended to limit the scope of the invention.

EXAMPLE 1

Effect of Compounds on Expression of Genes Associated with Senescence

The following compounds were obtained from Ligand Pharmaceuticals (San Diego, Calif.). LGD1550 is a pan-RAR agonist. LGD1268 is a pan-RXR agonist. LG100815 is a pan-RAR antagonist. LG101208 is a pan-RXR antagonist. The LG100815 compound is a specific RAR antagonist that binds to RAR, but fails to activate its Retinoic Acid Responsive Element (RARE)-dependent transcription transactivation function (see Lee et al, Mol. Cell Biol. 19: 1973-1980 (1999)). Also used was all-trans retinoic acid (RA), the most commonly used RAR agonist. To determine how these compounds affect the expression of retinoid-inducible growth inhibitory genes EPLIN, UBD, IGFBP3 and TGFB1, as well as TRIM31 gene that contains a putative RARE element in its promoter, MCF-7 cells (subline MCF-7 3′SS6) were treated with individual compounds or their combinations for 2 days, and RNA was extracted by standard procedures. In the initial assays, gene expression was analyzed by semi-quantitative reverse transcription-PCR (RT-PCR), as described in Dokmanovic et al., Cancer Biology & Therapy 1:24-27 (2002). In subsequent assays, the initial results were confirmed, expanded and quantitated by real-time RT-PCR (QPCR), using Applied BioSystems 7900HT real-time PCR instrument. β-actin was used as a normalization standard. The primers used for Q-PCR of the corresponding genes are listed in Table 1.

	TABLE 1
	Gene Name	Primer Sequences
	IGFBP3	sense	5′-TTGCACAAAGACTGCCAAG-3′
		antisense	5′-AATCAGTTCACCACAAACAG-3′
	TRIM31	sense	5′-CATGAGGGAACGGAAGCGGG-3′
		antisense	5′-AACGAGCTTCTTGAGATCGT-3′
	UBD	sense	5′-AATGCTTCCTGCCTCTGTGT-3′
		antisense	5′-CGCTGTCATATGGGTTGGCA-3′
	EPLIN-β	sense	5′-AGAAGCCGCCCATTCACTGT-3′
		antisense	5′-CTGATAGGTGGGGACACAGT-3′
	TGFBI	sense	5′-TGCGACTAGCCCCTGTCTAT-3′
		antisense	5′-GTGGTGCATTCCTCCTGTAG-3′
	β-actin	sense	5′-CTTCCTGGGCATGGAGTC-3′
		antisense	5′-TGTTGGCGTACAGGTCTTTG-3′

cDNA was prepared by reverse transcription with random primer using 4 μg total RNA. 5 μl SYBR Green PCR Master Mix (Applied Biosystems) was mixed with 50 μg cDNA and 0.8 pmol of gene-specific primers and brought up to 10 μl with ultra pure H₂O in 384-well optical plates. For amplification, reaction mixtures were heated for 2 min at 50° C. and 10 min at 95° C., followed by 40 cycles of two-step PCR consisting of 15 sec at 95° C. and 1 min at 60° C., to construct dissociation curves and verify that single PCR products were obtained. PCR products were also analyzed by gel electrophoresis to confirm that a single product of the expected size was amplified. Serial cDNA dilutions were used for primer validation experiments to demonstrate that both target and reference genes had equal amplification efficiency according to the standard curve method. The comparative CT method for relative quantitation of gene expression described by Applied Biosystems was used to determine expression levels for target genes. Experiments were carried out in triplicate for each data point. Sequence Detection Systems software version 2.1 (Applied Biosystems) and Microsoft Excel were used for data analysis.

The results of a representative set of QPCR assays are shown in Table 2.

	TABLE 2
	Fold induction
Compounds/Genes	IGFBP3	EPLIN	UBD	TGFBI	TRIM31
RA (100 nM)	2.5 ± 0.5	3.1 ± 0.7	8.5 ± 2.8	6.5 ± 1.4	34.4 ± 3.9
LGD1550 (100 nM)	4.2 ± 2.0	4.2 ± 0.8	9.5 ± 0.2	5.1 ± 0.6	42.7 ± 1.3
LGD1550 (1 μM)	3.7 ± 0.7	3.2 ± 0.3	7.7 ± 1.1	3.5 ± 0.4	30.3 ± 0.7
LGD1550 (10 μM)	2.8 ± 1.6	4.9 ± 2.5	7.3 ± 1.1	5.6 ± 0.1	45.0 ± 1.4
LG100815 (1 μM)	2.1 ± 1.1	3.8 ± 0.9	4.6 ± 0.6	4.3 ± 0.7	7.7 ± 4.1
LG100815 (10 μM)	2.0 ± 0.5	3.2 ± 1.7	2.8 ± 0.6	4.4 ± 0.4	11.7 ± 4.4
LG100815 (1 μM) + RA	2.4 ± 1.1	1.7 ± 0.5	8.0 ± 1.5	6.4 ± 2.1	30.3 ± 3.3
(100 nM)
LG100815 (10 μM) + RA (100 nM)	1.9 ± 0.5	0.9 ± 0.2	2.2 ± 0.1	3.7 ± 0.9	10.0 ± 4.6
LGD1268 (100 nM)	1.2 ± 0.5	1.2 ± 0.2	1.0 ± 0.1	1.9 ± 1.1	1.3 ± 0.4
LGD1268 (1 μM)	0.9 ± 1.1	1.0 ± 0.1	0.8 ± 0.2	2.2 ± 1.0	1.4 ± 0.7
LGD1268 (10 μM)	1.2 ± 0.4	1.4 ± 0.2	1.0 ± 0.2	2.4 ± 0.3	3.8 ± 0.7
LG101208 (10 μM)	1.0 ± 0.2	0.8 ± 0.1	1.0 ± 0.1	0.9 ± 0.2	1.0 ± 0.5

The results of this analysis show the following. The pan-RAR agonist LGD1550 induced all five genes to approximately the same extent as RA. The pan-RXR agonist LGD1268 induced only one of five genes (TGFB1) but the extent of induction was much lower than the effect of RA or LGD1550. These findings indicate that retinoid-inducible gene expression is activated primarily through RAR.

Whereas the pan-RXR antagonist LG101208 had no effect on gene expression, the pan-RAR antagonist LG100815, surprisingly, induced the expression of all five genes. The magnitude of induction by LG100815 was similar to or slightly lower than that of RA or LGD1550 for EPLIN, IGFBP3, TGFB1 and UBD, but 3-4 fold lower for TRIM31 (the only gene that contains RARE in its promoter). When LG100815 was combined with RA, it decreased the induction of gene expression by RA to the levels that were similar to or (in the case of EPLIN) lower than the levels observed with LG100815 alone, with the biggest decrease from RA-induced levels observed for TRIM31. This finding was consistent with the notion that LG100815, while sharing with RA the ability to induce gene expression, also partially antagonizes the inducing effect of RA.

EXAMPLE 2

Effect of LG100815 on RARE-Dependent Induction of Transcription

To confirm the ability of LG100815 to antagonize RARE-dependent induction of transcription analysis of firefly luciferase expression from a RARE-containing artificial promoter DR5 (Stratagene, catalog number 240119) was performed. Cells were plated to the density of 3×10⁵ in P60 24 hrs before transient transfection. DR5 reporter plasmid (4 μg) was mixed with the SV40-driven-Renilla luciferase control plasmid (0.04 μg) and transfected using Lipofectamine Plus (Life Technologies/Invitrogen, Carlsbad, Calif.) as described by the manufacturer. 3 hrs after transfection, cells were rinsed three times with PBS, trypsinized and replated in a 12-well plate to the density of 5×10⁴ cells per well. Retinoid agonists and antagonists were added 48 hrs later, and the luciferase assay was performed after another 24 hrs.

FIG. 1 shows the results of DR5-luciferase transient transfection assays, carried out in the presence of LGD1550, RA, and LG100815, alone or in pairwise combinations. 100 nM concentrations of RA or LGD1550 agonists strongly activated the RARE-containing promoter approximately 50-fold, whereas 10 μM of LG100815 antagonist (the concentration used in the literature for maximal effect) produced an order of magnitude weaker (4.2-fold) induction. On the other hand, the addition of LG100815 to RA or RAR agonist LGD1550 diminished the induction of transcription by the latter compounds 2.5-3 times. These results confirm that LG100815 is inefficient in stimulating RARE-dependent transcription relative to RAR agonists and that it antagonizes the effect of RAR agonists on such transcription.

EXAMPLE 3

Induction of Senescence in Treated Cells

Induction of senescence-associated growth-inhibitory genes by the RAR agonist LGD1550 and the RAR antagonist LG100815 (Table 2) suggests that these compounds may also be able to induce senescence in the treated cells. To test this, we have analyzed the effects of different compounds on the growth of MCF-7 cells, as measured by the cell number after 7 days exposure to the compounds (FIG. 2) and on the fraction of cells expressing the senescence-associated β-galactosidase activity (SA-β-gal), a marker of senescence, after 7-day treatment with the compounds (FIG. 3).

