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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

• the invention generally concerns methods of evaluating biological activities of agents, e.g., whether the agents inhibit or activate cellular signal transduction pathways such as TGF- ⁇ and activin signaling pathways and other pathways mediated by Smad proteins.
• TGF- ⁇ Transforming growth factor ⁇
• TGF- ⁇ superfamily encompasses over 50 evolutionarily conserved members that are found in all metazoan organisms.
• BMPs bone morphogenetic proteins
• GDFs growth and differentiation factors
• MIS Mullerian inhibitory substance
• GDNF glial cell line-derived neurotropic factor
• TGF- ⁇ Members of the TGF- ⁇ family exert a wide range of biological effects on a variety of cell types. They have been implicated in normal physiology as well as a variety of diseases. For example, TGF- ⁇ is involved in morphogenesis (epithelial-to-mesenchymal transformation), control of cell growth, chemotaxis, and extracellular matrix formation and maintenance (e.g., in wound repair of both soft and hard tissues), development and function of immune cells (lymphocytes, macrophages, and dendritic cells), and hematopoietic homeostasis.
• morphogenesis epidermal growth
• chemotaxis e.g., in wound repair of both soft and hard tissues
• extracellular matrix formation and maintenance e.g., in wound repair of both soft and hard tissues
• immune cells lymphocytes, macrophages, and dendritic cells
• hematopoietic homeostasis hematopoietic homeostasis.
• TGF- ⁇ plays a role
• diseases include, for example, chronic inflammatory disorders, fibrotic disease, scar formation, carcinogenesis, parasitic infections, and autoimmune diseases.
• TGF- ⁇ as well as other members of the superfamily represent important targets for the development of novel therapeutic agents.
• TGF- ⁇ family members initiate their cellular action by binding to cell surface receptors that possess intrinsic kinase activity in the cytoplasmic domain.
• the receptors initiate signaling events that ultimately lead to changes in gene expression.
• a group of several receptor-activated proteins named Smads function to transduce the signal directly from the TGF- ⁇ superfamily receptor kinases to the nucleus.
• Smads are phosphorylated on C-terminal serines by the type I TGF- ⁇ receptor. Upon phosphorylation, Smads undergo heterodimerization with the common mediator Smad4. The resulting complex translocates into the nucleus where it regulates target genes.
• Smad2 and Smad3 are the principal transducers of signals from TGF- ⁇ and activin receptors (Massague et al. (2000) EMBO J., 19:1745-1754). Other pathways can also modulate the activity of Smad2 and Smad3 (see, e.g., Yakymovych et al. (2001 ) FASEB J., 15:553-555; and Kretzschmar et al. (1999) Ras. Genes Dev., 13:804-816).
• Nuclear Smad3/Smad4 and Smad2/Smad4 complexes bind either directly to DNA consensus sites or via a variety of coactivators, corepressors, and transcription factors which brings about the multifaceted patterns of gene regulation attributed to the TGF- ⁇ .
• the Smad3/Smad4 complexes bind DNA directly at a consensus CAGA motif (also known as "CAGA box") (Zawel et al. (1998) MoI. Cell, 1 :611-617; Dennler et al.
• Smad-binding elements such as CAGA boxes can confer TGF- ⁇ inducibility to heterologous promoters (see, e.g., Zawel et al. (1998) MoI. Cell, 1 :611-617; Dennler et al. (1998) EMBO J., 11:3091-3100; Jonk et al. (1998) J. Biol. Chem., 273:21145-21152; Johnson et al.
• PAI-1 Plasminogen activator inhibitor type 1
• the PAI-1 promoter includes three CAGA boxes and was found to be inducible by TGF- ⁇ in vitro and in vivo (Dong et al. (1996) supra).
• the baseline in vivo expression of PAI-1 is nearly undetectable (Dichek (1989) In Vitro Cell. Dev. Biol., 25:289-292).
• the invention provides an assay system for evaluating pharmacodynamic properties of compounds that modulate Smad3-mediated gene regulation, and the TGF- ⁇ /activin signaling pathway, in particular.
• the invention further provides a reporter construct responsive in vivo to a member of the TGF- ⁇ superfamily (e.g., TGF- ⁇ or activin) and comprises a tandem of N Smad-binding elements operably linked to a reporter gene, wherein N is an integer greater than 12 and less than 51.
