Method for identifying hormonally modulated genes

Abstract

This invention provides methods of identifying hormone-regulated traits in a cell. The methods involve cultivating the cell as a graft in two different hormonal environments and determining whether expression of the trait differs in the two grafts.

What is claimed is:  
     
         1 . A method for identifying a hormone-regulated trait comprising the steps of: 
 a) exposing grafts of a biological material to different hormonal environments in first and second non-human animals of different reproductive states, and    b) detecting a difference in expression of a trait in the biological material between the first and second animals;    whereby detecting a difference identifies the trait as a hormone-regulated trait.    
     
     
         2 . The method of  claim 1  wherein the hormonal environments are natural hormonal environments and the non-human animals are same species, non-human mammals.  
     
     
         3 . The method of  claim 2  wherein the first non-human mammal is a sexually mature female and second non-human mammal is a sexually mature male.  
     
     
         4 . The method of  claim 2  wherein the first and second non-human mammals differ in a state of sexual maturity.  
     
     
         5 . The method of  claim 2  wherein the first and second non-human mammals are females differing in a reproductive status selected from cycling, pregnant or pseudo-pregnant.  
     
     
         6 . The method of  claim 3  wherein the biological material comprises human benign or malignant neoplastic cells from a laryngeal cancer, a breast cancer, an ovarian cancer, a uterine cancer, a cervical cancer, an endometrial cancer, a vaginal cancer, a testicular cancer, a prostate cancer, a leiomyoma or a benign prostate hyperplasia.  
     
     
         7 . The method of  claim 3  wherein the biological material comprises human endocrine cells or human exocrine cells from gall bladder, adrenal gland, pancreas, thyroid, salivary gland, pituitary gland, hypothalamus, ovary or testis.  
     
     
         8 . The method of  claim 3  wherein the biological material comprises hormonally responsive human cells selected from adipocytes, osteoblasts, osteoclasts, chondrocytes, hematopoietic cells, cells from lymphoid or myeloid cell lines, lymphoreticular cells, cells from neural tissue, cells from hair follicles, cells from sebaceous glands, cells from bladder tissue, cells from male or female reproductive tract tissue or cells from mammary gland tissue.  
     
     
         9 . The method of  claim 3  wherein the biological material comprises hormonally responsive human cells selected from endometrial tissue, endometriotic tissue, pelvic floor tissue or vaginal tissue.  
     
     
         10 . The method of  claim 3  wherein the trait is expression of a gene.  
     
     
         11 . The method of  claim 10  wherein the step of detecting a difference in expression of a gene comprises performing subtractive hybridization on cDNA isolated from the biological material from the first and second animals, and identifying a cDNA species that is either more abundant or less abundant in the biological material from one animal compared with the other.  
     
     
         12 . The method of  claim 11  further comprising the step of isolating the cDNA species.  
     
     
         13 . The method of  claim 3  wherein the trait is protein expression, post-translational protein modification or protein secretion.  
     
     
         14 . The method of  claim 3  wherein the trait is expression of a structural or functional trait of a cell or a tissue.  
     
     
         15 . The method of  claim 14  wherein the trait is rate of cell cycling, anchorage dependence of cells, tumor staging or histochemical staining.  
     
     
         16 . The method of  claim 3  wherein the non-human mammals are genetically immunocompromised.  
     
     
         17 . The method of  claim 3  wherein the non-human mammals are chemically immunocompromised or immunocompromised from exposure to radiation.  
     
     
         18 . The method of  claim 3  wherein the non-human animals are rodents.  
     
     
         19 . The method of  claim 3  wherein the non-human animals are nude, athymic mice.  
     
     
         20 . The method of  claim 3  wherein the step of exposing comprises exposing human tumor tissue in the non-human mammal for a time sufficient for the tumor to become palpable.  
     
     
         21 . A model system for identifying hormone-regulated traits comprising at least two non-human, same species animals of different reproductive states, each animal hosting a graft of a biological material, whereby the grafts are exposed to different hormonal environments.  
     