The RAR agonist LGD1550 inhibited the cell growth and induced SA-β-gal to an extent similar to that of RA, demonstrating that RAR stimulation is sufficient to induce senescence. The RXR agonist LGD1268 did not inhibit cell growth and did not induce the senescent phenotype; in fact, LGD1268 treatment produced a modest but reproducible increase in cell growth. The RXR antagonist LG101208 had no effect on the cell growth or the senescent phenotype. The RAR antagonist LG100815 produced both growth inhibition and the induction of the senescence marker; at the highest concentration (10 μM), its effects were similar to those of RA and LGD1550. Hence, an RAR antagonist that inhibits transactivation of RARE-dependent transcription but stimulates the expression of senescence-associated growth-inhibitory genes, induces cell growth arrest and senescence in MCF-7 breast carcinoma cells.

EXAMPLE 4

RAR Agonist and Antagonist Produce Similar Effects on Global Gene Expression

To determine the effects of the RAR agonist and antagonist on the expression of essentially all the human genes, MCF-7 cells were treated with 100 nM of RAR agonist LGD1550 or 10 μM of RAR antagonist LG100815, concentrations that provide maximal induction of gene expression according to Q-PCR assays (as discussed above). Cells were treated for 24, 48 or 72 hrs, and total RNA from the untreated or treated cells was isolated using Qiagen's RNeasy Total RNA Isolation Kit. For gene expression profiling, RNA samples were provided to the microarray service facility of the Wadsworth Center Genomics Institute, which carried out biotinylated target preparation and hybridization with Affymetrix U 133 2.0 Plus oligonucleotide microarrays containing 56,000 probe sets representing 48,500 human transcripts. The hybridization signals were normalized using GCRMA procedure and analyzed using GeneSpring software (Silicone Genetics). The results of the analysis (FIG. 4a) showed good concordance among different time points (e.g. 85-93% of genes induced or inhibited ≧1.5-fold on day 2 were also induced or inhibited ≧1.2-fold, respectively, on day 3). The effects of the agonist and the antagonist also agreed with the results of the earlier analysis of the effects of RA on MCF-7 cells, with all 13 genes previously shown by RT-PCR to be induced in that system (Dokmanovic et al., 2002) also showing induction in the present microarray analysis (FIG. 4b).

Strikingly, the effects of the RAR agonist and the RAR antagonist were exceedingly similar. 74% of the genes showing ≧1.5-fold induction and 77% of the genes showing ≧1.5-fold inhibition by the antagonist were also induced or inhibited, respectively, at least 1.3-fold by the agonist, and vice versa (77% and 69%, respectively). FIG. 5 plots (on the log scale) the maximal changes in gene expression (at any time point) produced by the agonist versus those produced by the antagonist for 11,729 probe sets that showed >1.3-fold changes in gene expression after treatment with either the agonist or the antagonist. The effects of the agonist and antagonist on gene expression show highly significant correlation. The regression through all the data points has an r squared value of 0.6955 with n=11729 (Student's t test yields p<<0.0001). The regression line has a slope of 1.027+/−0.006 (FIG. 5), indicating that the RAR agonist and the RAR antagonist have the same effect on the majority of the affected genes. The similarity of the overall effects of the agonist and the antagonist on gene expression agrees with the results of QPCR analysis of selected genes (see Table 2 above) but contrasts with an order of magnitude weaker effect of the antagonist on RARE-dependent transcription (see FIG. 1).

On the other hand, 316 genes showing the strongest (>5-fold) induction or inhibition by RAR ligands were significantly more responsive to the agonist than to the antagonist (at p<<0.0001), with the regression slope increasing to 1.297+/−0.038 (this translates to approximately 2-fold stronger average effect of the agonist relative to the antagonist) (FIG. 5). Whether preferential induction of the most responsive genes by the agonist could indicate the presence of RARE sequences in the corresponding promoters was considered. Indeed, the gene showing the strongest induction by the agonist (FIG. 5) encodes RA-metabolizing enzyme CYP26A1 (induced 220-fold by the agonist and 23.5-fold by the antagonist), which was reported to contain two synergistically acting RARE sequences in its promoter (Loudig et al., 2005). The effects of the agonist and the antagonist on the expression of 40 human genes, identified by Balmer and Blomhoff (Balmer and Blomhoff, 2005) as containing canonical and evolutionarily conserved RARE sequences in their promoters was examined. Only seven of these 40 genes were induced ≧1.5-fold in MCF-7 cells by the RAR agonist and just three genes were induced by the antagonist. RARE-containing genes induced by both ligands showed 3-5 fold stronger response to the agonist than to the antagonist (FIG. 4c). In particular, HOXA1 was induced 95-fold by the agonist but only 18-fold by the antagonist, HOXA4 was induced 7.8-fold by the agonist and 2.5-fold by the antagonist, and RBP1 was induced 4.4-fold by the agonist and 1.5-fold by the antagonist. Hence, RARE-containing genes that are responsive to RAR ligands in MCF-7 cells indeed show stronger response to the agonist than to the antagonist. However, when promoter sequences of ten randomly chosen genes that show preferential induction by the agonist for the presence of putative RARE sequences were examined (this analysis was carried out using MatInspector program as previously described (Dokmanovic et al., 2002)), only one of ten promoters was found to contain putative RARE sequences. Therefore the majority of genes showing preferential response to the agonist do not contain RARE sequences in their promoters. Without wishing to be bound to any particular theory, this finding can be explained as follows. The agonist induces a small number of genes that have RARE elements in their promoter, whereas the antagonist is less efficient in inducing such genes. Some of these RARE-containing genes in their turn cause the activation of a number of other genes, which don't contain RARE elements. The latter genes, which are also preferentially induced by the agonist, can therefore also be regarded as RARE-dependent, despite the absence of RARE in their promoters.

RNA or protein products of genes that are induced to a similar level by both the RAR agonist and the RAR antagonist can be used as reporters in screening for compounds with properties similar to LG0100815. RNA or protein products of genes from this group (or promoter constructs for such genes) can be used as reporters in screening for compounds that mimic the effect of retinoids. Table 3, as shown below, lists a set of 508 genes chosen as preferred reporters. These genes were selected by being strongly (at least 2-fold) induced by both the agonist and the antagonist relative to untreated cells and showing no more than two-fold difference between their induction by the agonist and the antagonist. 62 genes listed at the top of Table 3 are particularly preferred reporters, as they are most strongly (at least 4-fold) induced by both the agonist and the antagonist. Table 4, as shown below, lists a set of 53 genes that can be used as preferred markers to discriminate between RARE-dependent and RARE-independent induction of transcription. These genes were chosen by being induced at least 3-fold by the agonist relative to untreated cells and also showing at least 4 times stronger induction by the agonist than by the antagonist. FIG. 6 plots the maximal fold induction of gene expression produced by the agonist versus that produced by the antagonist for the genes listed in Table 3 (particularly preferred reporters only) and in Table 4, with the corresponding trend lines. The relative effects of a tested compound on the genes in Table 3 and Table 4 should indicate whether the compound behaves as a RAR agonist or RAR antagonist. For example, one can select matching pairs of genes from Table 3 and Table 4 that are induced by the agonist to approximately the same degree. A compound that mimics the effects of the agonist should induce such genes to a similar level, whereas a compound that behaves like an antagonist should induce the gene from Table 3 to a much greater degree than the gene from Table 4.