• N is an integer greater than 12 and less than 51.
• N equals 25 ⁇ K, wherein K is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12.
• N equals 25.
• the Smad-binding element comprises a CAGA box having the nucleotide sequence X1X 2 X 3 -CAG AC-X 4 (SEQ ID NO:1 , with the proviso that X 1 is A or G, X 2 is G or T, X 3 is C, A, G or T, and X 4 is A or C, all independently of each other.
• each CAGA box comprises the nucleotide sequence AGCCAGACA (SEQ ID NO:2).
• CAGA are positioned in tandem so that each of the 9-bp CAGA box sequences is separated by 3-6 bps from the next one.
• the invention further provides vectors containing reporter constructs of the invention; cells and animals carrying the reporter constructs of the invention; and cell and tissue extracts prepared from these cells and animal tissues.
• the animal is a transgenic mouse carrying a luciferase transgene operably linked to a tandem of twenty-five CAGA boxes.
• a viral vector carrying a reporter transgene operably linked to a tandem of twenty-five CAGA boxes is introduced into a non-transgenic mouse by a systemic injection.
• the invention provides a method of evaluating Smad-mediated signaling in an animal carrying a reporter construct of the invention.
• the method includes determining the amount of the reporter gene expressed, and optionally stimulating reporter gene expression in the animal, for example, by administering a member of the TGF- ⁇ superfamily to the animal and/or by inducing endogenous production of a member of the TGF- ⁇ superfamily in the animal.
• the reporter gene is expressed at a detectable level for at least 6 hours following the administration of the member of the TGF- ⁇ superfamily.
• the invention provides a method of evaluating the effect of a test compound or composition on Smad-mediated signaling (Smad- mediated gene regulation).
• the method includes administering the test compound or composition to the animal of carrying a reporter construct of the invention and determining the expression level of the reporter gene.
• the expression level of the reporter gene indicates the effect of the test compound or the composition on the Smad-mediated signaling.
• the method of evaluating the effect of a test compound or composition optionally includes stimulating reporter gene expression in the animal, for example, by administering a member of the TGF- ⁇ superfamily to the animal and/or by inducing endogenous production of a member of the TGF- ⁇ superfamily in the animal.
• the reporter gene is expressed at the detectable level starting within 2 hours following the induction for a period of at least 6 hours with the maximal expression level at around 30 times the baseline level.
• the administration of the test compound is performed before, concurrently with, or after the stimulation of reporter gene expression.
• Figure 1 depicts the phylogenetic tree of the TGF- ⁇ superfamily. The sequences compared are human unless noted: (c) C. elegans; (x) Xenopus; (m) murine; (b) bovine; (z) zebrafish; (d) Drosophila; (s) sea urchin.
• SEQ ID NO:1 is a generic sequence of the CAGA box XIX 2 X S -CAGAC-X 4 , wherein Xi is A or G, X 2 is G or T, X 3 is C, A, G or T, and X 4 is A or C, all independently of each other.
• SEQ ID Nos:2-9 are examples of CAGA boxes present in the various promoters of human genes as listed in Table 1.
• SEQ ID NO:10 and SEQ ID NO:11 are oligonucleotides containing 6 SBEs uses in the preparation of (CAGA) 25 reporter construct. DETAILED DESCRIPTION OF THE INVENTION
• SBE Smad-binding element
• SBEs include but are not limited to CAGA boxes which are found in natural and synthetic TGF- ⁇ /activin responsive promoters, such as for example, 3TP (Wrana et al. (1992) Cell, 71 :1003-1014), PAI-1 (Zonneveld et al. (1988) Prot. Natl. Acad. Sci.
• Zawel et al. identified another 8-bp palindromic sequence GTCTAGAC (SEQ ID NO:3) as a Smad-binding element (SBE) (Zawel et al. (1998) MoI. Cell, 1 :611-617; United States Patent No. 6,100,032).
• Suitable assays for determining the binding of a polynucleotide to Smads include but are not limited to gel-shift assays (EMSA) with a Mad-homology 1 (MH1 ) domain and/or full-length Smads, as well as antibody supershift assays and TGF- ⁇ -inducible cell-based reporter gene assays.
• ESA gel-shift assays
• MH1 Mad-homology 1
• antibody supershift assays and TGF- ⁇ -inducible cell-based reporter gene assays.