     
         22 . The model system of  claim 21  wherein the hormonal environments are natural hormonal environments and the non-human animals are non-human mammals.  
     
     
         23 . The model system of  claim 22  at least one non-human mammal is a sexually mature female and at least one non-human mammal is a sexually mature male.  
     
     
         24 . The model system of  claim 22  wherein at least two non-human mammals differ in a state of sexual maturity.  
     
     
         25 . The model system of  claim 22  wherein at least two non-human mammals are females differing in a reproductive status selected from cycling, pregnant or pseudo-pregnant.  
     
     
         26 . The model system of  claim 22  wherein the biological material comprises human benign or malignant neoplastic cells from a laryngeal cancer, a breast cancer, an ovarian cancer, a uterine cancer, a cervical cancer, an endometrial cancer, a vaginal cancer, a testicular cancer, a prostate cancer, a leiomyoma or a benign prostate hyperplasia.  
     
     
         27 . The model system of  claim 22  wherein the biological material comprises human endocrine cells or human exocrine cells from gall bladder, adrenal gland, pancreas, thyroid, salivary gland, pituitary gland, hypothalamus, ovary or testis.  
     
     
         28 . The model system of  claim 22  wherein the biological material comprises hormonally responsive human cells selected from adipocytes, osteoblasts, osteoclasts, chondrocytes, hematopoietic cells, cells from lymphoid or myeloid cell lines, lymphoreticular cells, cells from neural tissue, cells from hair follicles, cells from sebaceous glands, cells from bladder tissue, cells from male or female reproductive tract tissue or cells from mammary gland tissue.  
     
     
         29 . A method for identifying a trait whose expression is regulated by a hormone-regulated gene, the method comprising the steps of: 
 a) exposing a graft of biological material comprising the hormone-regulated gene to a natural hormonal environment in a first non-human animal, the environment comprising a hormone that up-regulates or down-regulates expression of the gene;    b) exposing a graft of the biological material to the hormonal environment in a second non-human animal, wherein cells of the material are provided with means for down-regulating expression of the gene if the hormone up-regulates its expression, or means for up-regulating expression of the gene if the hormone down-regulates its expression; and    c) detecting whether a trait is differently expressed in the graft from the first animal compared with the second animal;    whereby detecting a difference identifies the trait as regulated by the hormone-regulated gene.    
     
     
         30 . The method of  claim 29  wherein the means for up-regulating comprises an expression vector comprising an expression control sequence operative in the cells and operatively linked with a nucleotide sequence encoding the gene, and the means for down-regulating comprises an antisense polynucleotide that specifically hybridizes with a nucleotide sequence of the gene or its promoter.  
     
     
         31 . The method of  claim 30  wherein the expression control sequence is inducible or repressible.  
     
     
         32 . A method for determining whether a first gene regulates expression of a second, hormone-regulated gene, the method comprising the steps of: 
 a) exposing a graft of biological material comprising the first and second genes to a natural hormonal environment in a first non-human animal, wherein the hormonal environment comprises a hormone that regulates expression of the second hormone-regulated gene;    b) exposing a graft of the biological material to the same hormonal environment in a second non-human animal, wherein the cells are provided with means for up-regulating or down-regulating expression of the first gene; and    c) detecting whether the second gene is differently expressed in the first animal and the second animal;    whereby detecting a difference provides a determination that the first gene regulates expression of the second, hormone-regulated gene.    
     
     
         33 . The method of  claim 32  wherein the means for up-regulating comprises an expression vector comprising an expression control sequence operative in the cells and operatively linked with a nucleotide sequence encoding the gene, and the means for down-regulating comprises an antisense polynucleotide that specifically hybridizes with a nucleotide sequence of the gene or its promoter.  
     
     
         34 . The method of  claim 33  wherein the expression control sequence is inducible or repressible.  
     