TABLE 3
Genes showing strong induction by both RAR agonist and RAR antagonist.
	Maximal effect of	Maximal effect of	Relative
	agonist	antagonist	maximal
		Affymetrix probe	Fold	Raw	Fold	Raw	induction
Genbank ID	Gene name	ID	induction	signal	induction	signal	(antag/agon)
Particularly preferred
NM_014059	RGC32	218723_s_at	49.56	458.52	33.97	313.09	0.69
AI693140	LOC283824	213725_x_at	17.58	248.67	18.59	261.87	1.06
NM_000623	BDKRB2	205870_at	21.40	559.77	16.36	426.27	0.76
AF146343	NR5A2	208343_s_at	28.17	391.17	14.95	207.89	0.53
AL353944	RUNX2	232231_at	14.40	349.06	14.21	343.00	0.99
AI935586		235350_at	15.09	349.00	11.32	260.91	0.75
NM_003999	OSMR	205729_at	10.63	74.54	11.70	82.16	1.10
AF052094	EPAS1	200878_at	18.63	5620.46	10.25	3081.12	0.55
NM_006329	FBLN5	203088_at	10.09	70.00	19.01	131.99	1.88
NM_024430	PSTPIP2	219938_s_at	13.08	182.67	9.79	136.87	0.75
NM_005564	LCN2	212531_at	13.27	19375.20	9.42	13703.50	0.71
X68285	GK	215977_x_at	14.75	152.27	9.06	93.02	0.61
AL359338	CMYA5	233520_s_at	16.94	369.96	8.54	185.81	0.50
NM_000584	IL8	202859_x_at	14.36	139.56	8.24	80.15	0.57
AF228422	NMES1	223484_at	8.21	1084.34	8.69	1148.89	1.06
BE500942	C6orf155	226810_at	7.95	280.01	8.18	286.84	1.03
NM_012449	STEAP	205542_at	7.41	109.11	10.73	157.35	1.45
NM_003128	SPTBN1	200672_x_at	13.66	184.68	7.37	99.28	0.54
AW444617	DCDC2	222925_at	8.21	561.12	7.16	487.38	0.87
AI953847	IBRDC2	228153_at	12.34	794.25	6.74	432.13	0.55
NM_016235	GPRC5B	203632_s_at	11.99	360.51	6.54	197.00	0.55
NM_006763	BTG2	201236_s_at	8.31	724.17	6.54	567.74	0.79
NM_015577	RAI14	202052_s_at	10.63	91.09	6.50	55.48	0.61
BF939996		231098_at	6.87	52.25	6.31	47.76	0.92
NM_004591	CCL20	205476_at	7.59	152.64	6.14	123.50	0.81
NM_018302	FLJ11017	219450_at	10.81	437.79	5.83	235.07	0.54
AI655057	RIT1	239843_at	5.71	71.05	6.32	78.33	1.11
NM_001415	EIF2S3	205321_at	6.48	1267.17	5.68	1106.51	0.88
AI918054		1555893_at	5.65	46.92	5.63	46.83	1.00
AB028976	SAMD4	212845_at	7.89	119.13	5.60	84.24	0.71
U58515	CHI3L2	213060_s_at	5.46	67.16	8.42	103.54	1.54
NM_003979	GPCR5A	203108_at	9.37	2383.55	5.40	1367.87	0.58
NM_024087	ASB9	205673_s_at	7.53	195.71	5.35	138.28	0.71
AI948503	ABCC4	203196_at	8.41	220.20	5.33	139.04	0.63
AL161725	DOCK8	232843_s_at	8.08	243.37	5.17	155.26	0.64
M55580	SAT	210592_s_at	9.95	4553.85	5.08	2313.75	0.51
U73778	COL12A1	231766_s_at	5.00	125.94	8.63	216.58	1.73
AB062292	CTNNB1	1554411_at	5.30	101.79	4.92	94.18	0.93
AL031680	PARD6B	214827_at	4.94	109.64	4.85	107.18	0.98
W46388	SOD2	215223_s_at	4.90	1590.32	4.83	1566.99	0.98
BE207758	ARRB1	222912_at	6.51	73.19	4.76	53.36	0.73
NM_001970	EIF5A	201123_s_at	4.66	2495.21	4.68	2498.82	1.01
NM_005204	MAP3K8	205027_s_at	4.66	224.27	6.04	289.39	1.30
NM_014322	OPN3	219032_x_at	6.88	318.00	4.62	212.55	0.67
AI888150		228494_at	4.59	36.28	7.42	58.67	1.62
AW264102	FAM43A	227410_at	4.59	130.51	4.57	129.32	1.00
BF575213	MGC5618	221477_s_at	5.35	727.20	4.46	605.90	0.83
L39833	KCNAB1	210078_s_at	7.54	77.61	4.44	45.44	0.59
NM_006275	SFRS6	206108_s_at	4.74	365.70	4.43	340.91	0.94
AL021977	MAFF	36711_at	4.68	146.54	4.40	137.09	0.94
M34421	PSG9	209594_x_at	4.35	53.49	4.99	61.09	1.15
AB046692	AOX1	205082_s_at	4.36	97.94	4.34	97.10	1.00
AA156240	RAI3	212444_at	7.59	752.15	4.31	427.30	0.57
AL035689	NCOA7	225344_at	5.36	1202.13	4.24	953.11	0.79
Z21533	HHEX	215933_s_at	6.67	146.87	4.16	91.23	0.62
M63310	ANXA3	209369_at	6.74	1062.27	4.15	652.48	0.62
AA195485	ZKSCAN1	225221_at	4.24	1791.93	4.14	1742.53	0.98
U22178	MSMB	210297_s_at	4.11	60.62	4.72	69.21	1.15
NM_001710	BF	202357_s_at	5.43	968.70	4.11	733.54	0.76
AW025141		228400_at	4.25	87.08	4.04	82.34	0.95
U49396	P2RX5	210448_s_at	5.46	31.21	4.01	22.60	0.74
AB037925	NFKBIZ	223218_s_at	5.61	1705.70	4.01	1221.80	0.72
Other preferred
AF145712	NRP1	210510_s_at	6.89	363.13	3.96	208.02	0.58
AA826324	RASEF	235144_at	7.35	559.95	3.93	298.12	0.53
AA807060	LOC283357	235151_at	3.90	20.80	4.21	22.47	1.08
AW341649	TP53INP1	225912_at	4.84	1086.95	3.90	872.37	0.81
AW501195	FBXW2	241736_at	4.40	32.52	3.88	28.63	0.88
AC004522	ZNF36	214900_at	4.90	297.50	3.87	234.13	0.79
AW052084	WIPI49	213836_s_at	7.51	392.30	3.87	200.83	0.51
AI809961	PFAAP5	221899_at	3.82	52.97	3.96	54.69	1.04
BE502982	YPEL2	227020_at	3.80	368.71	4.13	398.97	1.09
AI129310	C13orf18	44790_s_at	3.97	134.75	3.79	128.24	0.96
AF278532	NTN4	223315_at	7.44	711.60	3.77	359.24	0.51
AL391688	SYTL4	227703_s_at	3.75	140.75	3.83	142.98	1.02
BF576053	CFL2	224663_s_at	3.74	77.99	3.78	78.46	1.01
AI608725	ICAM1	202637_s_at	5.94	198.71	3.67	122.86	0.62
AF345568	GPR81	224131_at	6.53	40.70	3.61	22.42	0.55
AL120021	LOC339924	226158_at	3.57	316.12	3.91	346.54	1.10
AA653300	ZNF36	214670_at	5.24	939.28	3.57	636.23	0.68
AI201594		43511_s_at	4.78	269.49	3.57	200.51	0.75
NM_016548	GOLPH2	217771_at	6.67	62.11	3.55	32.87	0.53
AI569872	FZD4	229441_at	3.65	54.08	3.54	52.19	0.97
L25541	LAMB3	209270_at	3.54	66.53	3.49	65.60	0.99
NM_018191	RCBTB1	218352_at	6.11	197.41	3.47	111.66	0.57
AI810244	MGC7036	227983_at	4.75	384.25	3.46	278.58	0.73
AB032963	ATP8B2	226771_at	4.89	573.51	3.43	400.38	0.70
BC005297	KMO	211138_s_at	3.39	55.36	6.35	103.79	1.87
AI271418	LOC150763	228198_s_at	5.91	284.69	3.38	162.22	0.57
BG036668	C9orf3	212848_s_at	3.36	153.82	3.51	160.13	1.04
AA928542	LOC91137	226831_at	3.36	141.02	3.35	140.25	1.00
NM_003410	ZFX	207920_x_at	3.82	214.68	3.35	187.37	0.88
NM_022969	FGFR2	203638_s_at	3.34	190.95	4.08	232.45	1.22
AI926697	Gup1	228568_at	3.37	31.46	3.34	30.70	0.99
N30649	SQSTM1	213112_s_at	6.30	175.09	3.33	92.25	0.53
NM_013445	GAD1	206670_s_at	4.56	104.39	3.33	75.96	0.73
AW339310	DTNA	227084_at	3.91	190.10	3.31	160.36	0.85
NM_005534	IFNGR2	201642_at	4.54	623.28	3.29	449.84	0.72
NM_000846	GSTA2	203924_at	5.65	291.96	3.27	169.31	0.58
NM_022873	G1P3	204415_at	5.92	2666.37	3.27	1467.82	0.55
BF342851	D2S448	212012_at	3.59	98.45	3.27	89.15	0.91
NM_014398	LAMP3	205569_at	3.25	40.50	3.45	42.83	1.06
AL117653	MITF	226066_at	4.10	110.70	3.22	87.07	0.79
AW263086	KIAA1961	228250_at	3.21	56.47	4.03	70.59	1.25
AI734993	OACT1	227379_at	5.22	701.28	3.20	427.30	0.61
NM_006542	SPHAR	206272_at	3.19	86.06	3.66	98.12	1.15
AL049699	RWDD2	213555_at	3.18	154.52	3.19	154.57	1.00
U37546	BIRC3	210538_s_at	3.90	120.59	3.17	98.17	0.81
AI133452	FGG	226621_at	3.38	377.64	3.17	354.43	0.94
AV700030	IL6R	226333_at	3.14	55.50	3.73	65.78	1.19
BF513121		226189_at	3.13	87.02	4.32	119.55	1.38
BE880703	SGPP1	223391_at	3.83	1629.37	3.12	1320.37	0.81
AW444761	CDKN2B	236313_at	3.17	45.67	3.11	44.61	0.98
BG231554		243179_at	3.10	50.04	4.51	72.43	1.45
AW006750	DRE1	221986_s_at	3.78	219.28	3.09	178.93	0.82
X83493	FAS	215719_x_at	3.08	31.58	3.28	33.44	1.06
AK000004	FGD3	227811_at	5.70	312.89	3.08	168.55	0.54
D42043	RAFTLIN	212646_at	3.08	45.61	3.60	52.97	1.17
BF510581	BTBD11	228570_at	3.08	52.05	3.30	55.79	1.07
AU144565	EPB41L4A	228256_s_at	3.99	67.18	3.07	51.46	0.77
BE545756	ADD3	201034_at	4.44	330.66	3.06	226.51	0.69
NM_147174	HS6ST2	1552767_a_at	3.05	245.34	5.02	401.74	1.64
BF983406	HNRPH1	213470_s_at	3.05	574.43	3.08	578.32	1.01