• TGF- ⁇ -inducible cell-based reporter gene assays are described in the Examples and also, for example, in Zawel et al. (1998) MoI. Cell, 1 :611-617; United States Patent No. 6,100,032; Transforming Growth Factor-Beta Protocols, ed. Howe, P.H., Humana Press, 2000.
• CAGA box refers to an SBE that comprises at least (a) the 5-bp palindromic sequence CAGAC (including the corresponding inverted nucleotide sequence GTCTG).
• tandem of SBEs refers to a positioning of SBEs within a nucleotide sequence, in which SBEs are separated by no more than 35 bps.
• transfection is used interchangeably with the terms “transduction” and “transformation” and refers to the intracellular introduction of a polynucleotide.
• modulating and its cognates refer to either reducing/inhibiting or increasing/stimulating/activating any biological activity associated with a specified biological process (e.g., the binding of Smad3 to DNA, inhibition of TGF- ⁇ ) or any biological activity (unless otherwise stated) associated with a specified molecule (e.g., inhibition of TGF- ⁇ by a test compound).
• biological activity refers to a function or set of functions (or the effect to which the function is attributed to) performed by a molecule in a biological system, which may refer to in vivo or in vitro systems, depending on the context.
• binding and its cognates mean that two molecules form a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity. Nonspecific binding usually has a low affinity with a moderate to high capacity. Typically, the binding is considered specific when the affinity constant K a is higher than 10 6 M “1 , or preferably higher than 10 8 M "1 . If necessary, nonspecific binding can be reduced without substantially affecting specific binding by varying the binding conditions. Such conditions are known in the art, and a skilled artisan using routine techniques can select appropriate conditions. II. Compositions and Methods
• a viral vector carrying a reporter transgene and twenty-five tandem CAGA boxes was introduced into mice by a tail vein injection.
• the expression of the reporter was then stimulated by peritoneal injection of TGF- ⁇ .
• Some mice expressing the reporter transgene were dosed with inhibitors prior to the TGF- ⁇ stimulation.
• the present invention is based, in part, on the discovery and demonstration that the TGF- ⁇ stimulation resulted in robust and sustainable reporter expression that can be selectively inhibited by TGF- ⁇ pathway inhibitors. Accordingly, the invention provides an assay system for evaluating pharmacodynamic properties of compounds that modulate Smad-mediated gene regulation, and the TGF- ⁇ /activin signaling pathway, in particular.
• the invention provides a reporter construct responsive in vivo to a member of the TGF- ⁇ superfamily (e.g., TGF- ⁇ or activin) and comprises a tandem of N Smad-binding elements operably linked to a reporter gene, wherein N is an integer greater than 12 and less than 51 , greater than 15 and less than 51 ; greater than 15 and less than 45; greater than 15 and less than 33; greater than 15 and less than 27; greater than 15 and less than 21; greater than 21 and less than 51 ; greater than 21 and less than 45; greater than 21 and less than 33; greater than 21 and less than 27; greater than 27 and less than 51 ; greater than 27 and less than 51 ; greater than 27 and less than 45; greater than 27 and less than 33; greater than 33 and less than 51 ; greater than 33 and less than 45; greater than 45 and less than 51 ; greater than 27 and less than 33, or N equals 22, 23, 24, 25, 26, 27, or 28.
• N is an integer greater than 12 and less than 51 , greater than 15 and less than 51 ;
• the Smad-binding element comprises a CAGA box having the nucleotide sequence X 1 X 2 X 3 -CAGAC-X 4 (SEQ ID NO:1 ), with the proviso that X 1 is A or G, X 2 is G or T, X 3 is C, A, G, or T, and X 4 is A or C, all independently of each other.
• each CAGA box comprises the nucleotide sequence AGCCAGACA (SEQ ID NO:2).
• the Smad-binding element comprises a CAGA box having the nucleotide sequence XiX 2 X 3 -CAGAC-X 4 (SEQ ID NO:1 ), with the proviso that X ⁇ is A or G, X 2 is G or T, X 3 is C, A, G or T, and X 4 is A or C, all independently of each other.
• each CAGA box comprises the nucleotide sequence AGCCAGACA (SEQ ID NO:2).
• a Smad-binding element comprises the nucleotide sequence GTCTAGAC (SEQ ID NO:3).