     
         35 . A method for detecting a differential effect of a drug on animals of at least two different reproductive states, the method comprising the steps of: 
 a) providing at least two immunocompromised, non-human, same species animals of different reproductive states serving as hosts for grafts of hormonally responsive biological material;    b) administering to the animals equivalent dosage regimens of the drug and    c) recording a statistically significant (p<0.05) difference in the effect of the dosage regimen on a trait of the biological material in the animals.    
     
     
         36 . The method of  claim 35  wherein the hormonal environments are natural hormonal environments and the non-human animals are non-human mammals.  
     
     
         37 . The method of  claim 36  wherein at least one mammal is a sexually mature female and at least one animal is a sexually mature male.  
     
     
         38 . The method of  claim 36  wherein at least two mammals differ in a state of sexual maturity.  
     
     
         39 . The method of  claim 36  wherein at least two mammals are females differing in a reproductive status selected from cycling, pregnant or pseudo-pregnant.  
     
     
         40 . A method for determining reproductive-state-specific dosages of a pharmaceutical comprising the steps of: 
 a) providing at least two immunocompromised, non-human, same species animals of different reproductive states serving as hosts for grafts of hormonally regulated, diseased, human tissue;    b) administering to the animals equivalent dosage regimens of the pharmaceutical; and    c) determining the efficacy of the pharmaceutical in treating the diseased tissue in each of the animals of the set.    
     
     
         41 . The method of  claim 40  wherein the hormonal environments are natural hormonal environments and the non-human animals are non-human mammals.  
     
     
         42 . The method of  claim 41  wherein at least one mammal is a sexually mature female and at least one mammal is a sexually mature male.  
     
     
         43 . The method of  claim 41  wherein at least two mammals differ in a state of sexual maturity.  
     
     
         44 . The method of  claim 43  wherein at least two mammals are females differing in a reproductive status selected from cycling, pregnant or pseudo-pregnant.  
     
     
         45 . A method for determining whether an agent modulates expression of a disease-associated, hormone regulated trait, wherein the trait is expression of a gene, comprising the steps of: 
 (a) exposing diseased biological material comprising a disease-associated, hormone regulated gene to a hormonal environment in an animal that regulates expression of the gene;    (b) contacting the biological material with the agent;    (c) measuring the amount of expression of the gene; and    (d) determining whether the measured amount is different than an amount of expression of the gene in the cell under control conditions in which the biological material is not exposed to the agent;    whereby a difference between the measured amount and the amount under control conditions indicates that the agent modulates expression of the gene.    
     
     
         46 . The method of  claim 45  wherein the step of exposing comprises exposing the cell to the hormonal environment in an animal in vivo.  
     
     
         47 . The method of  claim 46  wherein the hormonal environment is a natural hormonal environment.  
     
     
         48 . The method of  claim 45  wherein the agent is an agent that modulates the action of a hormone that regulates expression of the gene.  
     
     
         49 . The method of  claim 48  wherein the agent modulates the action of estrogen or testosterone.  
     
     
         50 . The method of  claim 49  wherein the agent is an estrogen analog.  
     
     
         51 . The method of  claim 45  wherein the step of measuring comprises measuring an amount of mRNA transcribed from the gene.  
     
     
         52 . The method of  claim 51  wherein the step of measuring comprises an amount of protein encoded by the gene.  
     
     
         53 . The method of  claim 46  further comprising the steps of: 
 identifying a second hormone regulated trait;  
 measuring the amount of expression of the second trait in the animal; and  
 determining whether the measured amount of expression of the second trait is different than an amount of expression of the second trait in the animal under control conditions in which the biological material is not exposed to the agent.  
 
     
     
         54 . The method of  claim 53  further comprising the steps of: 
 determining the degree of difference between the measured amounts of gene expression to provide a degree of gene expression modulation;  
 determining the degree of difference between the measured amounts of expression of the trait to provide a degree of trait expression modulation; and  
 determining whether the degree of gene expression modulation is different than the degree of trait expression modulation;  
 whereby a difference indicates whether the agent preferentially modulates expression of the gene.