NM_000416	IFNGR1	202727_s_at	3.04	906.08	3.09	921.34	1.02
NM_017410	HOXC13	219832_s_at	5.55	1217.29	3.04	664.86	0.55
D28124	NBL1	37005_at	3.97	241.19	3.04	183.39	0.76
NM_014965	OIP106	202080_s_at	3.30	167.17	3.03	152.45	0.92
U84138	RAD51L1	210255_at	3.12	64.11	3.01	61.69	0.97
U78168	RAPGEF3	210051_at	3.01	16.71	3.04	16.62	1.01
AI923675	MGC19764	238430_x_at	4.51	218.01	3.00	145.32	0.67
AL831862		1558142_at	2.98	45.42	3.25	49.03	1.09
W65369	SETDB2	235338_s_at	2.98	56.85	3.22	61.45	1.08
U53823	OCLN	209925_at	3.52	394.57	2.97	333.44	0.84
BC001427	SLC35C1	222647_at	4.70	566.64	2.95	353.12	0.63
BE671084	ARHGAP26	205068_s_at	4.80	60.32	2.94	36.82	0.61
AF327722	NARG1	222837_s_at	2.93	110.20	3.14	117.55	1.07
N80935		49111_at	3.90	159.63	2.92	118.92	0.75
BE349147	CPD	201941_at	2.91	128.64	3.45	152.64	1.19
AF251053	BEX2	224367_at	5.29	954.18	2.90	520.98	0.55
NM_018418	SPATA7	219583_s_at	2.90	65.43	4.42	99.44	1.53
NM_000632	ITGAM	205786_s_at	2.89	102.19	3.07	108.38	1.06
NM_021980	OPTN	202074_s_at	3.95	403.35	2.89	294.45	0.73
NM_152569	C9orf66	1552755_at	4.88	358.49	2.89	211.05	0.59
AB040897	RANBP10	221809_at	2.88	60.91	2.98	62.86	1.04
U73844	ELF3	210827_s_at	4.89	1553.45	2.88	911.04	0.59
AI289311	JUB	225806_at	3.02	423.47	2.88	401.35	0.95
NM_003060	SLC22A5	205074_at	4.96	511.28	2.88	295.07	0.58
BE500977	SULF1	212354_at	2.87	360.11	2.94	368.65	1.02
NM_001144	AMFR	202203_s_at	2.89	58.57	2.87	57.97	0.99
AF307097	ZNF317	1555337_a_at	3.05	57.91	2.87	54.27	0.94
NM_006018	GPR109B	205220_at	3.39	33.99	2.86	28.59	0.84
BG109855		213169_at	2.86	43.73	3.80	58.18	1.33
AA128023	STARD13	213103_at	3.26	76.17	2.85	66.50	0.88
AA017721		214046_at	3.35	290.74	2.84	246.24	0.85
NM_003561	PLA2G10	207222_at	4.57	605.12	2.83	370.32	0.62
AK022549	CDW92	224595_at	2.83	1133.49	3.02	1203.29	1.07
AK025862	LRRC28	216450_x_at	2.89	114.24	2.82	110.84	0.98
NM_020639	RIPK4	221215_s_at	2.82	244.86	3.50	304.12	1.24
AY028896	CARD10	210026_s_at	2.82	161.03	2.95	167.14	1.04
NM_006290	TNFAIP3	202644_s_at	2.82	310.68	2.84	312.79	1.01
AI744280	NBEA	239010_at	2.90	27.11	2.82	26.24	0.97
AA485908	INSR	213792_s_at	4.88	130.76	2.82	75.14	0.58
BE501881		227565_at	3.22	134.99	2.82	117.63	0.87
AI023320		229243_at	3.18	23.57	2.81	20.72	0.88
C15005	GRPEL2	238427_at	3.30	38.73	2.81	32.96	0.85
AA128261	SAT2	225272_at	3.20	291.16	2.81	254.49	0.88
NM_001453	FOXC1	1553613_s_at	2.93	219.31	2.81	208.93	0.96
NM_018333	PRPF39	220553_s_at	3.03	229.07	2.80	210.95	0.92
AK025344		225443_at	2.80	71.28	3.01	76.60	1.07
AI356412	LYN	202625_at	2.80	215.02	2.99	228.85	1.07
BF218115	HIPK2	225368_at	3.67	415.92	2.79	314.85	0.76
AL157484		221861_at	3.60	136.58	2.79	105.25	0.77
NM_016044	FAHD2A	218504_at	4.38	796.55	2.79	504.75	0.64
BF983948	SRPRB	222532_at	2.78	483.56	3.15	544.60	1.13
AI660243	TMPRSS2	226553_at	4.46	823.88	2.78	505.81	0.62
NM_002886	RAP2B	214487_s_at	3.18	117.10	2.77	101.58	0.87
NM_001197	BIK	205780_at	2.97	539.38	2.76	502.33	0.93
NM_018013	FLJ10159	218974_at	2.75	48.91	3.58	63.43	1.30
AA191573	SYNJ2	212828_at	5.31	294.31	2.73	150.91	0.51
AK026921	SLC17A5	223441_at	2.73	112.31	2.85	115.81	1.05
NM_006895	HNMT	204112_s_at	2.73	213.78	3.05	238.34	1.12
AA576961	PHLDA1	217996_at	3.73	1335.09	2.72	969.32	0.73
AK026747	LOC54103	222150_s_at	4.52	151.66	2.72	90.79	0.60
AW511222		241879_at	3.57	362.20	2.71	275.99	0.76
NM_022748	TENS1	217853_at	3.60	948.52	2.71	711.72	0.75
AW028110	KIAA0500	213839_at	2.70	64.17	2.92	68.29	1.08
D87811	GATA6	210002_at	4.78	327.81	2.70	184.29	0.56
U10473	B4GALT1	216627_s_at	4.42	69.07	2.70	42.00	0.61
M25915	CLU	208792_s_at	3.42	2463.74	2.70	1935.88	0.79
NM_006705	GADD45G	204121_at	3.85	140.57	2.69	98.43	0.70
AI982758		228328_at	4.44	308.45	2.69	186.11	0.61
NM_006399	BATF	205965_at	2.68	570.94	2.94	622.78	1.10
AB004064	TMEFF2	224321_at	3.41	124.71	2.67	97.26	0.78
AF070596	LOC57146	213272_s_at	2.67	199.45	3.76	280.60	1.41
NM_002341	LTB	207339_s_at	2.66	181.30	2.72	185.06	1.02
NM_014456	PDCD4	202731_at	2.66	1097.30	2.81	1162.07	1.06
BF970340	LOC339448	238010_at	2.66	95.57	2.87	102.85	1.08
NM_003174	SVIL	202565_s_at	2.68	773.23	2.66	767.29	0.99
AI758950	SLC26A7	239006_at	3.24	38.62	2.65	31.46	0.82
NM_017420	SIX4	231797_at	3.41	47.47	2.64	36.62	0.77
AB051495	KIF21A	226003_at	4.17	3244.69	2.64	2045.86	0.63
AP001743	ANKRD3	234730_s_at	2.64	41.03	3.05	47.26	1.16
AI700506	RAD52	236042_at	2.64	22.22	3.01	25.28	1.14
AI963605		230269_at	5.22	84.87	2.63	42.86	0.50
AW628045	CXorf23	232087_at	3.45	66.01	2.63	50.07	0.76
AV720650	KIAA0888	235048_at	2.82	108.27	2.62	100.32	0.93
AW265065		235079_at	2.96	51.84	2.62	45.83	0.89
AL049385	KLHL5	232297_at	3.99	169.17	2.62	110.45	0.66
NM_007107	SSR3	217790_s_at	2.61	245.65	3.05	285.83	1.17
BE788984	GALM	235256_s_at	2.91	84.42	2.61	75.33	0.90
AI357376	NEDD4L	212445_s_at	3.82	287.63	2.60	195.39	0.68
AA532655	FLJ39739	229872_s_at	3.71	501.92	2.59	349.11	0.70
NM_020169	LXN	218729_at	3.60	11822.71	2.59	8491.81	0.72
BF690020	EEF1D	213087_s_at	4.74	117.76	2.58	63.87	0.55
AA133285		230383_x_at	2.58	42.20	3.65	59.46	1.41
AF183569	ARTS-1	209788_s_at	3.83	242.63	2.58	162.69	0.67
BE620832	CPEB4	224831_at	2.58	65.65	2.64	66.93	1.02
AL118571	C10orf74	235016_at	3.27	127.47	2.57	98.89	0.79
AL096776	RHOU	223168_at	2.57	220.76	3.25	277.82	1.26
AI829724	MEF2D	225641_at	2.66	49.54	2.57	47.56	0.97
BF979668	C9orf41	241781_at	2.57	15.92	3.67	22.75	1.43
AFFX-r2-Bs-phe-5		AFFX-r2-Bs-phe-	2.57	81.83	2.68	84.08	1.04
		5_at
BF432550	MYO1B	212364_at	3.66	2326.05	2.56	1621.27	0.70
BG491844	JUN	201464_x_at	2.58	487.30	2.56	483.10	1.00
NM_003411	ZFY	207247_s_at	2.65	70.76	2.56	68.22	0.97
AL136865	ZNF38	223424_s_at	2.56	210.65	2.71	223.37	1.06
NM_016205	PDGFC	218718_at	3.39	35.80	2.55	26.88	0.75
U86755	ADAM17	205746_s_at	2.55	103.29	2.65	107.03	1.04
NM_005766	FARP1	201911_s_at	4.07	1092.20	2.55	680.63	0.63
AI344311	PLDN	224883_at	2.60	123.10	2.54	120.42	0.98
AL110191	DSIPI	208763_s_at	2.84	1914.54	2.54	1705.15	0.90
NM_023039	ANKRA2	218769_s_at	2.54	158.41	2.56	159.49	1.01
AW269686	RAP2B	238622_at	2.54	57.07	3.60	80.69	1.42
NM_020995	HPR	208470_s_at	2.54	41.48	2.71	44.30	1.07
J03068	APEH	201283_s_at	4.54	56.00	2.53	31.31	0.56
BF940761		229427_at	3.50	119.92	2.53	86.34	0.72
NM_005110	GFPT2	205100_at	2.53	42.76	2.90	49.11	1.15
AV688972		239314_at	2.53	48.47	2.94	56.06	1.16
AW973177		236150_at	2.53	70.24	2.90	79.84	1.15
NM_020240	CDC42SE2	1552613_s_at	3.01	162.93	2.52	135.73	0.84
AI400463	LOC255326	229428_at	2.51	56.43	3.35	74.90	1.33
NM_030751	TCF8	208078_s_at	4.59	114.57	2.51	61.84	0.55
AI139252	STAT3	225289_at	2.81	195.93	2.50	173.85	0.89
BE222389	PCDHB4	240317_at	2.50	96.31	2.71	103.68	1.09
AA769615	MGC2610	230434_at	3.35	753.00	2.49	556.99	0.74
BG252842	C6orf62	213875_x_at	2.58	558.60	2.49	536.27	0.97
AB004903	SOCS2	203372_s_at	2.53	109.80	2.48	107.94	0.98
BG054987	RHPN2	227196_at	3.09	915.59	2.48	735.43	0.80
AK000002	ABCC10	213485_s_at	2.48	219.84	3.01	267.10	1.21
AA094434	SLC8A1	241752_at	2.48	32.14	3.82	49.18	1.54
AL563572	MtFMT	235689_at	2.48	150.09	2.55	154.84	1.03