• the SBEs in the tandem are separated by 3-6 bps. In other embodiments, they are positioned so that the separation is no more than 35, 30, 25, 20, 15, 12, 9, or 7 bps.
• the reporter constructs of the invention comprise a "promoter” and a reporter gene.
• promoter refers to a regulatory element that directs the transcription of a nucleic acid to which it is operably linked.
• a promoter can regulate both rate and efficiency of transcription of an operably linked nucleic acid.
• a promoter may also be operably linked to other regulatory elements which enhance (“enhancers”) or repress ("repressors”) promoter-dependent transcription of a nucleic acid.
• operably linked refers to a nucleic acid placed in a functional relationship with another nucleic acid. A promoter may be positioned 5' (upstream) or 3' (downstream) of a transcription initiation site in the nucleic acid.
• a promoter may also encompass regions both 5' and 3' of the transcription initiation site of the operably linked nucleic acid.
• a reporter gene can be any measurable expression product, most typically a protein. Commonly used reporter genes include but are not limited to ⁇ -galactosidase, alkaline phosphatase, chloramphenicol acetyl transferase (CAT), DsRed, ⁇ -galactosidase, ⁇ -glucuronidase, green fluorescent protein (GFP), ⁇ -lactamase luciferase, LacZ, red fluorescent protein; or fusions (chimeras) thereof (see, e.g., Spergel et al. (2001 ) Prog.
• the reporter construct of the invention when introduced into a host cell or animal can be episomal, or chromosomally integrated as, for example, in transgenic animals carrying the reporter construct of the invention.
• the invention further provides vectors containing reporter constructs of the invention.
• vectors both viral and nonviral
• retroviral vectors which include vectors derived from Moloney murine leukemia virus (MoMLC), lentiviral vectors (see, e.g., Englund (2002) Dev. Brain Res., 134:123-141 ; Tamaki (2002) J. Neurosci.
• AAV adeno-associated viral
• HSV-1 herpes-simplex-1 viral
• Ad adenoviral vectors. Naked DNA, liposomes, and molecular conjugates can also be used.
• the level of transgene expression in eukaryotic cells is largely determined by the transcriptional promoter within the transgene expression cassette. Promoters that show long-term activity and are tissue- and even cell-specific are used in some embodiments. Nonlimiting examples of promoters include but are not limited to the cytomegalovirus (CMV) promoter (Kaplitt et al. (1994) Nat. Genet, 8:148-154), CMV/human ⁇ 3-globin promoter (Mandel et al. (1998) J. Neurosci., 18:4271-4284), GFAP promoter (Xu et al.
• CMV cytomegalovirus
• Virol., 72:5085-5092 or the bovine growth hormone (BGH) polyadenylation site.
• BGH bovine growth hormone
• pharmacological regulation of gene expression can be obtained by including various regulatory elements and drug-responsive promoters as described, for example, in Habermaet al. (1998) Gene Ther., 5:1604-16011 ; and Ye et al. (1995) Science, 283:88-91.
• the invention further provides cells carrying the reporter constructs of the invention.
• Cells can be primary cell cultures (e.g., mesenchymal cells (e.g., chondrocytes, fibroblasts, myocytes, etc.), stem cells (e.g., hematopoietic or neural stem cells), and other cells types. Suitable cells also include established clonal cell lines. Generally, most available cell lines can be transfected with the vectors of the invention. In some embodiments, the cell line is a hepatoma cell line, HepG2, a colorectal carcinoma cell line, CT26, a breast carcinoma cell line, Mx-1 , or a nonsmall cell lung tumor cell line, Calu-6. Examples of other commonly used cell lines also include COS cells, CHO cells, and other cell lines. Such cell lines are widely available commercially.
• C. Animals e.g., mesenchymal cells (e.g., chondrocytes, fibroblasts, myocytes, etc.
• the invention further provides animals carrying the reporter constructs of the invention and tissues (including organs and cells) derived from these animals.
• tissues include liver, kidney, skin, muscle, lung, brain, fat, spleen, testes, and gastrointestinal tract tissues.
• the animal is a non-transgenic rodent (e.g., a mouse) carrying a luciferase transgene operably linked to a tandem of twenty-five CAGA boxes which is introduced into the animal by systemic injection.
• rodent e.g., a mouse
• suitable animals include mammals, including but not limited to rats, rabbits, sheep, pigs, dogs, cats, monkeys, chimpanzees, and guinea pigs.