BF221852	LPP	202822_at	2.48	228.89	2.83	260.44	1.14
D85181	SC5DL	211423_s_at	3.59	1312.76	2.47	901.58	0.69
NM_000310	PPT1	200975_at	2.89	2584.14	2.47	2202.42	0.86
NM_024491	Cep70	219036_at	2.72	131.71	2.47	118.92	0.91
NM_016289	CAB39	217873_at	2.51	1808.99	2.47	1771.89	0.98
BF432276		242300_at	2.45	66.68	2.91	79.04	1.19
BE893995		234983_at	2.72	1368.09	2.45	1225.14	0.90
NM_016531	KLF3	219657_s_at	2.49	80.60	2.45	78.90	0.98
NM_014938	MONDOA	202519_at	3.10	132.86	2.44	104.04	0.79
AB029031	TBC1D1	212350_at	2.76	138.39	2.44	121.45	0.88
NM_017412	FZD3	219683_at	2.42	219.49	2.78	251.81	1.15
AL136924	RIN2	209684_at	2.42	72.94	3.14	94.20	1.30
AI652868		225811_at	2.42	54.00	3.79	84.11	1.57
AW117765	PEX13	1556009_at	3.96	493.45	2.41	299.46	0.61
AI810572	PGPEP1	237202_at	2.56	70.89	2.41	65.77	0.94
AW157094	ID4	209291_at	2.41	159.87	2.47	163.03	1.03
AI627850		230446_at	2.41	92.83	2.67	103.26	1.11
AV726376		238436_s_at	2.41	18.14	2.71	20.36	1.13
NM_001038	SCNN1A	203453_at	3.33	1077.74	2.40	766.55	0.72
AI494047		242898_at	2.48	40.19	2.40	38.32	0.97
NM_005059	RLN2	214519_s_at	2.39	509.31	3.19	678.67	1.33
NM_018360	CXorf15	219969_at	2.39	35.84	3.86	57.92	1.61
BE910071	CXorf39	242781_at	2.39	58.08	3.47	84.19	1.46
AI141556	BAIAP1	225474_at	2.49	118.05	2.38	112.27	0.96
NM_018638	ETNK1	219017_at	2.38	145.88	3.84	235.19	1.61
AB020663	DMXL2	212820_at	2.38	153.06	2.89	184.77	1.21
NM_030952	SNARK	220987_s_at	2.57	136.78	2.38	126.26	0.93
NM_014298	QPRT	204044_at	2.93	781.72	2.37	629.21	0.81
AA042983		227755_at	2.37	84.14	3.14	111.18	1.33
NM_013352	SART2	218854_at	2.37	60.04	2.94	74.25	1.24
BF115054	DKFZP564D166	224952_at	2.42	238.96	2.37	232.67	0.98
NM_016315	GULP1	204237_at	2.99	693.55	2.36	545.83	0.79
BF674052	VMP1	224917_at	4.34	2881.95	2.36	1563.97	0.54
NM_002801	PSMB10	202659_at	4.27	1030.67	2.36	568.29	0.55
AW628987		225522_at	3.21	266.24	2.36	194.86	0.73
U55936	SNAP23	209131_s_at	2.35	63.81	4.13	111.25	1.75
NM_003810	TNFSF10	202688_at	2.35	271.95	2.44	280.42	1.04
AI492376		231195_at	2.44	26.48	2.35	25.46	0.97
BG434174	SBLF	213413_at	2.35	16.59	2.69	18.91	1.14
NM_014840	ARK5	204589_at	2.35	600.19	2.70	688.00	1.15
AI823592	KIAA0423	213304_at	2.84	710.46	2.35	587.69	0.83
AW973842	C10orf46	227257_s_at	2.86	116.82	2.34	95.21	0.82
H17038		226612_at	2.34	140.16	3.89	231.77	1.66
AW043602	KIAA1946	227370_at	2.33	448.54	2.95	565.19	1.26
NM_005724	TM4SF8	200973_s_at	2.70	487.38	2.33	417.53	0.86
AB037730	KLHL13	227875_at	2.76	132.63	2.32	111.23	0.84
BG339050		225155_at	2.33	4861.54	2.32	4828.85	1.00
NM_022071	SH2D4A	219749_at	2.40	157.60	2.32	152.42	0.97
AW169333	CBWD1	229804_x_at	2.39	208.49	2.32	201.58	0.97
NM_001621	AHR	202820_at	2.92	475.93	2.32	376.43	0.79
NM_000821	GGCX	205351_at	2.47	100.74	2.32	94.15	0.94
AW196959	C14orf150	235025_at	2.93	245.06	2.32	193.14	0.79
AK026966	AK3	225342_at	2.32	94.53	2.57	105.14	1.11
AB046783	ALS2	226291_at	2.31	72.27	2.32	72.40	1.01
AI394529	PRKAG1	227527_at	2.32	100.11	2.31	99.00	0.99
AB023179	KIAA0962	212911_at	2.76	57.87	2.30	47.84	0.83
NM_006736	DNAJB2	202500_at	2.63	58.90	2.30	51.69	0.88
AW340595		227384_s_at	2.37	17.36	2.29	16.75	0.97
AI700633		212812_at	2.92	2650.99	2.29	2085.02	0.79
AW006441	MGC52110	226039_at	2.33	130.35	2.29	127.26	0.98
AA724565	MGC34732	237160_at	2.28	164.45	2.89	207.96	1.26
AW069729	ACPL2	226925_at	3.74	198.56	2.28	120.75	0.61
N79004	SIX1	228347_at	4.55	814.67	2.28	402.32	0.50
NM_020190	OLFML3	218162_at	2.28	86.09	2.40	90.78	1.05
NM_001206	KLF9	203543_s_at	2.35	86.60	2.28	83.26	0.97
AI279819	LOC400451	221880_s_at	4.10	941.69	2.28	521.07	0.56
AW592684	LIFR	227771_at	3.60	27.29	2.28	17.15	0.63
X96588	RYK	216976_s_at	2.27	329.39	2.76	398.01	1.21
AL136710	ANK3	209442_x_at	2.27	304.95	2.32	309.69	1.02
U77914	JAG1	216268_s_at	2.29	217.63	2.27	215.99	0.99
AW206602	ZSCAN2	231188_at	3.29	116.44	2.27	80.11	0.69
AW779917		230003_at	3.23	140.21	2.27	98.01	0.70
AL565362	SLC2A13	227176_at	2.43	77.10	2.27	71.58	0.93
AF285167	ABCA1	203505_at	2.26	229.07	2.43	245.26	1.07
AI984074	RPL7	239493_at	2.49	20.26	2.26	18.33	0.91
AU154321	KPNA6	212101_at	2.26	91.01	2.53	101.27	1.12
NM_018133	FLJ10546	218733_at	2.31	167.84	2.26	163.56	0.98
AF041209	MID1	210694_s_at	2.26	25.14	2.57	28.46	1.14
NM_002925	RGS10	204319_s_at	3.05	915.59	2.26	679.43	0.74
BF968057	IRF2BP2	224571_at	2.40	587.78	2.25	550.05	0.94
AI139990		231022_at	2.74	129.26	2.25	105.66	0.82
W73272	PDE8A	212522_at	4.47	1611.41	2.25	808.15	0.50
AA812993	LOC120376	228338_at	2.25	76.76	2.81	95.98	1.25
BC005352	TNFAIP8	210260_s_at	2.33	107.84	2.25	103.79	0.97
AF230929	ANXA9	210085_s_at	2.24	1089.22	2.27	1097.48	1.01
BE675337	GSN	214040_s_at	2.24	121.96	2.79	150.96	1.24
AF339824	HS6ST3	232276_at	2.71	65.50	2.24	53.88	0.83
AI799018	EPHA4	227449_at	2.24	57.87	2.91	74.96	1.30
NM_004703	RABEP1	203223_at	3.03	338.02	2.24	249.75	0.74
NM_003916	AP1S2	203300_x_at	2.69	46.41	2.23	38.20	0.83
AF131850	EI24	216396_s_at	2.23	326.91	2.63	385.74	1.18
BF109906		239153_at	2.81	236.51	2.23	186.98	0.79
NM_004114	FGF13	205110_s_at	2.93	542.42	2.23	413.32	0.76
NM_015400	SMAD3	218284_at	4.25	495.99	2.23	259.65	0.53
BF435123	MSI2	225237_s_at	2.28	286.52	2.23	278.13	0.98
BE889301		244007_at	2.54	19.88	2.23	17.34	0.88
NM_024610	HSPBAP1	219284_at	2.23	87.46	2.23	87.47	1.00
BG289443		244561_at	3.27	86.30	2.22	58.31	0.68
AB028869	BIRC5	210334_x_at	2.67	161.06	2.22	131.65	0.83
BG230586	SLC7A6	203578_s_at	2.56	60.43	2.22	52.05	0.87
AI631833		227396_at	2.59	369.66	2.22	315.46	0.86
AI554300	SERPINB1	213572_s_at	4.05	474.94	2.22	259.15	0.55
AI827990	SLC25A16	214140_at	2.94	38.56	2.22	28.94	0.75
AF115512	DNAJB9	1554462_a_at	2.22	170.86	2.26	174.08	1.02
NM_014782	ARMCX2	203404_at	2.21	77.36	2.84	98.80	1.28
BC005122	ARFGAP3	202211_at	2.54	203.27	2.21	175.93	0.87
BE973687	HES1	203394_s_at	2.96	984.23	2.21	731.42	0.75
AC007130	HIBADH	231955_s_at	2.61	406.74	2.21	342.56	0.85
AI659800	FLJ38725	228937_at	2.20	44.72	2.93	59.15	1.33
BE677761	MSCP	221920_s_at	2.63	32.97	2.20	27.56	0.84
BE465380	ARNT	231016_s_at	2.22	74.92	2.20	74.43	0.99
AV725364	MGC35048	225511_at	3.38	59.31	2.20	38.47	0.65
BC003637	DDIT3	209383_at	2.51	328.33	2.20	286.94	0.88
NM_002293	LAMC1	200771_at	4.18	869.01	2.20	455.00	0.53
BE897886	PIGF	212120_at	2.20	477.85	2.20	475.86	1.00
AL515381	CORO2A	227177_at	2.19	65.93	3.14	92.77	1.43
BC000758	C6orf80	209479_at	2.46	364.00	2.19	322.89	0.89
AJ271379	SPIN3	1555882_at	2.19	33.07	3.74	56.46	1.71
AA533109	LOC401431	229094_at	2.19	19.97	2.28	20.76	1.04
AK025446	CSIG	212019_at	2.19	31.86	2.54	36.85	1.16
AW450329		226381_at	2.26	196.84	2.19	190.07	0.97
AF225981	ATP2C1	209934_s_at	2.66	32.44	2.19	26.59	0.82
AF277181	LOC85028	223774_at	2.24	105.83	2.19	103.16	0.98
AK074366	ZNF621	1558620_at	2.19	40.94	2.90	54.13	1.33
T87542	WDFY2	227490_at	3.12	80.98	2.19	56.85	0.70
AU154740	STAF65(gamma)	201836_s_at	2.19	34.87	2.36	37.70	1.08
AL096842	MTUS1	212096_s_at	2.47	665.60	2.18	585.71	0.88
AA456973	PC4	221727_at	2.18	77.92	2.64	93.86	1.21
BC000185	CPT1A	210688_s_at	2.18	119.09	2.75	149.75	1.26