• 6,265,387 which describes a method for transfecting the mouse liver through an intravenous injection of hypertonic solution containing naked plasmid DNA. Additionally, delivery of retroviral vectors to the liver is described, for example, in Ferry et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8377-8381 and Kay et al. (1992) Hum. Gene Then, 3:641-647; delivery of adenoviral gene transfer vectors via the portal vein in rats is described, for example, in Rosefeld et al. (1991 ) Science, 252:431-434.
• the animal is a transgenic rodent (e.g., a mouse) carrying a luciferase transgene operably linked to a tandem of twenty-five CAGA boxes.
• transgenic rodent e.g., a mouse
• suitable animals include mammals, including but not limited to rats, rabbits, sheep, pigs, dogs, cats, monkeys, chimpanzees, and guinea pigs.
• mammals including but not limited to rats, rabbits, sheep, pigs, dogs, cats, monkeys, chimpanzees, and guinea pigs.
• Transgenic Mouse Methods and Protocols Hofker, M. H., van Deursen, J. (eds.) Humana Press, 2002
• Transgenesis Techniques Principles and Protocols, 2nd ed., Clark, A.R.
• the transgenics are generated by lentiviral transfection of single-cell mouse embryos as described, for example, in Lois et al. (2002) Science, 295:868-872. Briefly, lentivirus carrying the reporter transgene will be used to infect single-cell embryo and stably transmit the transgene to the zygote via integration into the genome. The modified embryos are then returned to foster mothers to complete development, and those born will be screened for the presence of transgene.
• transgenic animals can be produced using pronuclear microinjection as described, for example, in U.S. patent No. 4,873,191.
• the invention further provides cell and tissue extracts prepared from cells and animal tissues comprising the reporter constructs of the invention. Methods of making cell and tissue extracts are well known in the art and are described in the Examples and in the references cited.
• the invention provides a method of evaluating Smad-mediated signaling in an animal (non-transgenic or transgenic) carrying a reporter construct (including a vector) of the invention.
• the method includes determining the amount of the reporter gene expressed, and optionally stimulating reporter gene expression in the animal, for example, by administering a member of the TGF- ⁇ superfamily to the animal and/or by inducing endogenous production of a member of the TGF- ⁇ superfamily in the animal.
• the member of TGF- ⁇ superfamily which is administered to induce reporter gene expression, is TGF- ⁇ .
• TGF- ⁇ is TGF- ⁇ .
• Other suitable members of the TGF- ⁇ superfamily include but are not limited to members of TGF- ⁇ subfamily and members of activin subfamily as shown in Fig 1.
• TGF- ⁇ 1 - ⁇ 5 isoforms of TGF- ⁇
• TGF- ⁇ 1 - ⁇ 5 all of which are homologous among each other (60-80% identity), form homodimers of about 25 kDa, and act upon common TGF- ⁇ receptors (T ⁇ R-l, T ⁇ R-ll, T ⁇ R-IIB, and T ⁇ R-lll).
• TGF- ⁇ 1 , TGF- ⁇ 2, and TGF- ⁇ 3 are found in mammals.
• the structural and functional aspects of many members of the TGF- ⁇ superfamily are well known in the art. For a review of the TGF- ⁇ superfamily and Smad signaling pathways, see, for example, Cytokine Reference, eds. Oppenheim et al., Academic Press, San Diego, CA, 2001 , and Mehra et al. (2002) Biochem. Cell Biol., 80:605-622).
• administering is not limited to any particular delivery system and may include, without limitation, parenteral (including subcutaneous, intravenous, intramedullary, intraarticular, intramuscular, or intraperitoneal injection), rectal, topical, transdermal, or oral (for example, in capsules, suspensions, or tablets). Administration may occur in a single dose or in repeat administrations, and in any of a variety of physiologically acceptable salt forms, and/or with an acceptable pharmaceutical carrier and/or additive as part of a pharmaceutical composition (described earlier).
• Physiologically acceptable salt forms and standard pharmaceutical formulation techniques and excipients are well known to persons skilled in the art (see, e.g., Physician's Desk Reference (PDR) 2003, 57th ed., Medical Economics Company, 2002; and Remington: The Science and Practice of Pharmacy, eds. Gennado et al., 20th ed, Lippincott, Williams & Wilkins, 2000).
• PAI-1 plasminogen activator inhibitor I
• a member of the TGF- ⁇ family is administered at a dose of 0.1 ng/kg to 100 ⁇ g/kg.
• the exact dose can be readily determined by one ordinary skill in the art through mere routine experimentation.
• the reporter gene expression can also be stimulated by inducing endogenous production of TGF- ⁇ in the animal.
• TGF- ⁇ there are numerous conditions that lead to increased production of TGF- ⁇ .
• Such conditions include but are not limited to cancer (Dumont et al. (2000) Breast Cancer Res. 2:125-132), wound healing, inflammation, autoimmune disease, ischemia, atherosclerosis, reperfusion, fibrosis, xenograft, CNS injury, diabetic nephropathy (Chen et al. (2001 ) Ren. Failure, 23:471) and other renal disease (Cheng et al. (2002) Exp. Biol. Med., 227:943), including lupus nephritis, IgA nephropathy, etc.
• TGF- ⁇ For a review of the role of TGF- ⁇ in human disease, see, e.g., BIobe et al. (2000) New EnI. J. of Med. 342(18): 1350-1358).
• Certain animal cancer models (spontaneous, transgenic/knock-out, syngeneic) have been developed that are characterized by elevated levels of TGF- ⁇ production. Such models and conditions characterized by elevated levels of TGF- ⁇ are described, for example, in Border et al. (2004) New Engl. J. of Med., 331(19):1286-1292; Cheng et al. (2002) Exp. Biol. Med., 227:943.
• the amount of the reporter gene expressed can be determined by any suitable method.
• Expression levels at the RNA or at the protein level, can be determined using routine methods. Expression levels are usually scaled and/or normalized per total amount of RNA or protein in the sample and/or a control, which is typically a housekeeping gene such as actin or GAPDH. RNA levels are determined by quantitative PCR (e.g., RT-PCR), Northern blotting, or any other method for determining RNA levels, e.g., as described in Sambrook et al. (eds.) Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989, or Lodie et al. (2002) Tissue Eng., 8(5):739-751), or as described in the Examples.
• quantitative PCR e.g., RT-PCR
• Northern blotting or any other method for determining RNA levels, e.g., as described in Sambrook et al. (eds.) Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989, or
• Protein levels are determined using, Western blotting, ELISA, enzymatic activity assays, or any other method for determining protein levels, e.g., as described in Current Protocols in Molecular Biology (Ausubel et al. (eds.) New York: John Wiley and Sons, 1998).
• the expression levels are determined using either luciferase activity in cell/tissue extracts or by tissue or whole animal imaging.
• tissue imaging on living animals can be performed by fluorescence detection (Hoffman (2002) Lancet Oncol., 3:546-556; Tung et al. (2000) Cancer Res., 60:4953-4958), bioluminescence detection (Shi (2001 ) Proc. Nat. Acad. Sci. USA, 98:12754-12759; Luke et al. (2002) J. Virol., 76:12149-12161 , and U.S. Patent Nos.
• the maximal expression level of the reporter is observed within 24, 12, 10, 8, 6, 4, and 2 hours following induction and is expressed at a detectable level for at least 4, 6, 8, 10, 12, 18, 24, 36, or 48 hours.
• the maximal expression level of the reporter gene following TGF- ⁇ induction is at least 3, 5, 10, 20, 30, 50, or 100 times greater than the corresponding base level of the reporter gene expression prior to the induction.
• the reporter gene is expressed at the detectable level starting within 2 hours following the induction for a period of at least 6 hours with the maximal expression level at around 30 times the baseline level.
• the invention provides a method of evaluating the effect of a test compound or composition on Smad-mediated signaling.
• the method includes administering the test compound or composition to the animal of carrying a reporter construct of the invention; and determining the expression level of the reporter gene.
• the expression level of the reporter gene indicates the effect of the test compound or the composition on the Smad-mediated signaling.
• the method of evaluating the effect of a test compound or composition optionally includes stimulating reporter gene expression in an animal, for example, by administering a member of the TGF- ⁇ superfamily to the animal and/or by inducing endogenous production of a member of the TGF- ⁇ superfamily in the animal.
• the examples of a member of the TGF- ⁇ superfamily that can be administered include those shown in Fig. 1.