AL573722	FLJ90024	226239_at	2.30	95.20	2.18	89.69	0.95
NM_000332	ATXN1	203232_s_at	2.18	228.34	2.70	281.10	1.24
NM_004289	NFE2L3	204702_s_at	2.49	77.59	2.18	67.53	0.87
BE742268	SORT1	212797_at	2.59	192.09	2.18	160.59	0.84
AW271788	NDUFB2	226391_at	2.32	29.63	2.18	27.56	0.94
AK001291	NCKAP1	217465_at	2.18	132.65	2.25	136.30	1.03
NM_015516	TSK	218245_at	2.83	421.10	2.17	322.17	0.77
AB017493	KLF6	208961_s_at	2.17	54.62	2.53	63.42	1.17
NM_019094	NUDT4	206302_s_at	2.34	623.08	2.17	580.18	0.93
AI954660	C17orf27	225931_s_at	2.27	60.55	2.17	57.70	0.96
AK000445	HOXC9	231936_at	2.45	61.55	2.17	54.04	0.89
AA551142	PHACTR2	204048_s_at	2.85	748.78	2.17	566.64	0.76
AI819238	ID2	213931_at	2.64	24.73	2.17	20.17	0.82
BC040952	PIK3C2A	1569022_a_at	2.56	68.80	2.16	58.26	0.85
AB040875	SLC7A11	209921_at	2.47	107.04	2.16	93.19	0.87
AW242916	IL6ST	212196_at	2.16	473.64	2.19	482.40	1.02
AA100250	DHX57	213420_at	2.15	73.88	2.19	74.91	1.02
AI554514		229796_at	2.53	569.56	2.15	480.93	0.85
AV753204	MAP3K9	213927_at	2.40	74.22	2.15	66.51	0.90
BF000047		235736_at	2.15	60.05	2.75	76.95	1.28
NM_002178	IGFBP6	203851_at	2.60	211.46	2.14	173.63	0.83
AW137982	HOXA3	235521_at	2.14	26.88	2.39	29.94	1.11
AW006123	FBXO32	225803_at	2.14	148.96	2.26	156.52	1.05
BF589251		227776_at	2.14	42.69	2.71	53.91	1.27
AI458439		231929_at	2.63	132.82	2.14	107.61	0.81
AF029750	TAPBP	208829_at	4.00	1486.80	2.14	791.19	0.53
NM_020130	C8orf4	218541_s_at	2.85	40.30	2.13	30.03	0.75
NM_022459	XPO4	218479_s_at	2.89	42.90	2.13	31.14	0.74
AA044835	SLC35F5	225872_at	2.17	1064.66	2.13	1038.98	0.98
BF574430		235059_at	2.13	293.65	2.13	292.33	1.00
AW418666	HINT3	226537_at	2.12	111.52	2.38	125.09	1.12
AF151046	C3orf19	223787_s_at	2.12	80.75	2.63	99.88	1.24
NM_012382	OSRF	219421_at	2.31	58.43	2.11	52.97	0.91
U75667	ARG2	203946_s_at	2.11	15.09	2.18	15.34	1.03
AF087847	GABARAPL1	208869_s_at	3.71	50.82	2.11	28.84	0.57
NM_019058	DDIT4	202887_s_at	2.32	769.51	2.11	702.40	0.91
AF314544	TBL1XR1	222634_s_at	2.51	122.82	2.11	103.53	0.84
NM_004734	DCAMKL1	205399_at	2.11	65.08	2.33	71.58	1.10
BC019922	ZNF252	1558722_at	2.17	138.44	2.11	133.91	0.97
NM_138995	MYO3B	1552578_a_at	3.34	77.32	2.11	48.65	0.63
BC021215	FLJ11193	1552660_a_at	2.11	156.54	2.96	219.17	1.41
AF275800	MGC5306	222728_s_at	2.10	346.15	2.30	376.61	1.09
NM_004363	CEACAM5	201884_at	3.85	95.32	2.10	51.85	0.55
AI990326	MPHOSPH9	221965_at	2.10	14.01	2.65	17.56	1.26
AFFX-M27830_M		AFFX-	2.82	1856.84	2.10	1377.96	0.74
		M27830_M_at
AA196245	EXT2	202012_s_at	2.15	150.26	2.10	145.60	0.97
NM_019061	PIP3AP	220953_s_at	2.09	37.79	2.68	47.78	1.28
AB014540	SWAP70	209307_at	2.23	49.60	2.09	46.26	0.94
NM_004578	RAB4A	203582_s_at	2.13	778.46	2.09	766.06	0.98
AY007243	REG4	223447_at	2.09	228.34	2.17	236.02	1.04
AK096921		1558105_a_at	2.79	203.69	2.09	151.59	0.75
AA496034	LOC55971	227372_s_at	2.09	252.98	2.12	256.38	1.02
AK056658	FLJ32096	1555870_at	2.98	22.02	2.09	15.34	0.70
AA121673	ZNF281	228785_at	2.28	851.05	2.08	777.96	0.91
BF056095	TMEM42	226361_at	2.47	124.49	2.08	104.19	0.84
AL136855	DKFZp434K2435	223594_at	2.08	111.84	2.40	129.34	1.16
NM_007236	CHP	207993_s_at	2.32	73.89	2.08	65.93	0.90
AI432196	NR3C1	201865_x_at	2.08	406.15	2.22	433.24	1.07
NM_003670	BHLHB2	201170_s_at	3.46	329.44	2.07	196.56	0.60
AY038927	DUSP16	1555399_a_at	2.30	28.67	2.07	25.78	0.90
AA025858	CRTAP	227138_at	2.07	28.73	2.24	30.55	1.08
AI439752	SMAD5	235451_at	2.30	155.69	2.07	139.44	0.90
AV718192	TRIO	209012_at	2.07	38.34	2.13	39.37	1.03
BC002427	CASP2	209811_at	3.72	48.83	2.06	26.75	0.56
NM_002393	MDM4	205655_at	2.06	48.40	3.09	72.72	1.50
AW612407		230098_at	2.32	18.38	2.06	16.24	0.89
NM_005980	S100P	204351_at	2.33	1213.97	2.06	1068.60	0.88
NM_000375	UROS	203031_s_at	2.13	162.12	2.06	155.96	0.97
X16135	HNRPL	35201_at	2.16	513.42	2.06	485.74	0.95
BF699855	GALNT7	222587_s_at	2.51	270.16	2.05	218.08	0.82
BE378670	MGC9850	224857_s_at	2.58	921.93	2.05	731.54	0.80
NM_004414	DSCR1	208370_s_at	3.64	244.75	2.05	137.53	0.56
BC005127	ADFP	209122_at	2.05	465.12	2.12	479.62	1.04
N25562	PTK9	214008_at	2.05	45.29	2.13	46.77	1.04
AL136139	NEDD9	202149_at	2.24	1198.86	2.05	1094.66	0.92
NM_018222	PARVA	217890_s_at	2.17	160.38	2.05	150.35	0.94
BM968434	ZNF286	1557684_at	2.29	59.17	2.05	52.67	0.89
AI611074	CDKL5	227004_at	3.20	126.90	2.05	80.97	0.64
AA541479	MAP3K1	225927_at	2.05	640.64	2.06	640.12	1.00
NM_000456	SUOX	1553030_a_at	2.05	58.46	3.53	100.04	1.73
NM_007173	PRSS23	202458_at	2.05	2332.41	2.22	2514.53	1.08
NM_004090	DUSP3	201538_s_at	2.94	153.75	2.05	107.27	0.70
BF972755	VHL	1559227_s_at	2.05	51.65	2.67	67.60	1.31
AJ005866	SLC35D2	213083_at	2.73	256.38	2.04	191.08	0.75
AB002344	JMJD3	41386_i_at	2.48	49.96	2.04	40.94	0.82
AW361702	PSMD7	238738_at	2.04	45.42	2.33	51.59	1.14
AL137370	DKFZp434H2226	232893_at	2.04	17.21	2.17	18.33	1.06
NM_006291	TNFAIP2	202510_s_at	2.41	135.69	2.04	115.14	0.85
BF038869		235322_at	2.13	41.98	2.04	39.87	0.95
BG292233	INSIG1	201626_at	2.04	776.46	2.33	885.79	1.15
AP001745	PRDM15	225539_at	2.04	78.14	3.40	130.79	1.67
AU145950	TGFB2	228121_at	2.04	41.53	2.66	54.00	1.31
NM_003901	SGPL1	208381_s_at	2.14	38.45	2.03	36.40	0.95
AW467077	LOC284408	235779_at	2.03	110.63	2.16	117.06	1.06
AI039874	NQO1	201467_s_at	2.56	6006.97	2.03	4706.32	0.80
NM_020327	ACVR1B	208223_s_at	2.81	32.53	2.03	23.29	0.72
AW268719		236798_at	2.03	157.04	2.83	218.47	1.40
BC046206	ZNF26	1555325_s_at	2.03	64.93	3.67	116.69	1.81
AI184512	CTMP	229253_at	2.03	24.87	2.24	27.30	1.10
BG236163	KIAA1219	221738_at	2.03	38.37	2.95	55.53	1.45
AK023014	ARHGAP5	233849_s_at	2.03	102.71	2.13	107.30	1.05
BF197707	FLJ30656	212529_at	2.09	201.93	2.03	195.23	0.97
J02923	LCP1	208885_at	2.25	189.70	2.03	169.88	0.90
AK022014	AKAP13	222024_s_at	2.03	20.93	2.51	25.67	1.24
N25732	FOXO3A	204131_s_at	2.53	1325.26	2.02	1053.09	0.80
NM_022470	WIG1	219628_at	2.30	106.38	2.02	93.40	0.88
NM_019044	FLJ10996	219774_at	2.02	52.41	2.25	58.25	1.11
NM_018357	FLJ11196	218651_s_at	2.24	26.44	2.02	23.71	0.90
NM_013332	HIG2	218507_at	2.19	1320.58	2.02	1208.52	0.92
AW959427	DNCL2B	238116_at	2.02	23.80	2.69	31.70	1.34
AL121883	ARMCX3	222444_at	2.01	552.98	2.84	780.21	1.41
BG390493	STYX	244030_at	2.01	238.65	2.46	290.46	1.22
L78132	LGALS8	208935_s_at	2.79	300.96	2.01	213.68	0.72
NM_002194	INPP1	202794_at	2.01	30.38	2.09	31.59	1.04
BE965029	MICAL2	212473_s_at	2.01	223.12	2.23	248.15	1.11
BF444943	USP24	212381_at	2.01	64.87	2.16	69.36	1.07
NM_005952	MT1X	208581_x_at	3.10	2649.72	2.01	1719.45	0.65
AA337048	TEAD2	238323_at	2.81	124.69	2.01	87.88	0.72
AA001423	AMIGO	226718_at	2.40	108.27	2.01	89.33	0.84
NM_004129	GUCY1B2	220506_at	2.63	31.31	2.01	23.77	0.76
AI911972		230780_at	3.89	232.52	2.00	119.32	0.52
AL047052	LOC23117	235060_at	2.00	27.38	3.66	49.37	1.83
AI769569	MAML2	235457_at	3.92	32.21	2.00	16.47	0.51
J04152	TACSTD2	202286_s_at	2.17	10226.30	2.00	9388.66	0.92
CA442342		1556097_at	3.11	30.60	2.00	19.70	0.64
BC000576	QDPR	209123_at	2.02	244.71	2.00	242.32	0.99
AI567426	TLE3	212769_at	2.72	98.78	2.00	71.80	0.73
BE504242	LOC158402	236769_at	2.00	12.59	3.78	23.72	1.89