• the member of the TGF- ⁇ superfamily administered is a member of the TGF- ⁇ subfamily (e.g., TGF- ⁇ 1 , TGF- ⁇ 2, and TGF- ⁇ 3), a member of the activin subfamily activin (e.g., activin ⁇ A and activin ⁇ B).
• the administration of the test compound is performed before, concurrently with, or after the stimulation of the reporter gene expression. Stimulation of the TGF- ⁇ expression and the methods of determining the reporter gene expression levels are described above.
• Another method for detecting the in vivo efficacy of T ⁇ RI inhibitors is by using cell lines (e.g., CT26 colorectal carcinoma cells or CaIu-6, nonsmall ceil lung tumor cells) stably transfected with a reporter construct carrying the TGF- ⁇ inducible promoter (CAGA) 2S operably linked to a reporter gene (e.g., the firefly luciferase reporter (CAGA) 25 -fLuc)).
• CAGA TGF- ⁇ inducible promoter
• CAGA the firefly luciferase reporter
• stably transfected cells are injected into the animal (e.g., into the tail vein in rodents). These cells colonize and form tumors (e.g., CT26 colonize in the lungs).
• the expression of the reporter can then be induced by endogenous or exogenous TGF- ⁇ .
• the ability of compound to inhibit TGF- ⁇ -dependent response, and therefore Smad-mediated signaling is then assessed.
• the assessment can be accomplished non-invasively by monitoring the level of luciferase activity over time using, for example, the MS® biophotonic imaging system (Xenogen, Alameda, CA). This technique allows acquisition of multiple (“real-time”) measurements from the same live animal.
• a method the invention comprises determining reporter gene expression levels at varying time points in the same live animal.
• cells e.g., CT26, Calu-6, or Mx-1 cells
• a second reporter construct carrying the constitutively expressed SV40 promoter linked to the renilla luciferase reporter (SV40-rl_uc). Since the firefly luciferase and the renilla luciferase utilize different substrates without cross reactivity, dual tracking of cells' responsiveness to TGF- ⁇ and their growth in the same animal can be accomplished using the two different substrates consecutively.
• a method of the invention comprises determining a first and a second reporter gene's expression levels at varying time points in the same live animal.
• Example 1 In vivo transfection and inhibition studies
• the first generation adenoviral vector (E1 , E3 deleted, serotype 5) (CAGA) 25 -GL (Ad.(CAGA) 25 -GL) was created using 293 cells as described in Ng et al. (2000) Human Gene Therapy 11 :693-699. Purification was performed by double cesium chloride density equilibrium gradient centrifugation. The vector was stored at -8O 0 C in 10 mM Tris-HCI, 1 mM MgCI 2 , 10% v/v glycerol, pH 8.0.
• liver homogenates for luciferase activity assay frozen livers were pulverized using the BioPulverizerTM (BioSpec). Pulverized livers were homogenized in 500 ⁇ l of luciferase assay buffer (LucLiteTM, Perkin Elmer), subjected to one round of freeze-thaw and spun at 13,000 rpm for 5 minutes. Liver homogenates were analyzed immediately for luciferase activity using the LucLiteTM assay kit as per manufacturer's instructions or stored at -8O 0 C until use.
• BioPulverizerTM BioSpec
• Pulverized livers were homogenized in 500 ⁇ l of luciferase assay buffer (LucLiteTM, Perkin Elmer), subjected to one round of freeze-thaw and spun at 13,000 rpm for 5 minutes. Liver homogenates were analyzed immediately for luciferase activity using the LucLiteTM assay kit as per manufacturer's instructions or stored at -8O 0 C until use.
• oligos containing six SBEs as shown in SEQ ID NO:10 and SEQ ID NO:11 were ligated 5' to the adenoviral minimal late promoter and the GFP-luciferase chimeric gene.
• the SBE 25 -GL reporter construct was introduced as a transgene into the C57BL6/Balk/c background using both the conventional pronuclear injection method (Gordon et al. (1983) Methods Enzymol., 101 :411-433) and the recently developed lentiviral method (Lois et al. (2002) Science, 295: 868-872).
• TGF- ⁇ inducible SBE 25 -GL reporter transgene expression time course measured using the Xenogen I VISTM system is shown in Table 6. Similarly to the viral reporter vector, the SBE 25 -GL reporter transgene was induced by TGF- ⁇ in a time-dependent manner. Table 6

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• General Health & Medical Sciences (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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• 2005
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