TABLE 4
Genes showing strong preferential induction by the RAR agonist relative to the antagonist.
	Maximal effect of	Maximal effect of	Relative
	agonist	antagonist	maximal
		Affymetrix probe	Fold	Raw	Fold	Raw	induction
Genbank ID	Gene name	ID	induction	signal	induction	signal	(antag/agon)
CYP26B1	NM_019885	219825_at	179.84	1122.80	2.41	14.96	0.01
TRIM31	NM_007028	208170_s_at	35.70	280.19	1.89	14.81	0.05
APXL2	AA588854	239435_x_at	63.13	2943.72	4.40	204.15	0.07
CYP26A1	NM_000783	206424_at	220.23	2729.21	23.46	289.57	0.11
HOXA3	T89711	230080_at	10.38	219.24	1.11	23.33	0.11
SLC1A1	AW235061	213664_at	8.96	50.61	1.07	6.04	0.12
RNASE1	NM_002933	201785_at	49.39	706.93	5.99	85.44	0.12
HPS3	AI922198	227253_at	21.01	212.31	2.70	27.15	0.13
KRT4	X07695	213240_s_at	9.38	279.97	1.27	37.27	0.13
CP	AI684991	228143_at	10.88	57.74	1.52	8.09	0.14
	CA448125	1557050_at	8.90	128.10	1.37	19.60	0.15
IGFBP3	BF340228	212143_s_at	16.12	296.21	2.49	45.62	0.15
HSXIAPAF1	AA142842	228617_at	17.54	396.67	2.76	62.15	0.16
GBP3	AL136680	223434_at	7.32	169.25	1.18	27.20	0.16
TRIM31	X81006	215444_s_at	12.16	235.45	2.07	39.84	0.17
APXL2	AL138455	241935_at	5.82	227.53	1.01	39.19	0.17
CEACAM1	NM_001712	206576_s_at	16.23	236.29	2.81	40.79	0.17
	BF593636	228642_at	130.43	964.04	23.01	169.44	0.18
CP	AL556703	1558034_s_at	44.25	324.77	7.85	57.39	0.18
OAS2	NM_016817	204972_at	15.91	262.84	2.84	46.29	0.18
	CA448125	1557051_s_at	23.74	671.29	4.26	120.03	0.18
GBP1	BC002666	202269_x_at	5.99	47.16	1.10	8.60	0.18
CYP1A1	NM_000499	205749_at	7.33	35.09	1.35	6.45	0.18
LOC134548	AI744123	230238_at	5.56	37.93	1.03	7.02	0.19
IFIT4	AI075407	229450_at	16.59	2294.88	3.09	426.20	0.19
	AK024898	227306_at	11.73	166.77	2.20	31.15	0.19
HOXA1	S79910	214639_s_at	95.33	726.74	18.35	139.31	0.19
PCSK2	NM_002594	204870_s_at	6.32	67.97	1.24	13.27	0.20
	AW510657	228904_at	10.13	137.42	2.05	27.49	0.20
KDR	NM_002253	203934_at	8.75	135.14	1.78	27.43	0.20
HOXA13	BG289306	231786_at	4.87	33.91	1.01	6.99	0.21
LOC145757	AK056534	1558649_at	4.71	25.05	1.01	5.32	0.21
IFI44L	NM_006820	204439_at	7.95	36.49	1.71	7.84	0.22
GPX2	NM_002083	202831_at	38.21	1926.26	8.34	418.80	0.22
CP	NM_000096	204846_at	67.09	433.38	14.65	94.27	0.22
KIAA1359	AB037780	231941_s_at	11.07	193.70	2.46	42.96	0.22
SELE	NM_000450	206211_at	4.54	28.14	1.02	6.31	0.22
PDK4	AV707102	225207_at	4.76	44.76	1.08	10.14	0.23
ChGn	NM_018371	219049_at	13.08	216.76	3.01	50.01	0.23
OAS2	AI651594	228607_at	7.28	153.38	1.68	35.23	0.23
CEACAM1	X16354	209498_at	39.62	216.69	9.17	49.98	0.23
PDE5A	AB015656	1553175_s_at	7.55	152.03	1.76	35.26	0.23
CEACAM1	M76742	211883_x_at	4.51	146.82	1.05	34.16	0.23
	BF221547	227088_at	26.46	253.51	6.24	59.53	0.24
LOC121838	AI680459	232318_s_at	28.28	330.40	6.70	77.93	0.24
FLJ32115	AI051248	227450_at	173.10	2721.33	41.18	644.97	0.24
FLJ11127	NM_019018	219694_at	22.82	199.13	5.45	47.42	0.24
TRIM31	AF230386	210159_s_at	17.09	319.39	4.11	76.49	0.24
LOC129607	AI742057	226702_at	6.01	958.79	1.45	230.73	0.24
TGFBI	NM_000358	201506_at	53.01	6739.18	12.84	1625.97	0.24
RSAD2	AI337069	213797_at	7.54	130.76	1.83	31.61	0.24
PDE5A	NM_001083	206757_at	16.48	158.21	4.03	38.56	0.24
FLJ10901	NM_018265	219010_at	4.05	328.02	1.01	81.19	0.25

Claims

1. A method for inducing growth arrest of proliferating cells comprising contacting the cells with a retinoic acid receptor (RAR)-modulating compound that induces RARE-independent retinoid-responsive gene expression and that is inefficient in inducing RARE-dependent gene expression.

2. The method according to claim 1, wherein the proliferating cells are neoplastic cells.

3. The method according to claim 1, wherein the proliferating cells are in a human.

4. A method for identifying a RAR-modulating compound that induces growth arrest of proliferating cells and that induces RARE-independent retinoid-responsive gene expression and that is inefficient in inducing RARE-dependent gene expression comprising providing proliferating cells, contacting the cells with a test compound, determining the level of RARE-independent retinoid-responsive gene expression, determining the level of RARE-dependent gene expression and comparing the levels of RARE-independent retinoid-responsive gene expression with the level of RARE-dependent gene expression, wherein a test compound that increases the expression of RARE-independent retinoid-responsive genes relative to cells not treated with the test compound and that produces the highest ratio of the expression of RARE-independent retinoid-responsive genes to the expression of RARE-dependent genes, is determined to be a RAR-modulating compound that is inefficient in inducing RARE-dependent gene expression and induces cell growth arrest.

5. The method according to claim 4, wherein RARE-independent retinoid-responsive gene expression and RARE-dependent gene expression are determined by providing cells transfected with a first gene encoding a first detectable protein operatively linked to a promoter of a RARE-independent retinoid-responsive gene and a second gene encoding a second detectable protein that is different from and separately detectable in the presence of the first detectable protein operatively linked to a promoter of a RARE-dependent gene, measuring the levels of expression of the first and second detectable proteins, and comparing the levels of expression of the first and second detectable proteins.

6. The method according to claim 4, wherein the levels of RARE-independent retinoid-responsive gene expression and RARE-dependent gene expression is further compared to cells treated with a compound known to induce RARE-dependent gene expression.

7. The method according to claim 5, wherein the detectable protein is selected from the group consisting of firefly luciferase, Renilla luciferase, beta-galactosidase, chloramphenicol acetyltransferase, horseradish peroxidase, green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, fluorescent protein DsRed, alkaline phosphatase and immunologically detectable proteins or peptides.

8. The method according to claim 4, wherein RARE-independent retinoid-responsive gene expression and/or RARE-dependent gene expression is determined by quantitative reverse transcription PCR.

9. The method according to claim 4, wherein RARE-independent retinoid-responsive gene expression and/or RARE-dependent gene expression is determined by microarray analysis.

10. The method according to claim 4, wherein the compounds are derivatives of existing RAR agonists or antagonists.

11. The method according to claim 4, wherein the RARE-independent retinoid-responsive genes are selected from the group consisting of IGF-BP3, EPLIN, FAT10, βIG-H3, RGC32, NR5A2, BDKRB2, EPAS1, LOC283824, CMYA5, A1935586, GK, RUNX2, IL8, SPTBN1, LCN2, PSTPIP2, IBRDC2, GPRC5B, FLJ1017, RAI14, OSMR, FBLN5, SAT, GPCR5A, ABCC4, BTG2, DCDC2, NMES1, DOCK8, C6orf155, SAMD4, CCL20, ASB9, STEAP, BF939996, EIF2S3, RIT1, A1918054, CHI3L2 and COL12A1.

12. The method according to claim 4, wherein the RARE-dependent genes are selected from the group consisting of CYP26A1, CYP26B1, HOXA1, APXL2, RNASE1, CP, CA448125, HPS3, HSXIAPAF1, IFIT4, CEACAM1, IGFBP3, TCRGC2, TRIM31, AK024898, HOXA3, KRT4, SLC1A1, GBP3 and PCSK2.

13. A compound identified by the method of claim 4.

14. The method according to claim 5, wherein the levels of RARE-independent retinoid-responsive gene expression and RARE-dependent gene expression is further compared to cells treated with a compound known to induce RARE-dependent gene expression.

15. The method according to claim 8, wherein the levels of RARE-independent retinoid-responsive gene expression and RARE-dependent gene expression is further compared to cells treated with a compound known to induce RARE-dependent gene expression.

16. The method according to claim 9, wherein the levels of RARE-independent retinoid-responsive gene expression and RARE-dependent gene expression is further compared to cells treated with a compound known to induce RARE-dependent gene expression.